Free Calcium Concentration Research Articles

Free Calcium Concentration, Calpain-2 Activity, and Final Product Tenderness of Electrically Stimulated Beef

The objective of this study was to evaluate the effect of timing of electrical stimulation on free calcium concentration, calpain-2 activity, Warner-Bratzler shear force (WBSF), and consumer sensory analysis. Twenty-three beef steers were harvested and stimulated (S) using extra-low voltage or not stimulated (NS), at exsanguination and/or 1 h postmortem, resulting in 4 different stimulation treatments: NS-NS, NS-S, S-NS, or S-S. Samples were removed from the longissimus lumborum (LL) and semimembranosus (SM) for free calcium and calpain-2 analysis on days 1, 4, and 14 postmortem. WBSF and sensory analysis steaks were removed on day 4 and frozen (4 d) or aged to 14 d postmortem. Data were analyzed using the mixed model or generalized linear mixed model procedure of SAS (SAS Institute, Inc., Cary, NC), with significance determined at P < 0.05. There was a tendency for an aging-period-by-stimulation-treatment interaction for LL free calcium concentration (P = 0.05), and there was a significant difference between aging periods (P < 0.01). No difference was observed in free calcium concentration in the SM between stimulation treatments (P = 0.44); aging, however, significantly increased SM free calcium concentration (P < 0.01). Stimulation did not impact native calpain-2 activity in the LL (P = 0.71) or SM (P = 0.89). Stimulation treatment did not improve tenderness values for WBSF analysis for the LL (P = 0.69) or SM (P = 0.61) or consumer sensory analysis in the LL (P = 0.56) or SM (P = 0.36). A longer aging period tended to increase calpain-2 activity in the SM (P = 0.08), improve WBSF in the LL (P = 0.09), and significantly improve consumer tenderness scores in the SM (P < 0.01). In conclusion, the timing of electrical stimulation utilized in the current study tended to influence free calcium concentration in the LL but did not influence calpain-2 activity or beef tenderness. Aging, however, improved tenderness.

Transition dairy cow health is associated with first postpartum ovulation risk, metabolic status, milk production, rumination, and physical activity

Our objective was to determine the association of health status during the first 60 d in milk (DIM) and first postpartum ovulation risk, physical activities recorded by an activity monitor, and metabolic and milk measures in Holstein cows. Late-gestation heifers and close-up dry cows in 1 herd fitted with CowManager SensOors (Agis, Harmelen, the Netherlands) were enrolled in the study 3 wk before expected parturition to assess ear skin temperature and daily rumination, eating, inactivity, and activity times. Blood samples were collected at calving (d 0), and on d 3, 7, and 14 to assess concentrations of free fatty acids, β-hydroxybutyrate (BHB), calcium, glucose, and haptoglobin. In addition, weekly measures were conducted for body condition, body weight, and progesterone through 63 ± 3 DIM when ovulation was synchronized (GnRH-1 - 7 d - PGF2α - 24 h - PGF2α - 32 h - GnRH-2 - 16 h - artificial insemination). Disease diagnosed in 68 of 160 cows (42.5%) was distributed equally between primiparous (48.5%) and multiparous (51.5%) cows. Cows were classified as diseased when any case of metritis, digestive disorders, ketosis, hypocalcemia, calving problems, mastitis, or lameness occurred during the first 60 DIM. Odds of early ovulation by median postpartum d 33 was 1.92 times greater in healthy than diseased cows. Incidence of individual diseases included metritis (18.8%), digestive disorders (17.5%), ketosis (BHB >10 mg/dL; 11.9%), hypocalcemia (Ca <2.2 mmol/L; 10.6%), calving problems (6.3%), mastitis (3.1%), and lameness (3.1%). Odds of early ovulation were 2.48, 2.65, and 5.72 times greater in healthy cows compared with cows diagnosed with metritis, digestive disorders, or ketosis, respectively. Diseased compared with healthy cows had greater concentrations of free fatty acids, BHB, haptoglobin, greater rectal temperature, and lesser concentration of serum calcium on d 0, 3, 7, and 14 than healthy cows. Plasma glucose was not affected by health status, but was lesser in concentration on d 3, 7, and 14 compared with day of calving. Weekly (calving through 9 wk) body condition scores tended to be and weekly body weights were greater in healthy compared with diseased cows. Activity measures differed by health status during prepartum (d -14 through -1) and postpartum (d 0 through 20) periods except for eating time. Healthy cows spent less time being inactive during both periods compared with diseased cows and had greater postpartum rumination times than diseased cows. Mean daily milk yield during the first 14 wk in milk was greater in healthy than diseased cows by 2.1 ± 0.8 kg. We conclude that disease negatively affects early postpartum ovulation risk and is associated with measurable changes in periparturient physical activity and postpartum metabolic profiles.

Design and Application of a Rotatory Device for Detecting Transient Ca2+ Signals in Response to Mechanical Stimulation Using an Aequorin-Based Ca2+ Imaging System.

Elevation of the cytosolic free calcium ion (Ca2+ ) concentration ([Ca2+ ]cyt ) is one of the earliest responses to biotic and abiotic stress in plant cells. Among the various Ca2+ detection systems available, aequorin-based luminescence Ca2+ imaging systems provide a relatively amenable and robust method that facilitates large-scale genetic-mutant screening based on [Ca2+ ]cyt responses. Compared to that mediated by chemical elicitors, mechanical stimulation-induced elevation of [Ca2+ ]cyt is considerably more rapid, occurring within 10 s following stimulation. Therefore, its assessment using aequorin-based Ca2+ imaging systems represents a notable challenge, given that a time interval of ≥1 min is required to reduce the background light before operating the photon imaging detector. In this context, we designed a device that can rotate automatically within the confines of an enclosed dark box, and using this, we can record [Ca2+ ]cyt dynamics immediately after plants had been rotated to induce mechanical stimulation. This tool can facilitate the study of perception and early signal transduction in response to mechanical stimulation on a large scale based on [Ca2+ ]cyt responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of background luminance signals in aequorin-transformed Arabidopsis seedlings using a photon imaging detector Basic Protocol 2: Construction of the rotatory device Basic Protocol 3: Calcium measurement in Arabidopsis seedlings after rotatory stimulation Basic Protocol 4: Data analysis and processing.

Effects of Glycosaminoglycan from Urechis unicinctus on ADP-Induced Platelet Calcium and Membrane Glycoprotein Expressions in Rats

Background: Previous studies had shown that glycosaminoglycan (GAG) from Urechis unicinctus exerts an obvious antiplatelet aggregation effect. Objective: This study aims to investigate the effects of GAG from Urechis unicinctus on ADP-induced platelet calcium and membrane glycoprotein expressions in rats. Methods: Fura-2/AM fluorescence probe was used to measure intracytosolic free-calcium concentration ([Ca²⁺]_i) of platelets and calculate platelet calcium influx and release concentrations. Flow cytometry was used to detect the expressions of platelet membrane glycoproteins GPIIb/IIIa (PAC-1) and P-selectin (CD62P) in rats. Results: The results showed that the GAG from U. unicinctus significantly inhibited the release of platelet calcium (p < 0.01) and the expressions of platelet GPII b/IIIa and P-selectin (p < 0.01) induced by ADP in rats but had no significant effect on the influx of platelet calcium (p > 0.01). Conclusions: This study indicated that GAG may inhibit platelet activation and aggregation by reducing the release of Ca²⁺ and ADP-induced expression of platelet membrane glycoprotein in rats.

Calcium balance during direct acidification of milk for Mozzarella cheese production

Calcium is an important nutrient but also contributes to the texture, taste and functionality of most dairy products. The balance between micellar and free calcium in curd and serum phase during direct acidification of bovine milk was studied by changing organic acid type, pH and acidification temperature. Concentration of free calcium increased with decreasing pH and increasing temperature. Citric acid was more efficient than lactic acid at lowering free calcium concentration, while temperature effect was larger for acidification with lactic acid. These findings are rationalized by the stronger binding of calcium to citrate compared to lactate with an almost athermal binding for citrate and an endothermal binding for lactate. Curd yield decreased by decreasing pH as solubilization of colloidal calcium phosphate causes loss of minerals into the serum phase decreasing the concentration of both calcium and phosphate left in the curd, while acidification temperature had little effect on curd yield. These results are important to improve yield and tailor the properties and functionality of dairy products, such as for example Mozzarella cheese.

Sanguinarine Rapidly Relaxes Rat Airway Smooth Muscle Cells Dependent on TAS2R Signaling.

Excessive contraction of airway smooth muscle cells (ASMCs) is a hallmark feature of asthma. Intriguing, the activation of bitter taste receptor (TAS2R) in ASMCs can relax ASMCs. However, there is a lack of potent TAS2R agonists that can be used in asthma therapies since those tested agonists cannot relax ASMCs at the dose below a few hundred micromolar. Considering that sanguinarine (SA) is a bitter substance often used in small doses for the treatment of asthma in folk medicine, the present study was to determine the rapid relaxation effect of SA on ASMCs and to reveal the underlying mechanisms associated with TAS2R signaling. Here, cell stiffness, traction force, calcium signaling, cAMP levels, and the mRNA expression were evaluated by using optical magnetic twisting cytometry, traction force microscopy, Fluo-4/AM labeling, enzyme-linked immunosorbent assay (ELISA), and quantitative (q)RT-PCR, respectively. We found that 0.5 µM SA immediately decreased cell stiffness and traction force, which is comparable with the effect of 5 µM isoproterenol. In addition, 0.5 µM SA immediately increased intracellular free calcium concentration ([Ca2+]i) and decreased the mRNA expression of contractile proteins such as calponin and α-smooth muscle actin after the treatment for 24 h. Furthermore, SA-mediated decrease in cell stiffness/traction force and increase in [Ca2+]i were significantly blunted by inhibiting the TAS2Rs signaling. These findings establish the rapid relaxation effect of SA at low concentration (<1 µM) on cultured ASMCs depending on TAS2R signaling, indicating that SA might be developed as a useful bronchodilator in asthma therapy.

Lanthanum and Cerium Toxicity to the Freshwater Green Alga Chlorella fusca: Applicability of the Biotic Ligand Model.

The environmental risk assessment of rare earth elements (REEs) requires data on their potential toxicity. In the present study, the toxicity of lanthanum (La) and cerium (Ce) was studied in relation to metal speciation in solution. For both La and Ce, the use of organic ligands demonstrated that the calculated free ion concentration was a good indicator of toxicity. Whether in the absence or presence of organic ligands, when based on free ion concentrations, the obtained half-maximal effective concentrations were similar. When all generated data were pooled, Ce and La showed identical toxicity thresholds after 120 h of exposure with free ion concentration-based median effective concentration values (95% confidence intervals) of 0.48 (0.38-0.60) µM and 0.47 (0.36-0.61) µM for La3+ and Ce3+ , respectively. The inhibition of algal growth was also correlated with the intracellular lanthanide concentrations, regardless of the ligand used. Finally, increasing the ambient calcium concentration protected the test algae by reducing the amount of lanthanide internalized into the cells. These results suggest that, at constant pH (5.5), REE accumulation and toxicity are linked to the free ion concentration and ambient calcium concentration, as predicted by the biotic ligand model. Environ Toxicol Chem 2020;39:996-1005. © 2020 SETAC.

Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration.

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

Abnormal Sarcoplasmic Reticulum/Mitochondria Ca2+ Microdomains Impairs Mitochondrial Ca2+ in Cardiomyocytes in Aging

Mitochondrial (Mito) metabolism is regulated by free calcium concentration ([Ca2+]m) in the mitochondrial matrix. Cardiac function is impaired in aging and is associated with Mito disfunction. [Ca2+]m homeostasis depends on the Mito calcium uniporter complex and functional sarcoplasmic reticulum (SR)/Mito calcium microdomains. We hypothesized that Mito dysfunction could be the result of aging‐induced maladaptive changes in protein levels of key proteins of the SR/Mito microdomain resulting in deficient Mito Ca2+ uptake. Therefore, the objective of this work was to determine the protein levels of key members of the SR/Mito microdomain and analyze Mito Ca2+ handling as well as spatial SR‐Mito interaction using the proximity ligation assay. We measured protein levels by Western blot of MFN2, GRP75 and SERCA2a, in hearts of young (YH; 2–3 months) and old (OH; 19–22 months) mice. In addition, we analyzed Mito Ca2+ uptake, release and [Ca2+]m using Mitycam and Mito‐pericam. Our results showed that the SR‐Mito Ca2+ microdomain tethering proteins MFN2 and GRP75 were decreased in the OH by 55% and 60% respectively. SERCA2a, the key mediator of SR Ca2+ loading was also decreased by 41% in the OH. SR‐Mito interaction was dramatically diminished in OH. Consequently, Mito Ca2+ uptake and release as well as [Ca2+]m were significantly decreased. In vivo cardiac AAV‐based expression MFN2 improved SR‐Mito interaction. Adenoviral MFN2 expression in cardiomyocytes increased [Ca2+]m.In conclusion, SR/Mito interaction is impaired in the cardiomyocyte in aging. This results in abnormal Mito Ca2+ homeostasis and can be improved by expression of MFN2.Support or Funding InformationAuthors acknowledge funding from a Merit Grant from Veterans Affairs system I01BX003429. This work was supported by the National Institute on Aging, Grant T35 AG26757 (PI: Dilip V. Jeste, MD), and the Stein Institute for Research on Aging and the Center for Healthy Aging at the University of California, San Diego. Angelica Suarez was a 2018 UC San Diego MSTAR Student.

How to measure myofilament calcium sensitivity? Theory and practical applications

In the heart, as well as in smooth muscle cells (SMC) of blood vessels, contractility is mainly determined by the concentration of free calcium [Ca2+]i. in myoplasm. Changes in the sensitivity of myofilaments to calcium can be very important in the regulation of muscle contractility. Currently, it is precisely this way of influence on the contractile properties of the heart and vascular muscles that attracts much attention of pharmacologists. Studies of the nature of calcium sensitivity can be the fundamental basis for the development of a new generation of drugs that affect vascular tone or contractility of the heart through direct effects on the contractile apparatus or indirectly affecting signaling pathways that regulate vascular tone. The dependence of the magnitude of the contraction F from [Ca2+]i with a good degree of accuracy is described by the Hill equation F = Fmax * [Ca2+]in / (EC50n + [Ca2+]in), which is uniquely determined by three parameters: the maximum contraction Fmax, the concentration of half contraction EC50, and the Hill coefficient n. The main goal of the experiments to determine the changes in the calcium sensitivity of the contractile apparatus of the SMC during any external influences including conducting screening trials of potential drugs should be to obtain the dependencies of the contraction force F on [Ca2+]i. before and after its exposure. To correctly evaluate changes in the calcium sensitivity of myofilaments, it is necessary to measure at least changes in three parameters of the obtained dependences: the generalized affinity of myofilaments for [Ca2+]i, which is characterized as EC50, the sensitivity of myofilaments to calcium, or Fmax, and Hill coefficient. Optimal experimental design for assessing changes in the calcium sensitivity of the vascular contractile apparatus includes: recording the contractile activity of intact smooth muscles, recording the contractile activity of chemically skinned vascular preparations, as well as simultaneously measuring [Ca2+]i, and contractile force using fluorescent calcium indicators. The complex of these experiments will enable a step-by-step assessment of the ability of any external impact, including pharmacological intervention, to change the calcium sensitivity of the contractile apparatus of SMC and can serve as an effective tool for the initial screening of compounds claiming to be calcium sensitizers or desensitizers.

The Myrosinases TGG1 and TGG2 Function Redundantly in Reactive Carbonyl Species Signaling in Arabidopsis Guard Cells.

Myrosinase (β-thioglucoside glucohydrolase, enzyme nomenclature, EC 3.2.1.147, TGG) is a highly abundant protein in Arabidopsis guard cells, of which TGG1 and TGG2 function redundantly in abscisic acid (ABA)- and methyl jasmonate-induced stomatal closure. Reactive carbonyl species (RCS) are α,β-unsaturated aldehydes and ketones, which function downstream of reactive oxygen species (ROS) production in the ABA signalling pathway in guard cells. Among the RCS, acrolein is the most highly reactive, which is significantly produced in ABA-treated guard cells. To clarify the ABA signal pathway downstream of ROS production, we investigated the responses of tgg mutants (tgg1-3, tgg2-1 and tgg1-3 tgg2-1) to acrolein. Acrolein induced stomatal closure and triggered cytosolic alkalization in wild type (WT), tgg1-3 single mutants and in tgg2-1 single mutants, but not in tgg1-3 tgg2-1 double mutants. Exogenous Ca2+ induced stomatal closure and cytosolic alkalization not only in WT but also in all of the mutants. Acrolein- and Ca2+-induced stomatal closures were inhibited by an intracellular acidifying agent, butyrate, a Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA) and a Ca2+ channel blocker, LaCl3. Acrolein induced cytosolic free calcium concentration ([Ca2+]cyt) elevation in guard cells of WT plants but not in the tgg1-3 tgg2-1 double mutants. Exogenous Ca2+ elicited [Ca2+]cyt elevation in guard cells of WT and tgg1-3 tgg2-1. Our results suggest that TGG1 and TGG2 function redundantly, not between ROS production and RCS production, but downstream of RCS production in the ABA signal pathway in Arabidopsis guard cells.

Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y2R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca2+). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling.

Fractional-order mathematical model for calcium distribution in nerve cells

Calcium (Ca$$^{2+}$$) ions are known as a second messenger, involved in a variety of signalling process. It regulates various physiological processes like signal transduction, proliferation, etc. Calbindin-D$$_{28k}$$ reacts with free Ca$$^{2+}$$ ions and significantly lowers down the cytosolic free calcium concentration ([Ca$$^{2+}$$]) in nerve cell. Voltage-gated calcium channel (VGCC) works as an outward source of Ca$$^{2+}$$ which initiates and sustains Ca$$^{2+}$$ signalling process for the smooth functioning of the cells. An alteration in Ca$$^{2+}$$ signalling process leads to the early symptoms of Parkinson’s disease (PD). In this piece of work two major analyses has done. First, we have developed a mathematical model in the form of a fractional reaction–diffusion equation. Second, the obtained results are interpreted with a neuronal disorder, i.e., PD. Hence the physiological role of calbindin-D$$_{28k}$$ and VGCC in PD is analyze using mathematical model by incorporating all important parameters like diffusion coefficient, fluxes, etc. Appropriate initial and boundary conditions are used according to the biophysical conditions of the cell.

Curcumin diminishes cisplatin-induced apoptosis and mitochondrial oxidative stress through inhibition of TRPM2 channel signaling pathway in mouse optic nerve

Background: Cisplatin (CiSP), a chemotherapeutic agent, is widely used to treat several types of cancers. However, its clinical use is limited due to adverse side effects caused by excessive production of reactive oxygen species (ROS) and death of neurons. The transient receptor potential (TRP) melastatin 2 (TRPM2) cation channel is activated by ADP-ribose (ADPR) and ROS. The protective effect of curcumin (CURCU) against CiSP-induced apoptosis and mitochondrial ROS through inhibition of TRP channels in several types of neuron except optic nerve, was recently reported. The aim of the current study is to clarify the protective effect of CURCU on CiSP-induced mitochondrial oxidative injury and TRPM2 activation in the mice optic nerve and SH-SY5Y human derived neuronal cells.Material and methods: The SH-SY5Y cells and mice were divided into four groups: Control, CURCU, CiSP, and CURCU + CiSP. The mice were treated for 14 days and the cells were incubated with CiSP and CURCU for 24 h.Results: CURCU and PARP-1 inhibitor (PJ34) treatments ameliorated CiSP-induced mitochondrial membrane depolarization, mitochondrial and cytosolic ROS levels and neuronal death in the optic nerve. In the patch-clamp of SH-SY5Y cells and laser confocal microscopy experiments of optic nerve, CURCU and TRPM2 blocker treatments also decreased ADPR-induced TRPM2 currents and cytosolic free calcium ion (Ca2+) concentration, suggesting a suppression of Ca2+ influx and neuronal death.Conclusion: CURCU prevents CiSP-induced optic nerve oxidative injury and cell death by suppressing mitochondrial ROS production via regulating TRPM2 signaling pathways. CURCU may serve as a potential therapeutic target against CiSP-induced toxicity in the optic nerve of CiSP-treated patients.

Calcium Regulates Average Time and not Velocity a Thin Filament Moves

Data showing that the rate thin filaments switch on and off varies with Ca2+-activation potential in a motility assay challenge proposals for a graded change in thin filament velocity. Movement of fluorescently labeled thin filaments on a surface coated with myosin is reconstructed from a sequence of images captured by a camera operating at fixed spatial and temporal resolution. Thin filaments selected for velocity measurement appear to change position at constant rate while elapsed time and recording resolution are held constant. Individual thin filaments appear uniformly stationary (pause) or gliding lengthwise continuously at maximum velocity (run) in extreme Ca2+-activation potential conditions produced by adjusting the -log free calcium concentration (pCa) to pCa 8.0 or 4.0, respectively. In a narrow range of Ca2+-activation potential (pCa 6.6 - 5.6), measured velocity is intermediate and thin filaments switch abruptly between run or pause with random dwell time. Although dwell times too brief to resolve decrease measured velocity, a change in velocity is unaffected if the rate of switching remains constant for a given change in Ca2+-activation potential. Our results and analysis show that the average dwell time for a run grows longer as the velocity increases and that increases in Ca2+-activation potential produce compounding increases in dwell times for a run. Hence, declining likelihood of including undetected pauses in the sample run explains how the resolution limitation of the instrumentation could introduce intermediate values to the velocity measurement. In this regime, maximum velocity varies only with the density of favorably oriented, mechanically active myosin that regulatory units can access as the filament glides. The simplest conclusion consistent with our analysis is that a random regulatory unit permits maximum velocity for an average time determined by Ca2+-activation potential.

Preparation of sheep bone collagen peptide-calcium chelate using enzymolysis-fermentation methodology and its structural characterization and stability analysis.

In this study, enzymatic hydrolysis and Lactobacillus fermentation were used in combination to prepare collagen peptide with high free calcium content, followed by the addition of anhydrous ethanol to obtain peptide–calcium chelate. The optimal conditions for the fermentation of enzymatic hydrolysate (glucose 3%, inoculum size 6%, 24.5 h, 37 °C and pH 6.5) were determined by response surface methodology (RSM), under which a free calcium content of 2212.58 mg/100 g was obtained. The calcium–chelating capacity was 42.57 ± 0.09%. The results of ultraviolet absorption spectrum, Fourier transform infrared (FT-IR) spectra, differential scanning calorimeter (DSC), X-ray diffraction and amino acid analysis indicated that calcium could be chelated through carboxyl oxygen and amino nitrogen atoms of collagen peptides, forming peptide–calcium chelate. The chelate is stable at 30–80 °C of temperatures and during in simulated gastrointestinal digestion, which could promote calcium absorption in human. The test intended to provide a basis for developing a novel calcium supplement and promoting utilization of sheep bone.

Hippocalcin Distribution between the Cytosol and Plasma Membrane of Living Cells

Hippocalcin (HPCA) is a neuronal calcium sensor (NCS) protein that provides intracellular signaling via its Ca2+-dependent translocation from the cytosol to the plasma membrane. Although such translocation of HPCA and other NCS proteins is well established, no quantitative estimates of HPCA distribution between the cytosol and plasma membrane at the basal level of the intracellular free calcium concentration ([Ca2+]i) have been obtained. Thus, it is still unknown whether HPCA regulates its plasma membrane targets under resting conditions. In this work, we have evaluated this distribution in living cells at the basal level of [Ca2+]i, comparing HPCA spatial distribution with that of exogenously expressed membrane protein, EYFP-Mem. Using fluorescence recovery after photobleaching, we showed that the diffusion coefficient of fluorescently tagged HPCA in the dendrites of hippocampal neurons (~40 μm2/sec) is within the range of values typical of cytosolic proteins. The coefficient was about 10-fold higher than that for EYFP-Mem. Both results indicate that HPCA is mainly localized in the cytosol of the dendrites of hippocampal neurons. Next, direct calculations for HPCA fractions in the cytosol and plasma membrane at the basal level of [Ca2+]i demonstrated that the plasma membrane fraction of HPCA does not exceed 8%. Altogether, our results suggest that although being mainly cytosolic, HPCA may potentially signal its plasma membrane targets under resting conditions. New approaches used for obtaining such estimates can be applied for quantitative evaluation of NCS protein distribution in living cells under different experimental conditions and for the development of precise biophysical models of their signaling.

Basophil Stimulation and Signaling Pathways.

Despite the growing use of flow cytometry to analyze the functional characteristics of basophils, the intracellular signaling cascades that control their ability to elaborate various pro-allergic and inflammatory mediators and cytokines remain comparatively obscure. Additionally, some studies require the analysis of pro-allergic and inflammatory mediators, such as histamine, LTC4, and various basophil-derived cytokines (e.g., IL-4 and IL-13). Elucidation of intracellular signaling proteins by Western blotting, cytosolic free calcium concentration by spectrofluorophotometry, and detection of mediator releases, as well as analysis of gene expressions by RT-PCR, generally requires relatively large numbers of purified basophils. In selected assays, flow cytometry enables the analysis of relatively low cell numbers and purity for the expression of intracellular signaling proteins or measurement of cytosolic free calcium concentrations by basophil-specific gating strategies. Unfortunately, many aspects of signal transduction relevant to human basophils cannot be readily extrapolated from the use of basophil or mast cell lines. This chapter therefore focuses on how to employ primary human basophils for studying mediator releases and signaling characteristics.

Parallel Acceleration on Removal of Optical Mapping Baseline Wandering.

Optical mapping measurements on hearts stained with fluorescent dyes is imagining method widely accepted and recognized as a tool to study complex spatial-temporal dynamics of cardiac electro-physiology. One shortcoming of the method is baseline wandering in obtained fluorescence signals as signals relevant to transmembrane potential (Vm) change and free intracellular calcium concentration ([Ca]i +2), the two most used dyes, are calculated as a relative signal change in respect to the fluorescence baseline. These changes are small fractional changes often smaller than 10 %. Baseline fluorescence drifts due to dye photo-bleaching, heart contraction/movement artifacts, and stability of the excitation light source over time. Depending on experimental instrumentation, recording duration, signal to noise levels and study aims of the optical imagining, many research groups adopted their own techniques tailored to a specific experimental data. Here we present a technique based on finite impulse response (FIR) filters with paralleled acceleration implemented on GPUs and multi-core CPU, in MATLAB.