Range Of Calcium Concentrations Research Articles

New promising class of anode materials for Ca-ion battery: polyaromatic hydrocarbons

Using first-principles calculations we propose new anode materials for calcium-ion batteries, namely anthracene (AN), tetracene (TN) and pentacene (PN) crystals. We show that adsorption of calcium atoms on isolated AN, TN and PN molecules is energetically favorable, as well as intercalation into bulk crystals in a wide range of calcium concentrations. For all crystals, volume expansion during intercalation is less than 20% and for pentacene, it is less than 8%. We assess the diffusion and electronic properties of AN, TN and PN. We show that calcium diffusion barriers along the polyacene molecules are less than 0.45 eV, but calcium diffusion is limited by “jumps” between the molecules. Sequential band filling upon increasing levels of intercalation leads to the reentrant semiconducting-metallic-semiconducting behavior. Our results point to the promise of anthracene, tetracene, and pentacene as anodes for calcium-ion batteries.

Establishment of a reference interval for 12-hour fasted serum gastrin concentration in adult dogs.

Adherence to traditional 24-h fasting periods for serum gastrin concentration in dogs can be challenging and may delay the institution of therapies for suspected hypergastrinemia. Peer-reviewed publications regarding serum gastrin reference intervals (RI) are lacking. Hypercalcemia is associated with hypergastrinemia in people; limited data exist in dogs. The objective of the study was to generate a RI for a 12-h fasted serum gastrin concentration in dogs and to investigate whether correlations exist with age, weight, sex, and total calcium concentration. Fifty-five healthy adult dogs (>1 year of age). The screening included: medical history, physical examination, CBC (15 dogs), and serum chemistry (55 dogs). Gastrin was measured via a commercial radioimmunoassay. The RI for 12-h fasted serum gastrin concentration was calculated according to the recommendations of the American Society for Veterinary Clinical Pathology. Additionally, data were evaluated for correlation with selected variables. The RI for serum gastrin following a 12-h fasting period was 15.1-78.9 ng/L with 90% confidence intervals for the lower and upper limits of 14.0-22.9 and 68.3-83.0 ng/L, respectively. A generalized linear model did not detect significant relationships between gastrin and age (P = 0.48), sex (P = 0.30), weight (P = 0.93), or total calcium concentration (P = 0.84). A 12-h fasted serum gastrin concentration RI has been established. Given the limited range of serum calcium concentrations in our healthy study population, additional investigations are needed to determine the effects of hypercalcemia on serum gastrin concentrations in dogs and for any potential clinical consequences thereof.

Rapid detection of serum calcium based on supramolecular aggregates of a cationic perylene in vitro

The steady-state level of serum calcium is of great significance to human body. Accurate and rapid detection of serum calcium is a crucial component of clinical diagnosis and blood calcium census. Current detection of Ca2+ in living environment mainly focus on intracellular imaging, and there are few probes appropriate for serum calcium detection. In this work, we designed a fluorescence sensing system based on the indicator displacement assays (IDAs) strategy using supramolecular complex formed by 3,4,9,10-tetra-(4-trimethylaminobutoxy-carbonyl)-perylene (PBTMA) and casein phosphopeptides (CPPs). Due to the synergistic effect of non-covalent interactions, the complex can be induced by Ca2+ to disintegrate, thus realizing the visual and rapid detection of serum calcium. The quantitative range is 0.01–3.0 mM, while the limit of detection (LOD) and the visual detection limit are 8.4 μM and 0.1 mM, respectively. This method features good stability, selectivity and anti-interference. It can be used for serum samples (recovery rate: 103.4%–106.3%; relative standard deviation (RSD): 2.21%–3.46%). Furthermore, a portable device for rapid serum calcium measurement was designed, which could substantially improve the applicability of this method. To our knowledge, this is the first organic small molecule fluorescent probe capable of application in the range of serum calcium concentrations.

Calcium-dependent strength of the inner-ear cadherin-23 ectodomain.

Cadherin proteins are essential for multicellular organisms and are involved in multiple processes including calcium-dependent cell-cell adhesion and inner-ear mechanotransduction. Cadherin-23 is an integral component of a fibrous protein filament, called the tip link, that is ultimately responsible for converting the mechanical energy of sound into the opening and closing of a gated ion channel that triggers signaling through the auditory nerve. Proper functioning of the tip link depends on its mechanical strength, which is stabilized by interactions of calcium ions with nearby protein residues at discrete triplet binding sites between extracellular cadherin (EC) repeats or so-called linker regions. These binding sites have varying affinity for calcium occupancy, and different calcium configurations at a linker region increase the mechanical rigidity of the tip link to varying degrees. Here we examine the mechanical strength of cadherin-23 repeats across an ensemble of calcium occupancy states at sites labeled as site1, site2, and site3 (ordered by increasing Kd). We do this through a combination of all-atom steered molecular dynamics simulations and by constructing probability distributions for a configuration ensemble which we analyze by Markov chain Monte Carlo. Simulations predict an increase in the tip link's mechanical strength according to an increase in the number of occupied calcium-binding sites. Surprisingly, our analyses also reveal that for the range of cochlear calcium concentration (20 to 50 uM), the most likely configuration involved a single calcium ion at site3, followed by two calcium ions at site2 and site3, then three calcium ions across all sites, and lastly an empty linker region. We discuss these results in the context of tip-link mediated inner ear mechanotransduction.

Calcium is a noncompetitive inhibitor of DMT1 on the intestinal iron absorption process: empirical evidence and mathematical modeling analysis.

Iron absorption is a complex and highly controlled process where DMT1 transports nonheme iron through the brush-border membrane of enterocytes to the cytoplasm but does not transport alkaline-earth metals such as calcium. However, it has been proposed that high concentrations of calcium in the diet could reduce iron bioavailability. In this work, we investigate the effect of intracellular and extracellular calcium on iron uptake by Caco-2 cells, as determined by calcein fluorescence quenching. We found that extracellular calcium inhibits iron uptake by Caco-2 cells in a concentration-dependent manner. Chelation of intracellular calcium with BAPTA did not affect iron uptake, which indicates that the inhibitory effect of calcium is not exerted through intracellular calcium signaling. Kinetic studies performed, provided evidence that calcium acts as a reversible noncompetitive inhibitor of the iron transport activity of DMT1. Based on these experimental results, a mathematical model was developed that considers the dynamics of noncompetitive inhibition using a four-state mechanism to describe the inhibitory effect of calcium on the DMT1 iron transport process in intestinal cells. The model accurately predicts the calcein fluorescence quenching dynamics observed experimentally after an iron challenge. Therefore, the proposed model structure is capable of representing the inhibitory effect of extracellular calcium on DMT1-mediated iron entry into the cLIP of Caco-2 cells. Considering the range of calcium concentrations that can inhibit iron uptake, the possible inhibition of dietary calcium on intestinal iron uptake is discussed.

Hypoparathyroidism Associated with the DNA Variants in Non-Coding Sequence Region of Calcium-Sensing Receptor

A stable narrow range of extracellular calcium concentration in the blood is essential for life. The calcium-sensing receptor (CaSR), a member of the G protein-coupled receptors family, is required to adjust the set point of blood extracellular calcium concentration, thus regulate parathyroid hormone (PTH) secretion and renal calcium excretion. Loss or gain function of CaSR mutations may result in either hyper- or hypocalcaemia. The CaSR activating mutations increase its sensitivity to extracellular ionized calcium (Ca2+). As consequence, PTH synthesis and secretion are suppressed continuously at normal ionized calcium concentrations. Patients display hypocalcaemia, hyperphosphatemia and lower levels of PTH. Urinary calcium excretion is increased due to the decreased circulating inappropriately PTH level and the activation of the renal tubular CaSR. Therefore, CaSR becomes a good potential clinical therapeutic target for hypoparathyroidism treatment. In order to define new drugs and improve medical management of hypoparathyroidism patients, this study attempts to identify new CaSR variants and analyse in detail the functional change of these CaSR variants, thus better understand the molecular mechanism involved. The study is based on collected hypoparathyroidism patients in our clinical site. In the study we enrolled in 10 patients, obtained all their clinical results and DNA results from seven patients. Our results indicated that the effect of serum intact PTH level correlated to change of serum Ca2+ and phosphate level. The CaSR carrying newly identified DNA variants displayed strong phosphorylation of phospholipase C and mitogen-activated protein kinases. Although the size of clinical cases need to be accumulated, current cases have showed a tendency that the identified multiple DNA variants in CaSR gene revealed an effect on the diagnostic criterion of the hypocalcaemic syndrome. It is undeniable that our research has certain limitations. So far, we tested several DNA variants at the same time, further functional examination for individual DNA variant would largely help to be better understand the mechanisms of CaSR regulation on extracellular calcium concentration.

Decreased calcium concentration interferes with life history defense strategies of Ceriodaphnia cornuta in response to fish kairomone

AbstractDeclining calcium in freshwater lakes has become a serious threat to aquatic organisms, which may affect the anti‐fish defense of cladocerans with high calcium demand. To evaluate the effects of low calcium on life history strategies of cladocerans for defending against fish predation, we exposed Ceriodaphnia cornuta to eight concentrations of calcium ranging from 0.5 to 50.0 mg L−1 in the absence and presence of fish kairomone, and observed the changes in body surface, recorded the key life‐history traits, and calculated the degree of its induced defense response. Results showed that, on average, low calcium (0.5, 1.0, 1.5 mg L−1) significantly reduced survival, maternal body size at maturation, and total offspring per female by 66.7%, 8.0%, and 48.8%, respectively, compared with those exposed to normal calcium level (10.0 mg L−1). At the normal calcium level, fish kairomone shortened the time to maturation, reduced maternal body size at maturation, and increased total offspring by 15.7%, 11.0%, and 29.6%, respectively, compared with those not exposed to fish kairomone. In the presence of fish kairomone, the reproduction of C. cornuta was affected by a narrower range of calcium concentrations. Calcium concentrations and fish kairomone showed a significant interaction on maternal body size at maturation, total offspring, and body size of neonates, i.e., low calcium impaired the life history defenses involved in maternal development, but enhanced the defense traits involved in reproductive performance, which indicated the diversified interference effects of decreased calcium on the life history defense strategies of C. cornuta against fish predators.

Activation of platelet-rich plasma by pulse electric fields: Voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin.

Activated platelet-rich plasma (PRP) has been used in the clinical settings of wound healing and regenerative medicine, with activation typically induced by the addition of bovine thrombin. To eliminate issues with availability, cost and potential side effects associated with bovine thrombin, ex vivo PRP activation using pulse electric fields (PEF) has been proposed and demonstrated. The present study characterizes the effect of PEF voltage and pulse width, in combination with a range of calcium concentrations, on clot formation, growth factor release, and serotonin (5-HT) release from dense granules. The main findings are: 1) increasing calcium concentrations with most PEF conditions leads to increased levels of PDGF and 5-HT release; 2) whether EGF levels increase or decrease with increasing calcium concentration depends on the specific PEF parameters; 3) the pattern of PDGF and EGF levels in supernatants suggest that these molecules are localized differently within platelets; 4) significant levels of PDGF, EGF, and 5-HT can be released without inducing clot formation or hemoglobin release. In conclusion, voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin from PEF-activated PRP. Because growth factor requirements vary for different types of wounds and for wounds at different stages of healing, the unique balance of factors in supernatants of PEF-activated PRP may provide more clinically advantageous than the current standard of bovine thrombin-activated PRP.

Impact of Ca(II) on the aqueous speciation, redox behavior, and environmental mobility of Pu(IV) in the presence of EDTA

The impact of calcium on the solubility and redox behavior of the Pu(IV)-EDTA system was investigated using a combination of undersaturation solubility studies and advanced spectroscopic techniques. Batch solubility experiments were conducted in 0.1 M NaCl-NaOH-HCl-EDTA-CaCl2 solutions at constant [EDTA] = 1∙10−3 M, 1 ≤ pHm ≤ 11, and 1∙10−3 M ≤ [CaCl2] ≤ 2∙10−2 M. Additional samples targeted brine systems represented by 3.5 M CaCl2 and WIPP simulated brine. Redox conditions were buffered with hydroquinone (pe + pH ≈ 9.5) with selected samples prepared in the absence of any redox buffer. All experiments were performed at T = 22 °C under Ar atmosphere. In-situ X-ray absorption spectroscopy indicated that PuO2(ncr,hyd) was the solubility-controlling phase during the lifetime of all experiments and that aqueous plutonium was present in the +IV oxidation state across all experimental conditions except at pHm ≈ 1, where a small fraction of Pu(III) was also identified. Current thermodynamic models overestimate Pu(IV)-EDTA solubility in the absence of calcium by approximately 1–1.5 log10-units and do not describe the nearly pH-independent, increased solubility observed with increased calcium concentrations. The ternary Pu(IV)-OH-EDTA system without calcium was reevaluated using solubility data obtained in this work and reported in the literature. An updated thermodynamic model including the complexes Pu(OH)(EDTA)−, Pu(OH)2(EDTA)2−, and Pu(OH)3(EDTA)3− was derived. Solubility data collected in the presence of calcium follows a pH-independent trend (log m(Pu)tot vs. pHm), which can only be explained by assuming the formation of a quaternary complex, tentatively defined as CaPu(OH)4(EDTA)2−, in solution. The significant enhancement of plutonium solubility observed in the investigated brine systems supports the formation of a quaternary complex that is not outcompeted by Ca(EDTA)2−, even in concentrated CaCl2 solutions. Although the exact stoichiometry of the complex may need to be revisited, this new quaternary complex has a pronounced impact on plutonium predominance diagrams over a broad range of pH, pe, and calcium concentrations that are relevant to nuclear waste disposal.

Calcium-enhanced retention of humic substances by carbon nanotube membranes: Mechanisms and implication

Efficient removal of humic substances (HS) in natural water is of importance to drinking water treatment. In this study, the adsorption and retention of HS by multi-walled carbon nanotube (MWCNT) membranes were systematically investigated by dynamic filtration of synthetic and natural surface water. In the absence of Ca(II), HS were dominated by small, dissolved species albeit the varying pH. Accordingly, HS retention by the MWCNT layers only ranged from 15%-65% at the end of the filtration. In contrast, the presence of Ca(II) in the feed water partially transformed HS molecules into colloidal aggregates as found by light scattering analyses. Furthermore, molecular dynamics (MD) simulation results reveal that Ca(II) complexation with -COO- on MWCNT and humic acid (HA) not only leads to HA aggregation in the feed solution, but also promotes HA adsorption onto carboxylated MWCNT. The modeling results are consistent with the high retention of HS by the carboxylated MWCNT membrane, i.e., >90% for the synthetic model water and >85% for the natural water, at a moderate calcium concentration range of 0.5–2.0 mM. Considering the widespread presence of calcium in natural water, these findings suggested that carboxylated MWCNT has a potential for effective adsorptive filtration of HS in drinking water.

The effect of hydrophobic alkyl sidechains on size and solution behaviors of nanodiscs formed by alternating styrene maleamic copolymer

The development of amphipathic polymers, including various formulations of styrene-maleic acid (SMA) copolymers, has allowed the purification of increasing sizes and complexities of biological membrane protein assemblies in native nanodiscs. However, the factors determining the sizes and shapes of the resulting bio-nano particles remain unclear. Here, we show how grafting on short alkyl amine sidechains onto the polar residues leads to a broad set of nanoparticle sizes with improved solution behavior. The solubilization of lipid vesicles occurs over a wide range of pH levels and calcium concentrations, providing utility across the physiologically relevant range of solution conditions.Furthermore, the active SMA derivatives can contain strictly alternating monomers, which have inherently lower sequence polydispersity. Pronounced differences in the shapes of native nanoparticles were formed from Escherichia coli bacterial outer membrane containing PagP protein using methyl, ethyl and propylamine derivatives of styrene-maleic anhydride. In particular, the methylamine-substituted polymer forms smaller, monodispersed nanodiscs, while the longer alkyl derivatives form worm-like nanostructures. Thus the introduction of hydrophobicity onto the polar sidechains of amphipathic polymers has profound effects on morphology of native nanodisc, with shorter methyl moieties offering more uniformity and utility for structural biology studies.

Non-toxic and toxic Microcystis aeruginosa reduce the tolerance of Daphnia pulex to low calcium in different degrees: Based on the changes in the key life-history traits

Calcium decline and cyanobacterial blooms pose a serious threat to the crustacean zooplankton Daphnia, which has a high demand for calcium. In the present study, we exposed two different clones of Daphnia pulex to different combinations of calcium concentrations (0.1, 0.5, 1.0, 5.0, 10.0 mg L−1) and food types (100% Chlorella; 80% Chlorella and 20% non-toxic Microcystis; 80% Chlorella and 20% toxic Microcystis) for 16 days, recorded the key life-history traits, and then used an exponential rise function to fit the traits. Results showed toxic Microcystis and low calcium together negatively affected the survival, development, and reproduction of Daphnia. The negative effect of non-toxic Microcystis and low calcium only affected the development and reproduction. The survival time and reproductive performance increased exponentially with increasing calcium concentration and then approached an asymptotic maximum. Both non-toxic and toxic Microcystis reduced the asymptotic maximum of the reproductive performance. The rising rate at which they reached the asymptotes differed significantly among the three food types; i.e., the reproductive performance of Daphnia was affected in a wider range of calcium concentrations under bad food quality. The findings indicated that Microcystis impaired the tolerance of Daphnia to low calcium, which may cause serious consequences in freshwater ecosystems.

Stopped-Flow Calcium Kinetics of Hypertrophic Cardiomyopathy-Associated Troponin T Mutations

Hypertrophic cardiomyopathy (HCM) is a complex genetic disorder of the cardiac sarcomere affecting 1/500 individuals worldwide. The cardiac thin filament (CTF) protein, cardiac troponin T (cTnT), is known to play a critical role in the regulation of contraction, and mutations in cTnT often cause HCM. This study focuses on 4 HCM-causing, clinically relevant point mutations located within the cTnT N-terminus (R92L/Q/W and I79N) which are each associated with distinct phenotypes and disease severity. Despite this, they exhibit comparable changes in steady-state calcium sensitivity of both force generation and myosin ATPase activity. Thus, we monitored the rates of calcium association to and dissociation from IAANS-labeled, Cys-substituted cardiac troponin C in CTFs to explain the comparable changes in steady-state calcium sensitivity. We and others previously characterized mutation-specific differences in calcium dissociation rates for the R92 mutations in CTFs. I79N CTFs significantly increased the rate of calcium dissociation from 116.0±3.2s−1 measured in WT CTFs to 136.1±6.1s−1. The observed calcium association rates to WT, R92L, R92W, and I79N CTFs were measured over a range of calcium concentrations (2.5µM-20µM) and were found to be 9.88±2.051 x106 M−1s−1, 9.141±1.94 x106 M−1s−1, 31.37±2.948 x106 M−1s−1, and 28.2±2.736 x106 M−1s−1, respectively. Both I79N and R92W, but not R92L, significantly increased the rate of calcium association compared to WT CTFs. Furthermore, both I79N and R92W, but not R92L, trend with increased responsiveness to artificially evoked calcium transients compared to WT. These data show that in addition to changing the rate of calcium dissociation, mutations in cTnT can significantly alter the rate of calcium association to affect CTF calcium sensitivity. Together this suggests that alterations in CTF calcium kinetics may represent a highly mutation-specific mechanism of disease initiation and are thus a target for potential therapeutic intervention.

Omecamtiv mecarbil lowers the contractile deficit in a mouse model of nebulin-based nemaline myopathy.

Nemaline myopathy (NEM) is a congenital neuromuscular disorder primarily caused by nebulin gene (NEB) mutations. NEM is characterized by muscle weakness for which currently no treatments exist. In NEM patients a predominance of type I fibers has been found. Thus, therapeutic options targeting type I fibers could be highly beneficial for NEM patients. Because type I muscle fibers express the same myosin isoform as cardiac muscle (Myh7), the effect of omecamtiv mecarbil (OM), a small molecule activator of Myh7, was studied in a nebulin-based NEM mouse model (Neb cKO). Skinned single fibers were activated by exogenous calcium and force was measured at a wide range of calcium concentrations. Maximal specific force of type I fibers was much less in fibers from Neb cKO animals and calcium sensitivity of permeabilized single fibers was reduced (pCa50 6.12 ±0.08 (cKO) vs 6.36 ±0.08 (CON)). OM increased the calcium sensitivity of type I single muscle fibers. The greatest effect occurred in type I fibers from Neb cKO muscle where OM restored the calcium sensitivity to that of the control type I fibers. Forces at submaximal activation levels (pCa 6.0-6.5) were significantly increased in Neb cKO fibers (~50%) but remained below that of control fibers. OM also increased isometric force and power during isotonic shortening of intact whole soleus muscle of Neb cKO mice, with the largest effects at physiological stimulation frequencies. We conclude that OM has the potential to improve the quality of life of NEM patients by increasing the force of type I fibers at submaximal activation levels.

Extracellular calcium regulates the adhesion and migration of osteoclasts via integrin αv β 3 /Rho A/Cytoskeleton signaling.

Integrin αv β3 is a transmembrane integrin, which can initiate osteoclasts' attachment on bones, leading to downward signaling pathways and subsequent bone resorption. Different calcium concentrations have been reported to have an influence on the activation of integrin αv β3 . To elucidate the regulatory mechanism of extracellular calcium concentrations on osteoclasts, a controlled micro flow plate (M04S) was utilized in the ONIX flow control system to observe the osteoclasts' adhesion and migration in different calcium concentration media. Fluorescent staining is conducted to show the distribution of integrin αv β3 and cytoskeleton reorganization. In addition, western blots were performed to detect the expression of integrin αv β3 and its downstream signaling pathways related to bone resorption. Also, real-time reverse-transcription polymerase chain reaction data of transcription co-activator (YAP/TAZ) and hydrolytic enzymes (the matrix metalloproteinase 9 and cathepsin K) areevaluated. Our findings suggest that osteoclasts' migration and adhesion is better promoted at 0.5 mM than 1.2 mM, which can be partly explained by the induced cytoskeleton organization via integrin αv β3 /Rho GTPase. But the activation and nuclear localization of YAP/TAZ, and the secretion of hydrolytic enzymes were upregulated when the calcium concentration is at a higher level (1.2 mM). According to our study, there is a highpossibility that the migration and attachment of osteoclasts and subsequent osteoclastic bone resorption are regulated over a specific range of extracellular calcium concentration.

SERCA Activity Controls the Systolic Calcium Increase in the Nucleus of Cardiac Myocytes.

In cardiomyocytes, nuclear calcium is involved in regulation of transcription and, thus, remodeling. The cellular mechanisms regulating nuclear calcium, however, remain elusive. Therefore, the aim of this study was to identify and characterize the factors that regulate nuclear calcium in cardiomyocytes. We focused on the roles of (1) the cytoplasmic calcium transient (CaT), (2) the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and (3) intracellular calcium stores for nuclear calcium handling. Experiments were performed on rat ventricular myocytes loaded with Fluo-4/AM. Subcellularly resolved CaTs were visualized using confocal microscopy. The cytoplasmic CaT was varied by reducing extracellular calcium (from 1.5 to 0.3 mM) or by exposure to isoprenaline (ISO, 10 nM). SERCA was blocked by thapsigargin (5 μM). There was a strict linear dependence of the nucleoplasmic CaT on the cytoplasmic CaT over a wide range of calcium concentrations. Increasing SERCA activity impaired, whereas decreasing SERCA activity augmented the systolic calcium increase in the nucleus. Perinuclear calcium store load, on the other hand, did not change with either 0.3 mM calcium or ISO and was not a decisive factor for the nucleoplasmic CaT. The results indicate, that the nucleoplasmic CaT is determined largely by the cytoplasmic CaT via diffusion of calcium through nuclear pores. They identify perinuclear SERCA activity, which limits the systolic calcium increase in the nucleus, as a novel regulator of the nuclear CaT in cardiac myocytes.

MthK Channel Activity in Planar Tetraether Lipid Membranes

Archaeal ion channels have proven to be robust model systems for linking channel function with high-resolution structures. However, electrophysiological studies of these channels have relied on reconstitution of purified channels in E.coli-like bilayer membranes, whereas the native archaeal membrane environment often consists of monolayers containing tetraether lipids. Using the partially purified lipid fraction polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon S. acidocaldarius, as well as lipids extracted from the microvesicles (MV) secreted by S. acidocaldarius we have measured properties of planar membranes of tetraether lipids. These membranes exhibited a high resistance and stable capacitance much longer than membranes painted using diester lipids, with a lifetime up to 11 days for PLFE membranes. In contrast, bilayer membranes of POPC or DMPC exhibited more or less stable capacitances and lasted typically only up to 3 hours. We have compared activity of the archaeal potassium channel MthK (from Methanothermobacter thermoautotrophicum) in a tetraether lipid planar membrane system to values recorded in diester lipid bilayers. MthK is an ion channel that has been extensively characterized in conventional diester lipid membranes, and used as a model for calcium gated potassium channels. However, physiologically, the channel is embedded in a system partially composed of tetraether lipids. We have found that vesicle membranes containing purified MthK channel can fuse with tetraether planar membranes, to yield channel activity. We also quantified channel activity over a range of calcium concentrations, as well as channel response to antagonists, such as barium, in tetraether lipid planar membranes. (Supported by NSF)

High tension in sarcomeres hinders myocardial relaxation: A computational study.

Experiments have shown that the relaxation phase of cardiac sarcomeres during an isometric twitch is prolonged in muscles that reached a higher peak tension. However, the mechanism is not completely understood. We hypothesize that the binding of calcium to troponin is enhanced by the tension in the thin filament, thus contributing to the prolongation of contraction upon higher peak tension generation. To test this hypothesis, we developed a computational model of sarcomere mechanics that incorporates tension-dependence of calcium binding. The model was used to simulate isometric twitch experiments with time dependency in the form of a two-state cross-bridge cycle model and a transient intracellular calcium concentration. In the simulations, peak isometric twitch tension appeared to increase linearly by 51.1 KPa with sarcomere length from 1.9 μm to 2.2 μm. Experiments showed an increase of 47.3 KPa over the same range of sarcomere lengths. The duration of the twitch also increased with both sarcomere length and peak intracellular calcium concentration, likely to be induced by the inherently coupled increase of the peak tension in the thin filament. In the model simulations, the time to 50% relaxation (tR50) increased over the range of sarcomere lengths from 1.9 μm to 2.2 μm by 0.11s, comparable to the increased duration of 0.12s shown in experiments. Model simulated tR50 increased by 0.12s over the range of peak intracellular calcium concentrations from 0.87 μM to 1.45 μM. Our simulation results suggest that the prolongation of contraction at higher tension is a result of the tighter binding of Ca2+ to troponin in areas under higher tension, thus delaying the deactivation of the troponin.

Respective optimal calcium concentrations for proliferation on type I collagen fibrils in two keratinocyte line cells, HaCaT and FEPE1L-8.

Keratinocyte line cells HaCaT and FEPE1L-8 are used for skin model with type I collagen fibrils (gels). For this purpose, not only differentiation but also regulation of proliferation on type I collagen gels by exogenous calcium concentration is important. When exogenous calcium concentration is low, primary keratinocyte proliferation is repressed and eventually cells are induced to apoptosis on type I collagen gels. The apoptosis induced on type I collagen gels is suppressed by increasing calcium concentration in the medium. That is, higher exogenous calcium concentration is necessary for primary keratinocyte survival on type I collagen gels than for that on dish surface culture. Meanwhile much higher exogenous calcium causes cell differentiation and inhibition of proliferation. The optimal calcium concentrations for proliferation on type I collagen gels have not been clarified in keratinocyte line cells. HaCaT cells have a unique calcium sensitivity in comparison with primary keratinocytes, whereas FEPE1L-8 cells have a similar sensitivity to primary keratinocytes. In this study, we compared the effect of calcium concentrations on proliferation of HaCaT and FEPE1L-8 cells on type I collagen gels. On type I collagen gels, both line cells required higher calcium concentrations for proliferation than on dish surface. HaCaT cells proliferated better in a wider range of calcium concentrations than FEPE1L-8 cells.

Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.

Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions.