Effects Of Calcium Chelators Research Articles

Effects of Calcium Chelators on Colloidal Stability and Interfacial Activity of Egg Yolk Granules

Effects of Calcium Chelators on Colloidal Stability and Interfacial Activity of Egg Yolk Granules

Calcium chelator BAPTA‑AM protects against iron overload‑induced chondrocyte mitochondrial dysfunction and cartilage degeneration.

Osteoarthritis (OA) is a common joint disease that is characterized by cartilage degradation. Iron deposition in the joints is common during the pathogenic progression of OA and recent studies have indicated that iron overload is an important contributor to OA progression. Calcium chelators have been reported to inhibit iron influx via modulating transferrin receptor protein 1 internalization, and they have been identified as a potential approach to the treatment of iron overload-induced diseases. The aim of the present study was to investigate the effect of calcium chelators on the progression of iron overload-induced OA. Primary chondrocytes were treated with various concentrations of ferric ammonium citrate (FAC) to mimic iron overload in vitro, followed by co-treatment with the calcium chelator BAPTA acetoxymethyl ester (BAPTA-AM). Subsequently, intracellular iron levels, cell viability, reactive oxygen species (ROS) levels, mitochondrial function and morphological changes, as well as MMP levels, were detected using commercial kits. It was demonstrated that FAC treatment significantly promoted chondrocyte apoptosis and the expression of MMPs, and these effects were reversed by co-treatment with BAPTA-AM. Moreover, BAPTA-AM suppressed iron influx into chondrocytes and inhibited iron overload-induced ROS production and mitochondrial dysfunction. These results indicated that calcium chelators may be of value in the treatment of iron metabolism-related diseases and iron overload-induced OA progression.

The influence of cows stresses resistance on cheese yield and quality

The influence of cows stresses resistance on cheese yield and quality

Effects of calcium chelation on the neutralization of milk protein isolate and casein micelle reassembling.

Rehydration in an alkaline solution has been shown to improve the rehydration behaviour of milk protein isolate (MPI). In this study, the focus is on citric acid neutralization of MPI powder dissolved in alkaline solution. The results showed that alkalization induced more negative zeta-potential compared to MPI control, reducing it from -22.4mV to -32.6mV. Neutralization had a relatively similar zeta-potential value as alkalized sample. Sodium carbonate addition increased pH and caused a consequential reduction of ionic calcium in aqueous phase and, neutralization caused a small increase in ionic calcium. Soluble aggregate of κ-casein protein and whey protein was suggested in alkalization and neutralization process by non-reducing SDS-PAGE. In addition, neutralization kept a stable colloidal particle size for pHs decreased to pH 9,8 and 7; however, alkalization and neutralization created casein aggregates of larger colloidal particle size than primary casein micelle in control MPI.

Effect of calcium chelators on heat stability and heat-induced changes of milk microfiltered concentrates

In this work, the effect of different calcium chelators on the heat stability of fresh microfiltered concentrates was investigated, by measuring the heat coagulation time (HCT) and pH, particle size, turbidity and viscosity of the dispersions after heat treatment. Samples in all conditions tested were stable with no visible coagulation when heated at 120 °C, for at least 20 min, and the highest values of HCT were observed at the original pH, 6.7. A stability peak was observed when a combination of citrate and phosphate at 15 mEq L−1 was added to the concentrates. Higher concentration of chelating salts decreased the heat stability of the dispersions. These results clearly demonstrated that fresh milk protein concentrates could be employed as ingredients for the development of new and improved dairy protein drinks, and that their stability behaviour showed profound differences compared with that reported for reconstituted milk protein concentrates of similar composition.

Effects of calcium chelators on calcium distribution and protein solubility in rennet casein dispersions

This study investigated the effects of calcium chelating salts on calcium-ion activity (ACa++), calcium distribution, and protein solubility in model CaCl2 solutions (50mmolL−1) or rennet casein dispersions (15g/100g). Disodium phosphate and trisodium citrate at concentrations of 10 and 30mmolL−1 and at ratios of 1:0, 2:1, 1:1, 1:2 and 0:1 were added to both systems. The CaCl2 system, despite its simplicity, was a good indicator of chelating salt–calcium interactions in rennet casein dispersions. Adding trisodium citrate either alone or as part of a mixed chelating salt system resulted in high levels of dispersed “chelated” calcium; conversely, disodium phosphate addition resulted in lower levels, while the ACa++ decreased with increasing concentration of both chelating salts. Neither chelating salt produced high levels of soluble protein. Thus calcium chelating salts may play a more subtle role in modulating hydration during manufacture of casein-based matrices than simply solubilising calcium or protein.

Hyper-osmolarity and calcium chelation: Effects on cystic fibrosis mucus

A non-functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) leads to the disease cystic fibrosis (CF). Although the CFTR is expressed in multiple organs, pulmonary disease is the major cause of illness and death in patients with CF. Stagnant mucus, causing airway obstruction, bacterial overgrowth, persistent inflammation and tissue destruction characterizes the disease, but how the defect in CFTR function is coupled to the mucus phenotype is still controversial. We have recently shown that bicarbonate ions passing through CFTR are necessary for proper unfolding of the MUC2 mucin, thus highlighting the importance of bicarbonate ion transport via the CFTR and the ability of these ions to raise the pH and chelate calcium bound to the mucin as the important steps in forming normal mucus. In order to find potential CF treatments and expand our knowledge about the usefulness of bicarbonate as an active ingredient in formulations to alleviate mucus plugging, we used an Ussing-type chamber and explants from the F508del-CFTR mutant mouse ileum to test the effect of calcium chelators on mucus attachment, either in isolation or in combination with osmolytes such as mannitol or hypertonic saline. We found that increasing the concentration of bicarbonate, both alone or in combination with increased osmolarity of the solution, detached the otherwise attached CF mucus.

Effect of calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutions: The role of calcium-ion activity and micellar integrity

There is general consensus that calcium chelators enhance heat stability in milk. However, they increase the heat stability to considerably different extents. For this reason, the effect of various calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutions was investigated by measuring the heat coagulation time (HCT), together with changes in calcium-ion activity, viscosity, turbidity, and zeta potential before and after heating. Surprisingly, the weakest chelator, disodium uridine monophosphate (Na2UMP), gave the most pronounced increase in HCT. Stronger chelators, i.e., disodium hydrogen phosphate, trisodium citrate and sodium phytate, gave a lower HCT than Na2UMP. Sodium hexametaphosphate (SHMP) was the least effective heat stabilizer. Heat-induced changes in SHMP were the major cause for this reduced heat stability effect. Differences in HCT caused by the addition of the various calcium chelators could be attributed to the calcium-ion activity and state of the micellar structure before and during heating.

Effect of calcium chelators on physical changes in casein micelles in concentrated micellar casein solutions

The effect of calcium chelators on physical changes of casein micelles in concentrated micellar casein solutions was investigated by measuring calcium-ion activity, viscosity and turbidity, and performing ultracentrifugation. The highest viscosities were measured on addition of sodium hexametaphosphate (SHMP), because it cross-linked the caseins. For the weak calcium chelator disodium uridine monophosphate (Na 2UMP), physical changes in the solutions were negligible. Disodium hydrogen phosphate (Na 2HPO 4), trisodium citrate (TSC), and sodium phytate (SP) caused similar increases in viscosity, but had different effects on turbidity. The increase in viscosity was attributed to swelling of the casein micelles (i.e., increased voluminosity) at decreasing calcium-ion activity. The major decrease in turbidity was due to dissociation of the casein micelles. The extent of micellar dissociation was dependent on the type and concentration of calcium chelator. It seems that the micelles were dissociated in the order of SHMP ≥ SP > TSC > Na 2HPO 4 > Na 2UMP.

Polystyrene nanoparticle trafficking across MDCK-II

Polystyrene nanoparticles (PNP) cross rat alveolar epithelial cell monolayers via non-endocytic transcellular pathways. To evaluate epithelial cell type-specificity of PNP trafficking, we studied PNP flux across Madin Darby canine kidney cell II monolayers (MDCK-II). The effects of calcium chelation (EGTA), energy depletion (sodium azide (NaN 3) or decreased temperature), and endocytosis inhibitors methyl-β-cyclodextrin (MBC), monodansylcadaverine and dynasore were determined. Amidine-modified PNP cross MDCK-II 500 times faster than carboxylate-modified PNP. PNP flux did not increase in the presence of EGTA. PNP flux at 4°C and after treatment with NaN 3 decreased 75% and 80%, respectively. MBC exposure did not decrease PNP flux, whereas dansylcadaverine- or dynasore-treated MDCK-II exhibited ∼80% decreases in PNP flux. Confocal laser scanning microscopy revealed intracellular colocalization of PNP with clathrin heavy chain. These data indicate that PNP translocation across MDCK-II (1) occurs via clathrin-mediated endocytosis and (2) is dependent on PNP physicochemical properties. We conclude that uptake/trafficking of nanoparticles (NPs) into/across epithelia depends both on properties of the NPs and on the specific epithelial cell type. From the Clinical Editor This basic science study investigates the membrane translocation of polystyrene nanoparticles (PNP) via an epithelial cell monolayer utilizing canine kidney cells. The authors conclude that translocation occurs via clathrin-mediated endocytosis and is dependent on the physicochemical properties of the particular PNP.

Asynchronous release of GABA via tonic cannabinoid receptor activation at identified interneuron synapses in rat CA1

The influence of local circuit interneurons is thought to play an important role in adjusting synaptic strength via endogenous cannabinoid type 1 (CB1) receptors. Using paired whole-cell recordings, combined with double immunofluorescence and biocytin labelling in acute slices of rat CA1 at postnatal day 18-23, we investigated the properties of Cholecystokinin (CCK)-positive stratum radiatum local circuit interneuron connections that utilised CB1 receptors. Three types of synaptic connections were studied, lacunosum-moleculare-radiatum perforant path-associated (LM-R PPA) to Shaffer collateral-associated (SCA) interneurons, SCA-SCA interneurons and SCA-pyramidal cells. These three synapses were differentially under tonic reduction of inhibition that was blocked by the CB1 receptor inverse agonist AM-251 (10 microM), which enhanced IPSPs. The strength of tonic reduction of inhibition was correlated with asynchronous release which was apparent at connections among interneurons. AM-251 increased the ratio of synchronous to asynchronous release (synchronicity ratio), while the CB receptor agonist anandamide (14 microM) decreased the synchronicity ratio. Fast and slow calcium chelators (BAPTA-AM and EGTA-AM) also increased the synchronicity ratio, accelerated inhibitory time courses and reduced IPSP amplitudes. These data suggest that CB1 receptors at connections among interneuron synapses play a role in tonic suppression of inhibition and govern the asynchronous release of GABA, modulating the time windows of inhibition. Effects of calcium chelators suggest that asynchronous release is a result of a long-lasting presynaptic calcium transients and/or a large distance between calcium source and sensor of exocytosis. These properties of specialised inhibitory neurons may have important modulatory roles in controlling spike timing among local circuit interneurons.

Exocytosis of Post-Golgi Vesicles Is Regulated by Components of the Endocytic Machinery

Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanisms involved in fusion of these vesicles are not well understood. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium triggered. Release of luminal cargo can be accompanied by complete, partial, or no release of membrane cargo. Partial and no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis, and we find that these are the predominant mode of post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs because of premature closure of the fusion pore and is modulated by the activity of clathrin, actin, and dynamin. Our results demonstrate that these components of the endocytic machinery modulate the nature and extent of biosynthetic cargo delivery by post-Golgi vesicles at the cell membrane.

Effect of calcium chelators on the formation and oxidation of the slowly relaxing reduced plastoquinone pool in calcium-depleted PSII membranes. Investigation of the F0 yield

The F(0) fluorescence yield in intact photosystem II (PSII), Ca-depleted PSII (PSII(-Ca/NaCl)), and Mn-depleted PSII membranes was measured before and after dim light treatment (1-2 min), using flash-probe fluorescence and fluorescence induction kinetic measurements. The value of F(0) after the light treatment (F'(0)) was larger than F(0) in dark-adapted PSII membranes and depended on the appearance of the slowly relaxing, reduced plastoquinone pool (t(1/2) = 4 min) formed during preillumination, which was not totally reoxidized before the F'(0) measurement. In PSII(-Ca/NaCl) such a pool also appeared, but the F'(0) yield was even higher than in intact PSII membranes. In Mn-depleted PSII membranes, the pool did not form. Interestingly, the yield of F'(0) in Ca-depleted PSII membranes prepared using chelators (EGTA and citrate) or containing 5 mM EGTA was significantly lower than in PSII(-Ca/NaCl) samples prepared without chelators. These data indicate that chelators inhibit the reduction of Q(A) and Q(B) and formation of the slowly relaxing plastoquinone pool, or alternatively they increase the rate of its oxidation. Such an effect can be explained by coordination of the chelator molecule to the Mn cluster in PSII(-Ca/NaCl) membranes, rather than different amounts of residual Ca2+ in the membranes (with or without the chelator), since the remaining oxygen-evolving activity (approximately 15%) in PSII(-Ca/NaCl) samples did not depend on the presence of the chelator. Thus, chelators of calcium cations not only have an effect on the EPR properties of the S2 state in PSII(-Ca/NaCl) samples, but can also influence the PSII properties determining the rate of plastoquinone pool reduction and/or oxidation. The effect of some toxic metal cations (Cd, Cu, Hg) on the formation of the slowly relaxing pool in PSII membranes was also studied.

Lysosome repair enables host cell survival and bacterial persistence followingChlamydia trachomatisinfection

Chlamydiae are obligate intracellular bacteria that replicate within the confines of a membrane-bound vacuole termed the inclusion. The final event in the infectious process is the disruption of the inclusion membrane and release of a multitude of infectious elementary bodies, each capable of eliciting a new infection. Strains of the trachoma biovar of Chlamydia trachomatis are released from the host cell without concomitant host cell death. In this study, analysis of events associated with chlamydial egress revealed that the integrity of the host cell plasma membrane was compromised prior to the inclusion membrane. This disruption was accompanied by the appearance of LAMP-1 at the infected cell surface, implicating lysosome repair of plasma membrane lesions in response to infection. Analysis of the effects of calcium chelators and actin stabilizing agents, indicated calcium-induced actin depolymerization as a requisite to lysosome-plasma membrane fusion and host cell survival. A consequence of this lysosome-mediated repair process, was the retention of residual bacteria within the surviving host cell, providing a unique mechanism for intracellular persistence of C. trachomatis.

EGTA inhibits floral induction of Pharbitis nil via its influence on gas exchange properties of stomata

The purpose of the study was to determine inhibitory effect of calcium chelator; ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) on flowering of a short-day (SD) plant Pharbitis nil. It was found that 20 mM solution of EGTA applied on cotyledons of 5-d-old P. nil seedlings four hours before the start of 16-h-long induction night decreased the flowering response by 55% compared to the control plants not treated with this Ca2+ chelator. It also caused a very significant decrease of photosynthesis rate, transpiration rate and stomatal conductance both in light and darkness conditions. The results of this study confirm earlier hypothesis suggesting the effect of Ca2+ and its modulators on P. nil flowering is due to their influence on the stomata.

Effects of Ca2+/Calmodulin Mediated Signaling on Spore Germination and Appressorium Development in Colletotrichum falcatum Went

Colletotrichum falcatum Went, the causal agent of red rot of sugarcane produces a specialized infection structure called appressorium, for penetrating the host. Environmental cues like surface hydrophobicity and hardness tend to break the dormancy of the conidia and initiate conidial germination. Conidial attachment is generally stronger on hydrophobic surfaces, while hydrophilic surfaces do not permit conidial attachment of C. falcatum. In vitro studies on conidial germination and appressorium development were made to examine whether the Ca2+/calmodulin dependent pathways are involved in appressorium formation in C. falcatum. Effects of calcium chelator (EGTA), calcium channel blocker (methoxy verampamil), calmodulin antagonists (chloropromazine, phenoxy benzamine and W-7) and phospholipase C inhibitor (neomycin) were also examined to see whether they can impair conidial germination and appressorium development. All these chemicals were found to inhibit conidial germination and or appressorium formation ...

Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP

AbstractThrombin-mediated endothelial-cell release of von Willebrand factor (VWF) and P-selectin functionally links protease-activated receptors (PARs) to thrombosis and inflammation. VWF release can be stimulated by both Ca2+ and cAMP, and, although both VWF and P-selectin are found in Weibel-Palade bodies (WPBs), we found that their release could be differentially regulated. In these studies, human umbilical vein endothelial cells stimulated with cAMP or PAR2-AP led to a delayed release of VWF and significantly less P-selectin release compared with histamine, thrombin, or PAR1-AP. Dose-response studies revealed that PAR2-AP was significantly less efficacious in promoting the release of P-selectin compared with VWF. PAR2-AP–induced robust stimulation of intracellular Ca2+ coupled with a significantly greater inhibitory effect of calcium chelation on release of VWF compared with cell-surface expression of P-selectin, suggests an additional Ca2+-independent pathway involved in release of P-selectin. PAR2-AP failed to increase global cAMP levels; however, inhibition of protein kinase A led to a significant attenuation of PAR2-AP–mediated release of VWF. Confocal microscopy studies revealed that PAR2 and forskolin caused preferential release of a population of Weibel-Palade bodies (WPBs) consisting of only VWF. Thus, WPBs are pharmacologically and morphologically heterogeneous, and distinct granule populations are susceptible to differential regulation.

Calcium/Calmodulin-Dependent Signaling for Prepenetration Development inColletotrichum gloeosporioides

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.

Selective Effects of Calcium Chelators on Anterograde and Retrograde Protein Transport in the Cell

Calcium plays a regulatory role in several aspects of protein trafficking in the cell. Both vesicle fusion and vesicle formation can be inhibited by the addition of calcium chelators. Because the effects of calcium chelators have been studied predominantly in cell-free systems, it is not clear exactly which transport steps in the secretory pathway are sensitive to calcium levels. In this regard, we have studied the effects of calcium chelators on both anterograde and retrograde protein transport in whole cells. Using both cytochemical and biochemical analyses, we find that the anterograde-directed exit of vesicular stomatitis virus G protein and the retrograde-directed exit of Shiga toxin from the Golgi apparatus are both inhibited by calcium chelation. The exit of vesicular stomatitis virus G from a pre-Golgi compartment and the exit of Shiga toxin from an endosomal compartment are sensitive to the membrane-permeant calcium chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid-tetrakis (acetoxymethyl ester) (BAPTA-AM). By contrast, endoplasmic reticulum exit and endocytic internalization from the plasma membrane are not affected by BAPTA. Together, our data show that some, but not all, trafficking steps in the cell may be regulated by calcium. These studies provide a framework for a more detailed analysis of the role of calcium as a regulatory agent during protein transport.

Characterization of adhesive and aggregating functions of pig platelets: comparative studies with human platelets and differential effects of calcium chelation

We have characterized functional responses of pig platelets in citrate-anticoagulated blood and compared results with those of human platelets. Platelet aggregation was induced with known activating agents using citrated platelet-rich plasma. Adhesive and cohesive functions of platelets were evaluated using a perfusion flow system with whole anticoagulated blood (10 min, 800/s). To test the differential sensitivity to free calcium levels, two citrate concentrations (19 mM {standard} vs 13 mM) were used as anticoagulants. The ability of platelets to produce thromboxane A2 was also quantified, using radioimmunoassay. In the presence of 19 mM citrate concentration, pig platelets responded poorly to ADP and collagen, and did not respond to arachidonic acid and U46619. Pig platelets adhered well onto subendothelium, but failed to form aggregates on previously spreaded platelets. At 13 mM citrate concentration, the aggregating responses of pig platelets improved, but some of the aggregation patterns were still reversible. The lower citrate concentration had a dramatic impact on perfusion studies, where the formation of platelet aggregates was restored up to levels found in humans. Thromboxane levels in pig serum were only 20 to 30% of that found in humans. So, pig platelets possess functional properties that clearly differ from human platelets: they are more sensitive to the calcium chelating effects of citrate. Arachidonic acid metabolism and thromboxane amplification of platelet responses seem less preponderant in the pig species.