Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: More evidence for oxidative stress in vitiligo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schallreuter, K.U.; Gibbons, N.C.J.; Zothner, C.

Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10{sup -3} M H{sub 2}O{sub 2}. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10{sup -3}M H{sub 2}O{sub 2} oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H{sub 2}O{sub 2} utilising {sup 45}calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activitiesmore » were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H{sub 2}O{sub 2}-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo.« less

Demonstration of the existence of receptor-dependent calcium channels in the platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Avdonin, P.V.; Bugrii, E.M.; Cheglakov, I.B.

1987-01-01

Recently, with the new methodology of measuring calcium ion concentration in the cytoplasm with the aid of the fluorescent indicator, it has been shown that calcium is a second messenger, mediating the action of many hormones, neuromediators, and other extracellular factors. Another argument in support of the existence of receptor-dependent calcium channels is provided by data on the activation by agonists of the uptake of /sup 45/Ca by the cells. In all the studies cited, the conditions were such that the passage of Ca/sup 2 +/ through the potential-dependent channels was excluded. In this paper, evidence is presented for themore » existence of receptor-dependent calcium channels in the plasma membrane using human platelets as the objects. Two approaches were used. First, the authors determined the binding of /sup 45/Ca by the platelets. In this case, to determine whether /sup 45/Ca passes into the cytoplasm or is adsorbed on the membrane, the authors compared its uptake by simply washed platelets and by platelets in whose cytoplasm buffer capacity for calcium was artificially created with quin 2. The second approach was based on the data of Hallam and Rink, who showed that agonists that increase the calcium level in the platelets induce an intake of Mn/sup 2 +/ ions into the cell in a calcium-free medium.« less

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

NASA Astrophysics Data System (ADS)

Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

PubMed Central

Taft, W C; DeLorenzo, R J

1984-01-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

Mosla dianthera inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Hee; Kim, Sang-Hyun; Eun, Jae-Soon

2006-11-01

In this study, we investigated the effect of the aqueous extract of Mosla dianthera (Maxim.) (AEMD) on the mast cell-mediated allergy model and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as asthma, sinusitis and rheumatoid arthritis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. AEMD inhibited compound 48/80-induced systemic reactions in mice. AEMD decreased immunoglobulin E-mediated local allergic reactions, passive cutaneous anaphylaxis. AEMD attenuated intracellular calcium level and release of histamine from rat peritoneal mast cells activated by compound 48/80. Furthermore, AEMDmore » attenuated the phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-{alpha}, IL-8 and IL-6 secretion in human mast cells. The inhibitory effect of AEMD on the pro-inflammatory cytokines was nuclear factor-{kappa}B (NF-{kappa}B) dependent. AEMD decreased PMA and A23187-induced degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B. Our findings provide evidence that AEMD inhibits mast cell-derived immediate-type allergic reactions and involvement of pro-inflammatory cytokines and NF-{kappa}B in these effects.« less

Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lad, P.M.; Olson, C.V.; Grewal, I.S.

1986-03-05

Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components.more » Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.« less

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

PubMed

Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

2012-05-01

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xinxin; Zhang, Min; Takano, Tetsuo

Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membranemore » and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.« less

Phosphate Uptake from Phytate Due to Hyphae-Mediated Phytase Activity by Arbuscular Mycorrhizal Maize.

PubMed

Wang, Xin-Xin; Hoffland, Ellis; Feng, Gu; Kuyper, Thomas W

2017-01-01

Phytate is the most abundant form of soil organic phosphorus (P). Increased P nutrition of arbuscular mycorrhizal plants derived from phytate has been repeatedly reported. Earlier studies assessed acid phosphatase rather than phytase as an indication of mycorrhizal fungi-mediated phytate use. We investigated the effect of mycorrhizal hyphae-mediated phytase activity on P uptake by maize. Two maize ( Zea mays L.) cultivars, non-inoculated or inoculated with the arbuscular mycorrhizal fungi Funneliformis mosseae or Claroideoglomus etunicatum , were grown for 45 days in two-compartment rhizoboxes, containing a root compartment and a hyphal compartment. The soil in the hyphal compartment was supplemented with 20, 100, and 200 mg P kg -1 soil as calcium phytate. We measured activity of phytase and acid phosphatase in the hyphal compartment, hyphal length density, P uptake, and plant biomass. Our results showed: (1) phytate addition increased phytase and acid phosphatase activity, and resulted in larger P uptake and plant biomass; (2) increases in P uptake and biomass were correlated with phytase activity but not with acid phosphatase activity; (3) lower phytate addition rate increased, but higher addition rate decreased hyphal length density. We conclude that P from phytate can be taken up by arbuscular mycorrhizal plants and that phytase plays a more important role in mineralizing phytate than acid phosphatase.

Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwieterman, W.; Sorrentino, D.; Potter, B.J.

1988-01-01

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (V/sub O/) of (/sup 3/H)-oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound (/sup 3/H)oleate in the medium. V/sub O/ reached a maximum as the concentration of unbound oleate was increased and was significantly inhibited both by phloretin and by prior incubation ofmore » the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 ..mu..g/ml V/sub max/ was reduced from 2480 /plus minus/ 160 to 1870 /plus minus/ 80 pmol/min per 5 /times/ 10/sup 4/ adipocytes, with no change in K/sub m/. A basic kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.« less

Effect of evaporation and pasteurization in the biochemical and immunological composition of human milk.

PubMed

Braga, Lucylea P M; Palhares, Durval B

2007-01-01

To assess the effects of evaporation and pasteurization of human milk on its biochemical and immunological composition and on its osmolarity. The samples of mature human milk were categorized into four study groups: in natura human milk, pasteurized human milk, human milk evaporated at 70% of the baseline volume and human milk pasteurized and evaporated at 70%, with 12 different samples of milk in each group. The samples were used to determine the concentrations of sodium, potassium, calcium, phosphorus, magnesium, protein, fat, lactose, immunoglobulin A and osmolarity. The pasteurization of human milk did not show statistically significant changes in the concentration of sodium, potassium, calcium, phosphorus, magnesium, protein, fat, lactose, or in osmolarity; however, it showed remarkable reduction in the mean concentration of immunoglobulin A. Evaporation had a mean increase of 38% in the concentration of sodium, potassium, calcium, phosphorus, magnesium, protein, fat and lactose and mean reduction of 45% in the concentration of immunoglobulin A, without significant change in osmolarity in unprocessed milk. By evaporation at 70% of the baseline value of human milk, it is possible to obtain human milk that meets the nutritional requirements recommended for preterm infants, except for calcium and phosphorus.

Influence of antigen on immune complex behavior in mice.

PubMed

Finbloom, D S; Magilavy, D B; Harford, J B; Rifai, A; Plotz, P H

1981-07-01

To explore the possibility that the behavior of immune complexes can, under some circumstances, be directed by the antigen, we have studied the behavior of complexes of identical size made with the glycoproteins, orosomucoid (OR), and ceruloplasmin: or with their desialylated derivatives, asialo-orosomucoid (ASOR) and asialo-ceruloplasmin. Such desialylated proteins are rapidly removed from the circulation by a hepatic cell receptor for galactose, the sugar exposed upon removal of sialic acid. Mixtures of 125I-goat anti-ASOR with either ASOR or OR and mixtures of 125I-rabbit anti-OR with either ASOR or OR form complexes identically. The complexes were separated by density gradient centrifugation and injected intravenously into C3H mice. Blood clearance and hepatic uptake of the OR complexes and ASOR complexes were markedly different. T 1/2 for the goat OR complexes exceeded 300 min, whereas that for the ASOR complexes was 15 min. More detailed studies using rabbit complexes of various sizes revealed that light rabbit complexes behaved similarly to the goat complexes. The light rabbit OR complexes were cleared slowly, with only 18% found in the liver at 60 min, whereas the light rabbit ASOR complexes were cleared much more rapidly, with 62% found within the liver by 30 min. This rapid clearance was completely suppressed by a prior injection of a blocking dose of ASOR, which implies uptake by a galactose-mediated mechanism on hepatocytes. As the size of the rabbit complexes increased, so did the rate of Fc receptor-mediated clearance. Heavy rabbit OR complexes were cleared more rapidly than light OR complexes but not so rapidly as heavy ASOR complexes. The clearance and hepatic uptake of the heavy OR complexes were markedly suppressed by a prior injection of heat-aggregated gamma globulin, a known Fc receptor-blocking agent (45% hepatic uptake without and 6% with aggregated gamma globulin). The heavy rabbit ASOR complexes exhibited inhibition of blood clearance and hepatic uptake by both galactose receptor-blocking and Fc receptor-blocking agents. A blocking dose of ASOR reduced the hepatic uptake at 30 min from 75 to 49%, and heat-aggregated gamma globulin reduced it from 75 to 39%, which suggests that these heavy complexes were removed from the circulation by receptors both for the immunoglobulin and for the antigen. Cell separation studies and autoradiographs confirmed that those complexes cleared primarily by galactose-mediated mechanism were within hepatocytes, and those cleared by Fc receptors were within the nonparenchymal cells of the liver. It seems probable, therefore, the some antigen-antibody complexes may be removed from the circulation via receptors not only for immunoglobulin but also for antigen.

Overexpression of human mutated G93A SOD1 changes dynamics of the ER mitochondria calcium cycle specifically in mouse embryonic motor neurons.

PubMed

Lautenschläger, Janin; Prell, Tino; Ruhmer, Julia; Weidemann, Lisa; Witte, Otto W; Grosskreutz, Julian

2013-09-01

Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS. Copyright © 2013 Elsevier Inc. All rights reserved.

Cadmium and calcium uptake in the mollusc donax rugosus and effect of a calcium channel blocker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sidoumou, Z.; Gnassia-Barelli, M.; Romeo, M.

Donax rugosus, a common bivalve mollusc in the coastal waters of Mauritania, has been studied for trace metal concentrations as a function of sampling site (from South of Mauritania to the North of this country) and of season. In this paper, the uptake of cadmium was experimentally studied in the different organs of D. rugosus. Since metals such as cadmium, copper and mercury may alter calcium homeostasis, calcium uptake was also studied in the animals treated with cadmium. Since calcium is taken up through specific channels, it appears that metals inhibit Ca uptake by interacting with these channels in themore » plasma membrane. Cadmium and calcium have very similar atomic radii, thus cadmium may be taken up through the calcium channels, particularly through voltage-dependent channels. The uptake of cadmium and calcium by D. Rugosus was therefore also studied in the presence of the calcium channel blocker verapamil. 13 refs., 3 figs., 1 tab.« less

Stiff person syndrome associated anti-amphiphysin antibodies reduce GABA associated [Ca(2+)]i rise in embryonic motoneurons.

PubMed

Geis, C; Beck, M; Jablonka, S; Weishaupt, A; Toyka, K V; Sendtner, M; Sommer, C

2009-10-01

Autoantibodies to the synaptic protein amphiphysin play a crucial pathogenic role in paraneoplastic stiff-person syndrome. Impairment of GABAergic inhibition is the presumed pathophysiological mechanism by which these autoantibodies become pathogenic. Here we used calcium imaging on rat embryonic motor neurons to investigate whether antibodies to amphiphysin directly hinder GABAergic signaling. We found that the immunoglobulin G fraction from a patient with stiff-person syndrome, containing high titer antibodies to amphiphysin and inducing stiffness in rats upon passive transfer, reduced GABA-induced calcium influx in embryonic motor neurons. Depletion of the anti-amphiphysin fraction from the patient's IgG by selective affinity chromatography abolished this effect, showing its specificity for amphiphysin. Quantification of the surface expression of the Na(+)/K(+)/2Cl(2-) cotransporter revealed a reduction after incubation with anti-amphiphysin IgG, which is concordant with a lower intracellular chloride concentration and thus impairment of GABA mediated calcium influx. Thus, anti-amphiphysin antibodies exert a direct effect on GABA signaling, which is likely to contribute to the pathogenesis of SPS.

Calmodulins from Schistosoma mansoni: Biochemical analysis and interaction with IQ-motifs from voltage-gated calcium channels.

PubMed

Thomas, Charlotte M; Timson, David J

2018-05-17

The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins - SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCa v 1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dengue vascular leakage is augmented by mast cell degranulation mediated by immunoglobulin Fcγ receptors

PubMed Central

Syenina, Ayesa; Jagaraj, Cyril J; Aman, Siti AB; Sridharan, Aishwarya; St John, Ashley L

2015-01-01

Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection. DOI: http://dx.doi.org/10.7554/eLife.05291.001 PMID:25783751

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaba, D.M.; Metz, S.; Maze, M.

Transthoracic electric countershock can cause necrotic myocardial lesions in humans as well as experimental animals. The authors investigated the effect on postcountershock myocardial damage of pretreatment with prazosin, an alpha-1 antagonist; L-metoprolol, a beta-1 antagonist, and verapamil, a calcium channel-blocking agent. Twenty dogs were anesthetized with halothane and given two transthoracic countershocks of 295 delivered joules each after drug or vehicle treatment. Myocardial injury was quantitated 24 h following countershock by measuring the uptake of technetium-99m pyrophosphate in the myocardium. Elevated technetium-99m pyrophosphate uptake occurred in visible lesions in most dogs regardless of drug treatment. For each of four parametersmore » of myocardial damage there was no statistically significant difference between control animals and those treated with prazosin, metoprolol, or verapamil. These data suggest that adrenergic or calcium channel-mediated mechanisms are not involved in the pathogenesis of postcountershock myocardial damage.« less

Construction and evaluation of BSA-CaP nanomaterials with enhanced transgene performance via biocorona-inspired caveolae-mediated endocytosis

NASA Astrophysics Data System (ADS)

Ma, Xi-Xi; Gao, Han; Zhang, Ya-Xuan; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2018-02-01

Non-viral nanovectors have attracted much attention owing to their ability to condense genetic materials and their ease of modification. However, their poor stability, low biocompatibility and gene degradation in endosomes or lysosomes has significantly hampered their application in vivo and in the clinic. In an attempt to overcome these difficulties a series of bovine serum albumin (BSA)-calcium phosphate (CaP) nanoparticles were constructed. The CaP condenses with DNA to form nanocomplexes coated with a biomimetic corona of BSA. Such complexes may retain the inherent endocytosis profile of BSA, with improved biocompatibility. In particular the transgene performance may be enhanced by stimulating the cellular uptake pathway via caveolae-mediated endocytosis. Two methods were employed to construct and optimize the formulation of BSA-CaP nanomaterials. The optimized BSA-CaP-50-M2 nanoparticles prepared by our second method exhibited good stability, negligible cytotoxicity and enhanced transgene performance with long-term expression for 72 h in vivo even with a single dose. Determination of the cellular uptake pathway and Western blot revealed that cellular uptake of the designed BSA-CaP-50-M2 nanoparticles was mainly via caveolae-mediated endocytosis in a non-degradative pathway in which the biomimetic uptake profile of BSA was retained.

Construction and evaluation of BSA-CaP nanomaterials with enhanced transgene performance via biocorona-inspired caveolae-mediated endocytosis.

PubMed

Ma, Xi-Xi; Gao, Han; Zhang, Ya-Xuan; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2018-02-23

Non-viral nanovectors have attracted much attention owing to their ability to condense genetic materials and their ease of modification. However, their poor stability, low biocompatibility and gene degradation in endosomes or lysosomes has significantly hampered their application in vivo and in the clinic. In an attempt to overcome these difficulties a series of bovine serum albumin (BSA)-calcium phosphate (CaP) nanoparticles were constructed. The CaP condenses with DNA to form nanocomplexes coated with a biomimetic corona of BSA. Such complexes may retain the inherent endocytosis profile of BSA, with improved biocompatibility. In particular the transgene performance may be enhanced by stimulating the cellular uptake pathway via caveolae-mediated endocytosis. Two methods were employed to construct and optimize the formulation of BSA-CaP nanomaterials. The optimized BSA-CaP-50-M2 nanoparticles prepared by our second method exhibited good stability, negligible cytotoxicity and enhanced transgene performance with long-term expression for 72 h in vivo even with a single dose. Determination of the cellular uptake pathway and Western blot revealed that cellular uptake of the designed BSA-CaP-50-M2 nanoparticles was mainly via caveolae-mediated endocytosis in a non-degradative pathway in which the biomimetic uptake profile of BSA was retained.

Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS).

PubMed

Charoensin, Suphachai; Eroglu, Emrah; Opelt, Marissa; Bischof, Helmut; Madreiter-Sokolowski, Corina T; Kirsch, Andrijana; Depaoli, Maria R; Frank, Saša; Schrammel, Astrid; Mayer, Bernd; Waldeck-Weiermair, Markus; Graier, Wolfgang F; Malli, Roland

2017-01-01

Mitochondrial Ca 2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO • ) production. However, it is not entirely clear if the organelles support or counteract NO • biosynthesis by taking up Ca 2+ . The objective of this study was to verify whether or not mitochondrial Ca 2+ uptake influences Ca 2+ -triggered NO • generation by endothelial NO • synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO • probes, the geNOps, and Ca 2+ sensors to monitor single cell NO • and Ca 2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP 3 )-generating agonist. Mitochondrial Ca 2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca 2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca 2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca 2+ -triggered NO • increase independently of global cytosolic Ca 2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca 2+ sequestration and Ca 2+ -induced NO • signals. The positive correlation between mitochondrial Ca 2+ elevation and NO • production was independent of eNOS phosphorylation at serine 1177 . Our findings emphasize that manipulating mitochondrial Ca 2+ uptake may represent a novel strategy to control eNOS-mediated NO • production. Copyright © 2016. Published by Elsevier Inc.

The Hepatitis B Virus X Protein Elevates Cytosolic Calcium Signals by Modulating Mitochondrial Calcium Uptake

PubMed Central

Yang, Bei

2012-01-01

Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication. PMID:22031934

The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

NASA Astrophysics Data System (ADS)

Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration.

PubMed

Karaoglu Hanzatian, Denise; Schwartz, Annette; Gizatullin, Farid; Erickson, Jamie; Deng, Kangwen; Villanueva, Ruth; Stedman, Christopher; Harris, Cristina; Ghayur, Tariq; Goodearl, Andrew

2018-05-17

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.

Effects of calcium on hepatocyte iron uptake from transferrin, iron-pyrophosphate and iron-ascorbate.

PubMed

Nilsen, T

1991-10-16

Calcium stimulates hepatocyte iron uptake from transferrin, ferric-iron-pyrophosphate and ferrous-iron-ascorbate. Maximal stimulation of iron uptake is observed at 1-1.5 mM of extra-cellular calcium and the effect is reversible and immediate. Neither the receptor affinity for transferrin, nor the total amounts of transferrin associated with the cells or the rate of transferrin endocytosis are significantly affected by calcium. In the presence of calcium the rate of iron uptake of non-transferrin bound iron increases abruptly at approximate 17 degrees C and 27 degrees C and as assessed by Arrhenius plots, the activation energy is reduced in a calcium dependent manner at approx. 27 degrees C. At a similar temperature, i.e., between 25 degrees C and 28 degrees C, calcium increases the rates of cellular iron uptake from transferrin in a way that is not reflected in the rate of transferrin endocytosis. By the results of this study it is concluded that calcium increases iron transport across the plasma membrane by a mechanism dependent on membrane fluidity.

The effect of essential fatty acid deficiency on the stimulation of intestinal calcium transport by 1,25-dihydroxyvitamin D3.

PubMed

Kreutter, D; Matsumoto, T; Peckham, R; Zawalich, K; Wen, W H; Zolock, D T; Rasmussen, H

1983-04-25

The effect of altering the lipid composition of the brush-border membrane on the ability of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to stimulate calcium transport across the intestinal mucosa was examined by raising chicks on a vitamin D, essential fatty acid-deficient diet (-DEFAD) and measuring calcium absorption from duodenal sacs in situ and calcium uptake into brush-border membrane vesicles in vitro. Administration of 1,25-(OH)2D3 to -DEFAD and to -D control chicks led to the same increase in calcium transport in situ, whereas calcium transport in isolated brush-border membrane vesicles was not stimulated in the EFAD group, but responded normally in the control group. When the incubation temperature was increased to 34 degrees C, brush-border membrane vesicles from 1,25-(OH)2D3-treated essential fatty acid-deficient (+DE-FAD) chicks accumulated calcium at a faster rate than did vesicles from -DEFAD chicks. There was a marked decrease in the linoleic acid content and an increase in the oleic acid content of both the total lipid extract of the brush-border membrane as well as the phosphatidylcholine and phosphatidylethanolamine fractions, which could explain the temperature sensitivity of the in vitro system. When the diet of the EFAD chicks was supplemented with linoleic acid, the rate of calcium uptake into subsequently isolated vesicles from +DE-FAD chicks correlated with the amount of linoleic acid in the brush-border membranes. These results support the concept that the action of 1,25-(OH)2D3 on membrane lipid turnover and structure plays a critically important role in the 1,25-(OH)2D3-mediated cellular transport responses.

Tyrosol Suppresses Allergic Inflammation by Inhibiting the Activation of Phosphoinositide 3-Kinase in Mast Cells.

PubMed

Je, In-Gyu; Kim, Duk-Sil; Kim, Sung-Wan; Lee, Soyoung; Lee, Hyun-Shik; Park, Eui Kyun; Khang, Dongwoo; Kim, Sang-Hyun

2015-01-01

Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of κB kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders.

Amyotrophic lateral sclerosis immunoglobulins increase Ca2+ currents in a motoneuron cell line.

PubMed

Mosier, D R; Baldelli, P; Delbono, O; Smith, R G; Alexianu, M E; Appel, S H; Stefani, E

1995-01-01

The sporadic form of amyotrophic lateral sclerosis (ALS) is an idiopathic and eventually lethal disorder causing progressive degeneration of cortical and spinal motoneurons. Recent studies have shown that the majority of patients with sporadic ALS have serum antibodies that bind to purified L-type voltage-gated calcium channels and that antibody titer correlates with the rate of disease progression. Furthermore, antibodies purified from ALS patient sera have been found to alter the physiologic function of voltage-gated calcium channels in nonmotoneuron cell types. Using whole-cell patch-clamp techniques, immunoglobulins purified from sera of 5 of 6 patients with sporadic ALS are now shown to increase calcium currents in a hybrid motoneuron cell line, VSC4.1. These calcium currents are blocked by the polyamine funnel-web spider toxin FTX, which has previously been shown to block Ca2+ currents and evoked transmitter release at mammalian motoneuron terminals. These data provide additional evidence linking ALS to an autoimmune process and suggest that antibody-induced increases in calcium entry through voltage-gated calcium channels may occur in motoneurons in this disease, with possible deleterious effects in susceptible neurons.

Acellular pertussis vaccines for adolescents.

PubMed

Pichichero, Michael E; Casey, Janet R

2005-06-01

The epidemiology of pertussis is changing, with a clear increase in the number of cases diagnosed in adolescents and adults. This development has spurred studies and anticipated licensure of safer diphtheria, tetanus, acellular pertussis combined (Tdap) vaccines for this older population. Literature review. Tdap vaccines are safe and immunogenic when administered to adolescents and adults. Correlates of immunity to pertussis after Tdap vaccination have not been established, but various combinations of antibody to pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae) provide protection. The importance of the number of antigens in Tdap vaccines for protection against mild pertussis disease is unclear. Pertussis vaccination establishes herd immunity after sufficient uptake within communities and countries. As experience with TdaP vaccines has accumulated, a 1-2% occurrence of large, local injection-site reactions with all TdaP vaccine products have been observed for booster doses in children 4-6 years of age. The frequency of large local reactions appears lower in adolescents and adults. The pathophysiologic mechanisms for the local reactions are not established, but a majority appears to be immunoglobulin E-mediated-reactive edema, and a minority appears to be immunoglobulin G-mediated Arthus-type reactions. Tdap vaccines appear safe and immunogenic. The economic impact of pertussis provides a cost-benefit justification for widespread use of Tdap vaccine boosting in adolescents.

Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum.

PubMed

Paydar, Mehrak Javadi; Pousti, Abbas; Farsam, Hasan; Amanlou, Massoud; Mehr, Shahram Ejtemaei; Dehpour, Ahmad Reza

2005-11-01

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.

The interactive roles of zinc and calcium in mitochondrial dysfunction and neurodegeneration.

PubMed

Pivovarova, Natalia B; Stanika, Ruslan I; Kazanina, Galina; Villanueva, Idalis; Andrews, S Brian

2014-02-01

Zinc has been implicated in neurodegeneration following ischemia. In analogy with calcium, zinc has been proposed to induce toxicity via mitochondrial dysfunction, but the relative role of each cation in mitochondrial damage remains unclear. Here, we report that under conditions mimicking ischemia in hippocampal neurons - normal (2 mM) calcium plus elevated (> 100 μM) exogenous zinc - mitochondrial dysfunction evoked by glutamate, kainate or direct depolarization is, despite significant zinc uptake, primarily governed by calcium. Thus, robust mitochondrial ion accumulation, swelling, depolarization, and reactive oxygen species generation were only observed after toxic stimulation in calcium-containing media. This contrasts with the lack of any mitochondrial response in zinc-containing but calcium-free medium, even though zinc uptake and toxicity were strong under these conditions. Indeed, abnormally high, ionophore-induced zinc uptake was necessary to elicit any mitochondrial depolarization. In calcium- and zinc-containing media, depolarization-induced zinc uptake facilitated cell death and enhanced accumulation of mitochondrial calcium, which localized to characteristic matrix precipitates. Some of these contained detectable amounts of zinc. Together these data indicate that zinc uptake is generally insufficient to trigger mitochondrial dysfunction, so that mechanism(s) of zinc toxicity must be different from that of calcium. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Effects of calcium antagonists on isolated bovine cerebral arteries: inhibition of constriction and calcium-45 uptake induced by potassium or serotonin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wendling, W.W.; Harakal, C.

1987-05-01

The purpose of this study was to determine the mechanisms by which organic calcium channel blockers inhibit cerebral vasoconstriction. Isolated bovine middle cerebral arteries were cut into rings to measure contractility or into strips to measure radioactive calcium (/sup 45/Ca) influx and efflux. Calcium channel blockers (10(-5) M verapamil or 3.3 X 10(-7) M nifedipine) and calcium-deficient solutions all produced near-maximal inhibition of both potassium- and serotonin-induced constriction. In calcium-deficient solutions containing potassium or serotonin, verapamil and nifedipine each blocked subsequent calcium-induced constriction in a competitive manner. Potassium and serotonin significantly increased /sup 45/Ca uptake into cerebral artery strips duringmore » 5 minutes of /sup 45/Ca loading; for potassium /sup 45/Ca uptake increased from 62 to 188 nmol/g, and for serotonin from 65 to 102 nmol/g. Verapamil or nifedipine had no effect on basal /sup 45/Ca uptake but significantly blocked the increase in /sup 45/Ca uptake induced by potassium or serotonin. Potassium, and to a lesser extent serotonin, each induced a brief increase in the rate of /sup 45/Ca efflux into calcium-deficient solutions. Verapamil or nifedipine had no effect on basal or potassium-stimulated /sup 45/Ca efflux. The results demonstrate that verapamil and nifedipine block /sup 45/Ca uptake through both potential-operated (potassium) and receptor-operated (serotonin) channels in bovine middle cerebral arteries.« less

The effect of calcium on non-heme iron uptake, efflux, and transport in intestinal-like epithelial cells (Caco-2 cells).

PubMed

Gaitán, Diego Alejandro; Flores, Sebastian; Pizarro, Fernando; Olivares, Manuel; Suazo, Miriam; Arredondo, Miguel

2012-03-01

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of (55)Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.

Neurotrophin Therapy of Neurodegenerative Disorders with Mitochondrial Dysfunction

DTIC Science & Technology

2007-09-01

of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia - reperfusion injury . FASEB J 20: E2166-76. Martin E...anti-apoptotic proteins bax and bcl - 2 . Functionally the Ts16 mitochondria had no change in either calcium uptake capacity or superoxide generation...apoptotic proteins bax and bcl - 2 and of cytochrome c were measured by western blot. Bax is known to redistribute from the cytoplasm to the

ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

PubMed

Zhang, Xinxin; Tang, Xuexi; Wang, Ming; Zhang, Wei; Zhou, Bin; Wang, You

2017-08-01

UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m -2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO 4 3- uptake was more serious compared to NO 3 - uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca 2+ -ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca 2+ -ATPase suppression, and a relation between Ca 2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the glutathione cycle was involved in the response of marine microalgae to UV-B stimuli. Copyright © 2017 Elsevier B.V. All rights reserved.

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells.

PubMed

Portugal, Camila Cabral; Miya, Vivian Sayuri; Calaza, Karin da Costa; Santos, Rochelle Alberto Martins; Paes-de-Carvalho, Roberto

2009-01-01

Vitamin C is transported in the brain by sodium vitamin C co-transporter 2 (SVCT-2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT-2 and the uptake and release of [(14)C] ascorbate in chick retinal cells. SVCT-2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT-2 was expressed in cultured retinal neurons, but not in glial cells. [(14)C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium-free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate-stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l-beta-threo-benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [(3)H] D-aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2-Carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7-initroquinoxaline-2,3-dione (DNQX) or (5R,2S)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). However, DNQX, but not MK-801 or 2-amino-5-phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non-NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2-bis (2-aminophenoxy) ethane-N',N',N',N',-tetraacetic acid tetrakis (acetoxy-methyl ester) (BAPTA-AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT-2, and the release can be stimulated by NMDA or non-NMDA glutamate receptors.

The fate of calcium carbonate nanoparticles administered by oral route: absorption and their interaction with biological matrices

PubMed Central

Lee, Jeong-A; Kim, Mi-Kyung; Kim, Hyoung-Mi; Lee, Jong Kwon; Jeong, Jayoung; Kim, Young-Rok; Oh, Jae-Min; Choi, Soo-Jin

2015-01-01

Background Orally administered particles rapidly interact with biological fluids containing proteins, enzymes, electrolytes, and other biomolecules to eventually form particles covered by a corona, and this corona potentially affects particle uptake, fate, absorption, distribution, and elimination in vivo. This study explored relationships between the biological interactions of calcium carbonate particles and their biokinetics. Methods We examined the effects of food grade calcium carbonates of different particle size (nano [N-Cal] and bulk [B-Cal]: specific surface areas of 15.8 and 0.83 m2/g, respectively) on biological interactions in in vitro simulated physiological fluids, ex vivo biofluids, and in vivo in gastrointestinal fluid. Moreover, absorption and tissue distribution of calcium carbonates were evaluated following a single dose oral administration to rats. Results N-Cal interacted more with biomatrices than bulk materials in vitro and ex vivo, as evidenced by high fluorescence quenching ratios, but it did not interact more actively with biomatrices in vivo. Analysis of coronas revealed that immunoglobulin, apolipoprotein, thrombin, and fibrinogen, were the major corona proteins, regardless of particle size. A biokinetic study revealed that orally delivered N-Cal was more rapidly absorbed into the blood stream than B-Cal, but no significant differences were observed between the two in terms of absorption efficiencies or tissue distributions. Both calcium carbonates were primarily present as particulate forms in gastrointestinal fluids but enter the circulatory system in dissolved Ca2+, although both types showed partial phase transformation to dicalcium phosphate dihydrate. Relatively low dissolution (about 4%), no remarkable protein–particle interaction, and the major particulate fate of calcium carbonate in vivo gastrointestinal fluids can explain its low oral absorption (about 4%) regardless of particle size. Conclusion We conclude that calcium carbonate nanoparticles can act more actively with biological matrices in vitro and ex vivo, but that in vivo, their biological interactions and biokinetics are not affected by particle size. PMID:25848250

The fate of calcium carbonate nanoparticles administered by oral route: absorption and their interaction with biological matrices.

PubMed

Lee, Jeong-A; Kim, Mi-Kyung; Kim, Hyoung-Mi; Lee, Jong Kwon; Jeong, Jayoung; Kim, Young-Rok; Oh, Jae-Min; Choi, Soo-Jin

2015-01-01

Orally administered particles rapidly interact with biological fluids containing proteins, enzymes, electrolytes, and other biomolecules to eventually form particles covered by a corona, and this corona potentially affects particle uptake, fate, absorption, distribution, and elimination in vivo. This study explored relationships between the biological interactions of calcium carbonate particles and their biokinetics. We examined the effects of food grade calcium carbonates of different particle size (nano [N-Cal] and bulk [B-Cal]: specific surface areas of 15.8 and 0.83 m(2)/g, respectively) on biological interactions in in vitro simulated physiological fluids, ex vivo biofluids, and in vivo in gastrointestinal fluid. Moreover, absorption and tissue distribution of calcium carbonates were evaluated following a single dose oral administration to rats. N-Cal interacted more with biomatrices than bulk materials in vitro and ex vivo, as evidenced by high fluorescence quenching ratios, but it did not interact more actively with biomatrices in vivo. Analysis of coronas revealed that immunoglobulin, apolipoprotein, thrombin, and fibrinogen, were the major corona proteins, regardless of particle size. A biokinetic study revealed that orally delivered N-Cal was more rapidly absorbed into the blood stream than B-Cal, but no significant differences were observed between the two in terms of absorption efficiencies or tissue distributions. Both calcium carbonates were primarily present as particulate forms in gastrointestinal fluids but enter the circulatory system in dissolved Ca(2+), although both types showed partial phase transformation to dicalcium phosphate dihydrate. Relatively low dissolution (about 4%), no remarkable protein-particle interaction, and the major particulate fate of calcium carbonate in vivo gastrointestinal fluids can explain its low oral absorption (about 4%) regardless of particle size. We conclude that calcium carbonate nanoparticles can act more actively with biological matrices in vitro and ex vivo, but that in vivo, their biological interactions and biokinetics are not affected by particle size.

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

1989-01-01

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivatesmore » a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.« less

Calcium, zinc, and iron bioavailabilities from a commercial human milk fortifier: a comparison study.

PubMed

Etcheverry, P; Wallingford, J C; Miller, D D; Glahn, R P

2004-11-01

Adding human milk fortifiers (HMF) to human milk (HM) is one way of overcoming the nutrient deficits found in the latter. In this study, the bioavailabilities of calcium, zinc, and iron in S-26/SMA HMF added to HM were compared with those in HM fortified with various bovine milk proteins: alpha-lactalbumin, colostrum, caseinate, casein phosphopeptides, and whey protein concentrate. The bioavailability of each mineral was assessed using an in vitro digestion/Caco-2 cell culture model. Calcium and zinc uptake by the cells was traced with radioisotopes; iron uptake was assessed via cell ferritin levels. Samples were prepared on an equal protein content basis and with added calcium, but no zinc or iron was added. Results revealed that calcium uptake from HM + S-26/SMA was not different from any of the HM fortified with the bovine milk proteins, except for unfortified HM and HM + colostrum in which calcium uptake was significantly lower (-89 and -38%, respectively). Uptake of zinc and iron were significantly higher for HM + S-26/SMA than for the other HM + fortifiers.

Pharmacological interference with 123I-metaiodobenzylguanidine: a limitation to developing cardiac innervation imaging in clinical practice?

PubMed

Stefanelli, A; Treglia, G; Bruno, I; Rufini, V; Giordano, A

2013-05-01

(123)I-metaiodo-benzylguanidine (MIBG) scintigraphy is considered a valid imaging test to evaluate the cardiac sympathetic nervous system. However, scientific literature showed that some drugs are able to or are expected to interfere with MIBG uptake. Thirty years after introduction of the method and over 15 years since the appearance of the first document on pharmacological interference with MIBG, an update on this issue has become necessary. The aims of this review paper are: (1) to identify the pharmacological basis of interference of a variety of substances with MIBG uptake; and (2) to update the list of drugs that definitely interfere with MIBG on the grounds of evidence in the literature. A MEDLINE search was conducted. Scientific studies, case report and review articles were collected. Papers published demonstrating drugs interfering with MIBG uptake were evaluated. Drugs may interact with MIBG uptake by 5 mechanism: (1) type-1 uptake inhibition; (2) inhibition of active transport to vesicles; (3) competition in transport to vesicles; (4) depletion of neurosecretory vesicle content; (5) calcium-mediated mechanism. We find that drugs like cocaine, antidepressants, some antipsychotic, tramadol, labetalol, sympatho-mimetics, reserpine and some calcium antagonists (as diltiazem, verapamil and nifedipine) do interfere with MIBG uptake. On the other hand, we find that controversial data are available on scientific literature regarding digoxin and amiodarone. A compiled statement of MIBG interfering medicines is now recommended to help nuclear medicine physicians in clinical practice to avoid potential pitfalls and improve the efficacy of (123)I-MIBG scintigraphy as a diagnostic tool.

The effects of electromagnetic fields on B16-BL6 cells are dependent on their spatial and temporal character.

PubMed

Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

2017-04-01

Exposure to low intensity, low frequency electromagnetic fields (EMF) has effects on several biological systems. Spatiotemporal characteristics of these EMFs are critical. The effect of several complex EMF patterns on the proliferation of B16-BL6 mouse melanoma cells was tested. Exposure to one of these patterns, the Thomas-EMF, inhibited cell proliferation and promoted calcium uptake. The Thomas-EMF is coded from a digital-to-analog file comprised of 849 points, which provides power to solenoids and can be set to alter timing, intensity, and duration of variable EMF. Setting the point duration to 3 ms generated a time-varying EMF pattern which began at 25 Hz and slowed to 6 Hz over a 2.5 s repeat. Exposing B16-BL6 cells to Thomas-EMF set to 3 ms for 1 h/day inhibited cell proliferation by 40% after 5 days, while setting the point duration to 1, 2, 4, or 5 ms had no effect on cell proliferation. Similarly, exposing cells to Thomas-EMF set to 3 ms promoted a three-fold increase in calcium uptake after 1 h, while the other timings had no effect. Exposure to Thomas-EMF for as short as 15 min/day slowed cell proliferation, but exposure for 1 h/day was optimal. This corresponded to the effect on calcium uptake where uptake was detected after 15 min exposure and was maximal by 1 h of treatment. Studies show that the specific spatiotemporal character of EMF is critical in mediating their biological activities. Bioelectromagnetics. 38:165-174, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D.

PubMed

Rajasree, S; Umashankar, P R; Lal, A V; Sarma, P Sankara; Kartha, C C

2002-03-01

Several studies have demonstrated that excess of vitamin D3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D3 (1,25(OH)2 D3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH)2 D3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH)2D3 receptor and increased calcium uptake in smooth muscle cells of the arteries.

Possible mechanisms of stimulatory action of papaverine on calcium-uptake by rat uterine microsomal fraction.

PubMed

Koike, K; Takayanagi, I

1981-10-01

Effects of papaverine and cyclic AMP on Ca-uptake by the microsomal fraction from rat uterus were studied. Papaverine (3 x 10(-5) M) potentiated Ca-uptake by the microsomal fraction in the presence of potassium oxalate. However, cyclic AMP and MIX (3-isobutyl-1-methylxanthine; 1 mM), a potent phosphodiesterase inhibitor, did not influence Ca-uptake by the microsomal fraction in the presence of potassium oxalate. Cyclic AMP in concentrations of 10(-8) to 10(-4) M did not influence Ca-uptake by the microsomal fraction in the presence of potassium oxalate. In the absence of potassium oxalate, papaverine and Aspaminol (1,1,-diphenyl-3-piperidinobutanol hydrochloride), a nonspecific smooth muscle relaxant, inhibited Ca-uptake by the microsomal fraction and cyclic AMP had no influence on this uptake. These results suggest that papaverine potentiated Ca-uptake by membranes such as sarcoplasmic reticulum, in the presence of potassium oxalate and inhibited Ca-uptake by the plasma membrane-derived vesicles in the absence of potassium oxalate. These results suggest that relaxation of smooth muscle by papaverine is related to a cyclic AMP-independent mechanism as well as to a mechanism mediated via cyclic AMP.

The effect of amino acids on the intestinal absorption of immunoglobulins in the neonatal rat

PubMed Central

Bamford, D. R.; Donnelly, H.

1974-01-01

An in vitro preparation of 10-day-old rat intestine was used to examine the absorption of a number of amino acids and immunoglobulins. Evidence was obtained for the active absorption of alanine, leucine, methionine, histidine and lysine, but not for aspartic acid. A selective absorption of the homologous molecule was found in experiments where 131I-labelled rat and bovine IgG were presented to the ileum in 10-minute incubations. The greater uptake of rat IgG was unrelated to the relative rates of catabolism of the two molecules. Although the uptake of rat IgG was unaffected by 100 mM concentrations of neutral and acidic amino acids, the basic amino acids arginine and lysine significantly stimulated uptake. PMID:4854740

Altered mechanical properties of titin immunoglobulin domain 27 in the presence of calcium.

PubMed

DuVall, Michael M; Gifford, Jessica L; Amrein, Matthias; Herzog, Walter

2013-04-01

Titin (connectin) based passive force regulation has been an important physiological mechanism to adjust to varying muscle stretch conditions. Upon stretch, titin behaves as a spring capable of modulating its elastic response in accordance with changes in muscle biochemistry. One such mechanism has been the calcium-dependent stiffening of titin domains that renders the spring inherently more resistant to stretch. This transient titin-calcium interaction may serve a protective function in muscle, which could preclude costly unfolding of select domains when muscles elongate to great lengths. To test this idea, fluorescence spectroscopy was performed revealing a change in the microenvironment of the investigated immunoglobulin domain 27 (I27) of titin with calcium. Additionally, an atomic force microscope was used to evaluate the calcium-dependent regulation of passive force by stretching eight linked titin I27 domains until they unfolded. When stretching in the presence of calcium, the I27 homopolymer chain became stabilized, displaying three novel properties: (1) higher stretching forces were needed to unfold the domains, (2) the stiffness, measured as a persistence length (PL), increased and (3) the peak-to-peak distance between adjacent I27 domains increased. Furthermore, a peak order dependence became apparent for both force and PL, reflecting the importance of characterizing the dynamic unfolding history of a polymer with this approach. Together, this novel titin Ig-calcium interaction may serve to stabilize the I27 domain permitting titin to tune passive force within stretched muscle in a calcium-dependent manner.

FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

PubMed

Zhang, Shizhu; Zheng, Hailin; Long, Nanbiao; Carbó, Natalia; Chen, Peiying; Aguilar, Pablo S; Lu, Ling

2014-02-01

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

Intra-amniotic administration (Gallus gallus) of cicer arietinum and lens culinaris prebiotics extracts and duck egg white peptides affects calcium status and intestinal functionality

USDA-ARS?s Scientific Manuscript database

Calcium (Ca) is one of the most abundant inorganic elements in the human body and has many important physiological roles. Prebiotics and bioactive peptides are two important substances used to promote calcium uptake. However, the difference in mechanisms of the calcium uptake from these two suppleme...

Phosphate and calcium are required for TGFbeta-mediated stimulation of ANK expression and function during chondrogenesis.

PubMed

Oca, Paulina; Zaka, Raihana; Dion, Arnold S; Freeman, Theresa A; Williams, Charlene J

2010-08-01

The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGFbeta. The purpose of this study was to determine whether TGFbeta stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGFbeta increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na(+)/Pi channels Pit-1 and Pit-2, indicated that the stimulation of ANK expression by TGFbeta required the influx of phosphate, specifically by the Pit-1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGFbeta on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGFbeta. Since previous studies of endochondral ossification in the growth plate have shown that L-type calcium channels are essential for chondrogenesis, we investigated their role in the TGFbeta-stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L-type channel Cav1.2 (alpha(1C)) inhibited the TGFbeta stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGFbeta stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation.

Anti-proliferative activity of silver nanoparticles

PubMed Central

AshaRani, PV; Hande, M Prakash; Valiyaveettil, Suresh

2009-01-01

Background Nanoparticles possess exceptional physical and chemical properties which led to rapid commercialisation. Silver nanoparticles (Ag-np) are among the most commercialised nanoparticles due to their antimicrobial potential. Ag-np based cosmetics, therapeutic agents and household products are in wide use, which raised a public concern regarding their safety associated with human and environmental use. No safety regulations are in practice for the use of these nanomaterials. The interactions of nanomaterials with cells, uptake mechanisms, distribution, excretion, toxicological endpoints and mechanism of action remain unanswered. Results Normal human lung fibroblasts (IMR-90) and human glioblastoma cells (U251) were exposed to different doses of Ag-nps in vitro. Uptake of Ag-nps occurred mainly through endocytosis (clathrin mediated process and macropinocytosis), accompanied by a time dependent increase in exocytosis rate. The electron micrographs revealed a uniform intracellular distribution of Ag-np both in cytoplasm and nucleus. Ag-np treated cells exhibited chromosome instability and mitotic arrest in human cells. There was efficient recovery from arrest in normal human fibroblasts whereas the cancer cells ceased to proliferate. Toxicity of Ag-np is mediated through intracellular calcium (Ca2+) transients along with significant alterations in cell morphology and spreading and surface ruffling. Down regulation of major actin binding protein, filamin was observed after Ag-np exposure. Ag-np induced stress resulted in the up regulation of metallothionein and heme oxygenase -1 genes. Conclusion Here, we demonstrate that uptake of Ag-np occurs mainly through clathrin mediated endocytosis and macropinocytosis. Our results suggest that cancer cells are susceptible to damage with lack of recovery from Ag-np-induced stress. Ag-np is found to be acting through intracellular calcium transients and chromosomal aberrations, either directly or through activation of catabolic enzymes. The signalling cascades are believed to play key roles in cytoskeleton deformations and ultimately to inhibit cell proliferation. PMID:19761582

Effects of NMDA antagonists on picrotoxin-, low Mg2+- and low Ca2+-induced epileptogenesis and on evoked changes in extracellular Na+ and Ca2+ concentrations in rat hippocampal slices.

PubMed

Köhr, G; Heinemann, U

1989-01-01

The anticonvulsant properties of ketamine and 2-APV were compared on 3 types of convulsant activity in hippocampal area CA1: the 'picrotoxin-epilepsy,' the 'low magnesium epilepsy' and the 'low calcium epilepsy.' In particular the spontaneous activity, the synaptically evoked responses and the changes in [Ca2+]0 were examined, since in many cases of epilepsy, Ca2+ uptake into cells is enhanced. In normal medium, ketamine and 2-APV have nearly no effect on stimulus evoked decreases in [Ca2+]0, although they clearly depress NMDA-induced ionic changes. However, ketamine and 2-APV prevent to some extent the augmentation of stimulus-induced changes in [Ca2+]0, observed after treating slices with picrotoxin or Mg2+-free medium. This extra Ca2+ uptake is probably mediated by NMDA operated channels. Our findings also show that ketamine, like 2-APV, has a stronger anticonvulsant effect on the low Mg-than on the picrotoxin-induced epileptiform activity. Responses to iontophoretically applied NMDA are facilitated in the 'low calcium epilepsy' and can be selectively blocked by ketamine. Spontaneous epileptiform activity occurring in low calcium can be blocked by ketamine only when some synaptic transmission is still present.

The binding of calcium ions by erythrocytes and `ghost'-cell membranes

PubMed Central

Long, C.; Mouat, Barbara

1971-01-01

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400μg-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140μg-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca2+ concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte `ghost' cells, the membranes of which appear to be permeable to Ca2+ ions, can bind about 1000μg-atoms of calcium per `ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane. PMID:5124387

Effect of sepsis on calcium uptake and content in skeletal muscle and regulation in vitro by calcium of total and myofibrillar protein breakdown in control and septic muscle: Results from a preliminary study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benson, D.W.; Hasselgren, P.O.; Hiyama, D.T.

Because high calcium concentration in vitro stimulates muscle proteolysis, calcium has been implicated in the pathogenesis of increased muscle breakdown in different catabolic conditions. Protein breakdown in skeletal muscle is increased during sepsis, but the effect of sepsis on muscle calcium uptake and content is not known. In this study the influence of sepsis, induced in rats by cecal ligation and puncture, on muscle calcium uptake and content was studied. Sixteen hours after cecal ligation and puncture or sham operation, calcium content of the extensor digitorum longus (EDL) and soleus (SOL) muscles was determined with an atomic absorption spectrometer. Calciummore » uptake was measured in intact SOL muscles incubated in the presence of calcium 45 (45Ca) for between 1 and 120 minutes. Total and myofibrillar protein breakdown was determined in SOL muscles, incubated in the presence of different calcium concentrations (0; 2.5; 5.0 mmol/L), and measured as release into the incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Calcium content was increased by 51% (p less than 0.001) during sepsis in SOL and by 10% (p less than 0.05) in EDL muscle. There was no difference in 45Ca uptake between control and septic muscles during the early phase (1 to 5 minutes) of incubation. During more extended incubation (30 to 120 minutes), muscles from septic rats took up significantly more 45Ca than control muscles (p less than 0.05). Tyrosine release by incubated SOL muscles from control and septic rats was increased when calcium was added to the incubation medium, and at a calcium concentration of 2.5 mmol/L, the increase in tyrosine release was greater in septic than in control muscle. Addition of calcium to the incubation medium did not affect 3-MH release in control or septic muscle.« less

Calcium-dependent mitochondrial formation of species mediating DNA single strand breakage in U937 cells exposed to sublethal concentrations of tert-butylhydroperoxide.

PubMed

Guidarelli, A; Clementi, E; Sciorati, C; Cattabeni, F; Cantoni, O

1997-10-01

Treatment of U937 cells with a sublethal albeit DNA-damaging concentration of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca++ uptake and ruthenium red (RR), a polycation that inhibits the calcium uniporter of mitochondria, significantly reduced the extent of DNA cleavage generated by the hydroperoxide. Release of Ca++ from the ryanodine(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca++ uptake and elicited a parallel enhancement in DNA strand scission induced by tB-OOH that was prevented by both Ry and RR. DNA damage caused by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetics superimposable with those observed after treatment with H2O2. Cf enhanced the DNA-damaging effects of tB-OOH in permeabilized cells as well, and similar effects were observed upon addition of CaCl2. Cf did not further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl2. The enhancing effects of Cf were prevented by RR and ryanodine, whereas those mediated by exogenous calcium were prevented only by RR. DNA strand scission caused by tB-OOH alone or associated with Cf in the permeabilized cell system was severely inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid. The mechanism(s) whereby Ca++ promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesions was subsequently analyzed using intact as well as permeabilized cells. Hydrogen peroxide was identified to be one of these species.

Role of receptor-mediated endocytosis in the antiangiogenic effects of human T lymphoblastic cell-derived microparticles.

PubMed

Yang, Chun; Xiong, Wei; Qiu, Qian; Shao, Zhuo; Shao, Zuo; Hamel, David; Tahiri, Houda; Leclair, Grégoire; Lachapelle, Pierre; Chemtob, Sylvain; Hardy, Pierre

2012-04-15

Microparticles possess therapeutic potential regarding angiogenesis. We have demonstrated the contribution of apoptotic human CEM T lymphocyte-derived microparticles (LMPs) as inhibitors of angiogenic responses in animal models of inflammation and tumor growth. In the present study, we characterized the antivascular endothelial growth factor (VEGF) effects of LMPs on pathological angiogenesis in an animal model of oxygen-induced retinopathy and explored the role of receptor-mediated endocytosis in the effects of LMPs on human retinal endothelial cells (HRECs). LMPs dramatically inhibited cell growth of HRECs, suppressed VEGF-induced cell migration in vitro experiments, and attenuated VEGF-induced retinal vascular leakage in vivo. Intravitreal injections of fluorescently labeled LMPs revealed accumulation of LMPs in retinal tissue, with more than 60% reductions of the vascular density in retinas of rats with oxygen-induced neovascularization. LMP uptake experiments demonstrated that the interaction between LMPs and HRECs is dependent on temperature. In addition, endocytosis is partially dependent on extracellular calcium. RNAi-mediated knockdown of low-density lipoprotein receptor (LDLR) reduced the uptake of LMPs and attenuated the inhibitory effects of LMPs on VEGF-A protein expression and HRECs cell growth. Intravitreal injection of lentivirus-mediated RNA interference reduced LDLR protein expression in retina by 53% and significantly blocked the antiangiogenic effects of LMPs on pathological vascularization. In summary, the potent antiangiogenic LMPs lead to a significant reduction of pathological retinal angiogenesis through modulation of VEGF signaling, whereas LDLR-mediated endocytosis plays a partial, but pivotal, role in the uptake of LMPs in HRECs.

Calcium uptake in aquatic insects: influences of phylogeny and metals (Cd and Zn).

PubMed

Poteat, Monica D; Buchwalter, David B

2014-04-01

Calcium sequestration in the hypo-osmotic freshwater environment is imperative in maintaining calcium homeostasis in freshwater aquatic organisms. This uptake process is reported to have the unintended consequence of potentially toxic heavy metal (Cd, Zn) uptake in a variety of aquatic species. However, calcium uptake remains poorly understood in aquatic insects, the dominant invertebrate faunal group in most freshwater ecosystems. Here, we examined Ca uptake and interactions with heavy metals (Cd, Zn) at low ambient Ca levels (12.5 μmol l(-1)) in 12 aquatic insect species within Ephemerellidae (mayfly) and Hydropsychidae (caddisfly), two families differentially responsive to trace metal pollution. We found Ca uptake varied 70-fold across the 12 species studied. Body mass and clade (family) were found to significantly influence both Ca uptake and adsorption (P≤0.05). Zn and Cd uptake rate constants (ku) exhibited a strong correlation (r=0.96, P<0.0001), suggesting a shared transport system. Ca uptake failed to significantly correlate with either Zn or Cd ku values. Further, neither Zn nor Cd exhibited inhibitory effects toward Ca uptake. In fact, we saw evidence of modest stimulation of Ca uptake rates in some metal treatments. This work suggests that insects generally differ from other freshwater taxa in that aqueous Ca uptake does not appear to be compromised by Cd or Zn exposure. It is important to understand the trace metal and major ion physiology of aquatic insects because of their ecological importance and widespread use as ecological indicators.

Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

PubMed

Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

2016-04-21

Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

Serum killing of Ureaplasma parvum shows serovar-determined susceptibility for normal individuals and common variable immuno-deficiency patients.

PubMed

Beeton, Michael L; Daha, Mohamed R; El-Shanawany, Tariq; Jolles, Stephen R; Kotecha, Sailesh; Spiller, O Brad

2012-02-01

Many Gram-negative bacteria, unlike Gram-positive, are directly lysed by complement. Ureaplasma can cause septic arthritis and meningitis in immunocompromised individuals and induce premature birth. Ureaplasma has no cell wall, cannot be Gram-stain classified and its serum susceptibility is unknown. Survival of Ureaplasma serovars (SV) 1, 3, 6 and 14 (collectively Ureaplasma parvum) were measured following incubation with normal or immunoglobulin-deficient patient serum (relative to heat-inactivated controls). Blocking monoclonal anti-C1q antibody and depletion of calcium, immunoglobulins, or lectins were used to determine the complement pathway responsible for killing. Eighty-three percent of normal sera killed SV1, 67% killed SV6 and 25% killed SV14; greater killing correlating to strong immunoblot identification of anti-Ureaplasma antibodies; killing was abrogated following ProteinA removal of IgG1. All normal sera killed SV3 in a C1q-dependent fashion, irrespective of immunoblot identification of anti-Ureaplasma antibodies; SV3 killing was unaffected by total IgG removal by ProteinG, where complement activity was retained. Only one of four common variable immunodeficient (CVID) patient sera failed to kill SV3, despite profound IgM and IgG deficiency for all; however, killing of SV3 and SV1 was restored with therapeutic intravenous immunoglobulin therapy. Only the classical complement pathway mediated Ureaplasma-cidal activity, sometimes in the absence of observable immunoblot reactive bands. Copyright © 2011 Elsevier GmbH. All rights reserved.

Mast cell degranulation and calcium influx are inhibited by an Echinacea purpurea extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide.

PubMed

Gulledge, Travis V; Collette, Nicholas M; Mackey, Emily; Johnstone, Stephanie E; Moazami, Yasamin; Todd, Daniel A; Moeser, Adam J; Pierce, Joshua G; Cech, Nadja B; Laster, Scott M

2018-02-15

Native Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Today, various Echinacea spp. preparations are used primarily to treat upper respiratory infections. The goal of this study was to evaluate the effects of an ethanolic E. purpurea (L) Moench root extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea. A15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines. A15 inhibited ß-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100µM, respectively. Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition, A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FcεRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE 2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production. These findings suggest that Echinacea extracts and alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, suggesting new uses for this plant and its constituents. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasminogen promotes macrophage phagocytosis in mice

PubMed Central

Ganapathy, Swetha; Settle, Megan; Plow, Edward F.

2014-01-01

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg+/+ mice, Plg−/− mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg−/− mice compared with Plg+/+ mice. A gene array of splenic and hepatic tissues from Plg−/− and Plg+/+ mice showed downregulation of numerous genes in Plg−/− mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis. PMID:24876560

Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grasso, P.; Reichert, L.E. Jr.

1990-08-01

We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+more » influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.« less

Effect of parathyroid hormone on transport by toad and turtle bladder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sabatini, S.; Kurtzman, N.A.

1987-01-01

The authors recently demonstrated that parathyroid hormone (PTH) inhibited both vasopressin- and cyclic AMP-stimulated water transport in the toad bladder. This was associated with an increase in calcium uptake by isolated epithelial cells. They postulated that PTH exerts its action on H/sub 2/O transport by directly stimulating calcium uptake. The current study was designed to compare the effects of PTH and the calcium ionophore, A23187, on H/sub 2/O and Na transport and H..mu.. secretion in toad and turtle bladders. In toad bladder, PTH and A23187 decreased arginine vasopressin (AVP)-stimulated H/sub 2/O flow and short-circuit current (SCC) after 60 min serosalmore » incubation. In turtle bladder A23187 decreased SCC to 79.3 +/- 3.6% of base line (P < 0.05), and significantly decreased RSCC as well. PTH had no effect on SCC or H/sup +/ secretion in turtle bladders. Both PTH and A23187 increased /sup 45/Ca uptake in toad bladder epithelial cells; only A23187 increased /sup 45/Ca uptake in the turtle bladder. The different action of PTH in these two membranes, compared with that of the calcium ionophore, illustrates the selectivity of PTH on membrane transport. PTH increases calcium uptake and decreases transport only in a hormone-sensitive epithelium, whereas the ionophore works in virtually all living membranes. The mode of action of these two agents to increase calcium uptake is, therefore likely different.« less

Acquisition of Ca2+ and HCO3−/CO32− for shell formation in embryos of the common pond snail Lymnaea stagnalis

PubMed Central

O’Donnell, Michael J.; Grosell, Martin

2010-01-01

Embryos of the freshwater common pond snail Lymnaea stagnalis develop to hatch within 10 days under control conditions (22°C, Miami-Dade tap water) and this development is impaired by removal of ambient calcium. In contrast, embryos did not exhibit dependence upon an ambient HCO3−/CO32− source, developing and hatching in HCO3−/CO32−-free water at rates comparable to controls. Post-metamorphic, shell-laying embryos exhibited a significant saturation-type calcium uptake as a function of increasing ambient calcium concentration. However, changes in ambient bicarbonate concentration did not influence calcium or apparent titratable alkalinity uptake. There was a distinct shift from no significant flux in pre-metamorphic embryos to net uptake of calcium in post-metamorphic stages as indicated by an increased uptake from the micro-environment surrounding the egg mass and increased net uptake in 24-h, whole egg mass flux measurements. Furthermore, HCO3−/CO32− acquisition as measured by titratable alkalinity flux is at least partially attributable to an endogenous carbonate source that is associated with acid extrusion. Thus, calcium requirements for embryonic shell formation are met via uptake but HCO3−/CO32−, which is also necessary for shell formation is acquired in part from endogenous sources with no detectable correlation to ambient HCO3−/CO32− availability. PMID:20361194

Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings

NASA Technical Reports Server (NTRS)

Ward, M. R.; Tischner, R.; Huffaker, R. C.

1988-01-01

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.

Enhanced Mitochondrial Transient Receptor Potential Channel, Canonical Type 3-Mediated Calcium Handling in the Vasculature From Hypertensive Rats.

PubMed

Wang, Bin; Xiong, Shiqiang; Lin, Shaoyang; Xia, Weijie; Li, Qiang; Zhao, Zhigang; Wei, Xing; Lu, Zongshi; Wei, Xiao; Gao, Peng; Liu, Daoyan; Zhu, Zhiming

2017-07-15

Mitochondrial Ca 2+ homeostasis is fundamental to the regulation of mitochondrial reactive oxygen species (ROS) generation and adenosine triphosphate production. Recently, transient receptor potential channel, canonical type 3 (TRPC3), has been shown to localize to the mitochondria and to play a role in maintaining mitochondrial calcium homeostasis. Inhibition of TRPC3 attenuates vascular calcium influx in spontaneously hypertensive rats (SHRs). However, it remains elusive whether mitochondrial TRPC3 participates in hypertension by increasing mitochondrial calcium handling and ROS production. In this study we demonstrated increased TRPC3 expression in purified mitochondria in the vasculature from SHRs, which facilitates enhanced mitochondrial calcium uptake and ROS generation compared with Wistar-Kyoto rats. Furthermore, inhibition of TRPC3 by its specific inhibitor, Pyr3, significantly decreased the vascular mitochondrial ROS production and H 2 O 2 synthesis and increased adenosine triphosphate content. Administration of telmisartan can improve these abnormalities. This beneficial effect was associated with improvement of the mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase in the vasculature of SHRs. In vivo, chronic administration of telmisartan suppressed TRPC3-mediated excessive mitochondrial ROS generation and vasoconstriction in the vasculature of SHRs. More importantly, TRPC3 knockout mice exhibited significantly ameliorated hypertension through reduction of angiotensin II-induced mitochondrial ROS generation. Together, we give experimental evidence for a potential mechanism by which enhanced TRPC3 activity at the cytoplasmic and mitochondrial levels contributes to redox signaling and calcium dysregulation in the vasculature from SHRs. Angiotensin II or telmisartan can regulate [Ca 2+ ] mito , ROS production, and mitochondrial energy metabolism through targeting TRPC3. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages.

PubMed

Tabei, Yosuke; Sugino, Sakiko; Eguchi, Kenichiro; Tajika, Masahiko; Abe, Hiroko; Nakajima, Yoshihiro; Horie, Masanori

2017-08-19

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO 3 )-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO 3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO 3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO 3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO 3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO 3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO 3 particles. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of Reaction Pathway on the Extent and Mechanism of Uranium(VI) Immobilization with Calcium and Phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mehta, Vrajesh S.; Maillot, Fabien; Wang, Zheming

Phosphate addition to subsurface environments contaminated with uranium can be used as an in situ remediation approach. Batch experiments were conducted to evaluate the dependence of the extent and mechanism of uranium uptake on the pathway for reaction with calcium phosphates. At pH 4.0 and 6.0 uranium uptake occurred via autunite (Ca(UO2)(PO4)3) precipitation irrespective of the starting forms of calcium and phosphate. At pH 7.5, the uptake mechanism depended on the nature of the calcium and phosphate. When dissolved uranium, calcium, and phosphate were added simultaneously, uranium was structurally incorporated into a newly formed amorphous calcium phosphate solid. Adsorption wasmore » the dominant removal mechanism for uranium contacted with pre-formed amorphous calcium phosphate solids,. When U(VI) was added to a suspension containing amorphous calcium phosphate solids as well as dissolved calcium and phosphate, then removal occurred through precipitation (57±4 %) of autunite and adsorption (43±4 %) onto calcium phosphate. The solid phase speciation of the uranium was determined using X-ray absorption spectroscopy and laser induced fluorescence spectroscopy. Dissolved uranium, calcium, and phosphate concentrations with saturation index calculations helped identify removal mechanisms and determine thermodynamically favorable solid phases.« less

Near-infrared light-controlled regulation of intracellular calcium to modulate macrophage polarization.

PubMed

Kang, Heemin; Zhang, Kunyu; Wong, Dexter Siu Hong; Han, Fengxuan; Li, Bin; Bian, Liming

2018-04-21

Macrophages are multifunctional immune cells with diverse physiological functions such as fighting against infection, influencing progression of pathologies, maintaining homeostasis, and regenerating tissues. Macrophages can be induced to adopt distinct polarized phenotypes, such as classically activated pro-inflammatory (M1) phenotypes or alternatively activated anti-inflammatory and pro-healing (M2), to execute diverse and dynamic immune functions. However, unbalanced polarizations of macrophage can lead to various pathologies, such as atherosclerosis, obesity, tumor, and asthma. Thus, the capability to remotely control macrophage phenotypes is important to the success of treating many pathological conditions involving macrophages. In this study, we developed an upconversion nanoparticle (UCNP)-based photoresponsive nanocarrier for near-infrared (NIR) light-mediated control of intracellular calcium levels to regulate macrophage polarization. UCNP was coated with mesoporous silica (UCNP@mSiO 2 ), into which loaded calcium regulators that can either supply or deplete calcium ions. UCNP@mSiO 2 was chemically modified through serial coupling of photocleavable linker and Arg-Gly-Asp (RGD) peptide-bearing molecular cap via cyclodextrin-adamantine host-guest complexation. The RGD-bearing cap functioned as the photolabile gating structure to control the release of calcium regulators and facilitated the cellular uptake of UCNP@mSiO 2 nanocarrier. The upconverted UV light emission from the UCNP@mSiO 2 under NIR light excitation triggered the cleavage of cap and intracellular release of calcium regulators, thereby allowing temporal regulation on the intracellular calcium levels. Application of NIR light through skin tissue promoted M1 or M2 polarization of macrophages, by elevating or depleting intracellular calcium levels, respectively. To the best of our knowledge, this is the first demonstration of NIR light-mediated remote control on macrophage polarization. This photoresponsive nanocarrier offers the potential to remotely manipulate in vivo immune functions, such as inflammation or tissue regeneration, via NIR light-controlled macrophage polarization. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cell-Based Screen Identifies Human Interferon-Stimulated Regulators of Listeria monocytogenes Infection

PubMed Central

Eitson, Jennifer L.; Chen, Didi; Jimenez, Alyssa; Mettlen, Marcel; Schoggins, John W.; Alto, Neal M.

2016-01-01

The type I interferon (IFN) activated transcriptional response is a critical antiviral defense mechanism, yet its role in bacterial pathogenesis remains less well characterized. Using an intracellular pathogen Listeria monocytogenes (Lm) as a model bacterial pathogen, we sought to identify the roles of individual interferon-stimulated genes (ISGs) in context of bacterial infection. Previously, IFN has been implicated in both restricting and promoting Lm growth and immune stimulatory functions in vivo. Here we adapted a gain-of-function flow cytometry based approach to screen a library of more than 350 human ISGs for inhibitors and enhancers of Lm infection. We identify 6 genes, including UNC93B1, MYD88, AQP9, and TRIM14 that potently inhibit Lm infection. These inhibitors act through both transcription-mediated (MYD88) and non-transcriptional mechanisms (TRIM14). Further, we identify and characterize the human high affinity immunoglobulin receptor FcγRIa as an enhancer of Lm internalization. Our results reveal that FcγRIa promotes Lm uptake in the absence of known host Lm internalization receptors (E-cadherin and c-Met) as well as bacterial surface internalins (InlA and InlB). Additionally, FcγRIa-mediated uptake occurs independently of Lm opsonization or canonical FcγRIa signaling. Finally, we established the contribution of FcγRIa to Lm infection in phagocytic cells, thus potentially linking the IFN response to a novel bacterial uptake pathway. Together, these studies provide an experimental and conceptual basis for deciphering the role of IFN in bacterial defense and virulence at single-gene resolution. PMID:28002492

Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses.

PubMed

Richard, Audrey Stéphanie; Zhang, Adam; Park, Sun-Jin; Farzan, Michael; Zong, Min; Choe, Hyeryun

2015-11-24

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared.

The leukocyte receptor CD84 inhibits Fc epsilon RI-mediated signaling through homophilic interaction in transfected RBL-2H3 cells.

PubMed

Oliver-Vila, Irene; Saborit-Villarroya, Ifigènia; Engel, Pablo; Martin, Margarita

2008-04-01

Signaling through the high-affinity receptor for immunoglobulin E (Fc epsilon RI) results in the coordinated activation of tyrosine kinases, thus leading to calcium mobilization, degranulation, and leukotriene and cytokine synthesis. Here, we show that CD84, a member of the CD150 family of leukocyte receptors, inhibits Fc epsilon RI-mediated mast cell degranulation in CD84-transfected rat basophilic leukaemia-2H3 mast cell line cells (RBL-2H3) through homophilic interaction. There was no reduction in overall protein phosphorylation following IgE triggering in CD84 RBL-2H3 cells. Indeed, phosphorylation of Dok-1 and c-Cbl increased in CD84 RBL-2H3, suggesting that inhibition is mediated by these molecules. MAP kinase phosphorylation (ERK1/2, JNK and p38) and cytokine synthesis were impaired in CD84 RBL-2H3. This inhibitory mechanism was independent of SAP and SHP-2 recruitment. Interestingly, CD84 mutants in tyrosines (Y279F and DeltaY324) reversed this inhibitory profile. These data suggest that CD84 may play a role in modulating Fc epsilon RI-mediated signaling in mast cells. Thus, CD84 could play a protective role against undesired allergic and inflammatory responses.

Alcohol enhances oxysterol-induced apoptosis in human endothelial cells by a calcium-dependent mechanism.

PubMed

Spyridopoulos, I; Wischhusen, J; Rabenstein, B; Mayer, P; Axel, D I; Fröhlich, K U; Karsch, K R

2001-03-01

Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with physiologically occurring oxysterols on atherogenesis.

Ca 45 Uptake in Fracture Callus of Normal and Aminoacetonitrile-Treated Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolognani, L.; Ponseti, T. V.

1962-04-01

Calcium content and Ca 45 uptake were measured in the fracture callus of normal and AAN-treated rats. It appears that total calcium deposition and Ca 45 uptake are both higher in the young callus, 5 and 10 days after fracture, of the AAN-treated animals. By the 20th day, mineralization of the callus in both groups is similar.

Heavy metal cations permeate the TRPV6 epithelial cation channel.

PubMed

Kovacs, Gergely; Danko, Tamas; Bergeron, Marc J; Balazs, Bernadett; Suzuki, Yoshiro; Zsembery, Akos; Hediger, Matthias A

2011-01-01

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications. Copyright © 2010 Elsevier Ltd. All rights reserved.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

PubMed

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian; Kielian, Margaret

2016-07-15

The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

PubMed Central

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian

2016-01-01

ABSTRACT The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. PMID:27122589

Hyaluronan and calcium carbonate hybrid nanoparticles for colorectal cancer chemotherapy

NASA Astrophysics Data System (ADS)

Bai, Jinghui; Xu, Jian; Zhao, Jian; Zhang, Rui

2017-09-01

A hybrid drug delivery system (DDS) composed of hyaluronan and calcium carbonate (CC) was developed. By taking advantage of the tumor-targeting ability of hyaluronan and the drug-loading property of CC, the well-formed hyaluronan-CC nanoparticles were able to serve as a DDS targeting colorectal cancer with a decent drug loading content, which is beneficial in the chemotherapy of colorectal cancer. In this study, hyaluronan-CC nanoparticles smaller than 100 nm were successfully developed to load the wide-range anti-cancer drug adriamycin (Adr) to construct hyaluronan-CC/Adr nanoparticles. On the other hand, we also found that hyaluronan-CC/Adr nanoparticles can possibly increase the uptake ratio of Adr into HT29 colorectal cancer cells when compared with hyaluronan-free nanoparticles (CC/Adr) via the CD44 receptor-mediated endocytosis via competitive uptake and in vivo imaging assays. Note that both in vitro (CCK-8 assay on HT29 cells) and in vivo (anti-cancer assay on HT-29 tumor-bearing nude mice model) experiments revealed that hyaluronan-CC/Adr nanoparticles exhibited stronger anti-cancer activity than free Adr or CC/Adr nanoparticles with minimized toxic side effects and preferable cancer-suppression potential.

Mutation/SNP analysis in EF-hand calcium binding domain of mitochondrial Ca[Formula: see text] uptake 1 gene in bipolar disorder patients.

PubMed

Safari, Roghaiyeh; Salimi, Reza; Tunca, Zeliha; Ozerdem, Aysegul; Ceylan, Deniz; Sakizli, Meral

2016-06-01

Calcium signaling is important for synaptic plasticity, generation of brain rhythms, regulating neuronal excitability, data processing and cognition. Impairment in calcium homeostasis contributed to the development of psychiatric disorders such as bipolar disorder (BP). MCU is the most important calcium transporter in mitochondria inner membrane responsible for influx of Ca[Formula: see text]. MICU1 is linked with MCU and has two canonical EF hands that are vital for its activity and regulates MCU-mediated Ca[Formula: see text] influx. In the current study, we aimed to investigate the role of genetic alteration of EF hand calcium binding motifs of MICU1 on the development of BP. We examined patients with BP, first degree relatives of these patients and healthy volunteers for mutations and polymorphisms in EF hand calcium binding motifs of MICU1. The result showed no SNP/mutation in BP patients, in healthy subjects and in first degree relatives. Additionally, alignment of the EF hand calcium binding regions among species (Gallus-gallus, Canis-lupus-familiaris, Bos-taurus, Mus-musculus, Rattus-norvegicus, Pan-troglodytes, Homosapiens and Danio-rerio) showed exactly the same amino acids (DLNGDGEVDMEE and DCDGNGELSNKE) except in one of the calcium binding domain of Danio-rerio that there was only one difference; leucine instead of Methionine. Our results showed that the SNP on EF-hand Ca[Formula: see text] binding domains of MICU1 gene had no effect in phenotypic characters of BP patients.

Carpal spasm in a girl as initial presentation of celiac disease: a case report.

PubMed

Ramosaj-Morina, Atifete; Keka-Sylaj, A; Hasbahta, V; Baloku-Zejnullahu, A; Azemi, M; Zunec, R

2017-09-04

Celiac disease is an immune-mediated disorder elicited by ingestion of gluten in genetically susceptible persons. This disorder is characterized by specific histological changes of the small intestine mucosa resulting in malabsorption. This case was written up as it was an unusual and dramatic presentation of celiac disease. We report the case of a 3-year-old Albanian girl who presented at our clinic with carpal spasms and hand paresthesia. A physical examination at admission revealed a relatively good general condition and body weight of 10.5 kg (10 percentile). Carpal spasms and paresthesias of her extremities were present. Neuromuscular irritability was demonstrated by positive Chvostek and Trousseau signs. Blood tests showed severe hypocalcemia with a total serum calcium of 1.2 mmol/L (normal range 2.12 to 2.55 mmol/L), ionized calcium of 0.87 (normal range 1.11 to 1.30 mmol/L), and 24-hour urine calcium excretion of 9.16 mmol (normal range female <6.2 mmol/day). Among other tests, screening for celiac disease was performed: antigliadin immunoglobulin A, anti-tissue transglutaminase, and anti-endomysial immunoglobulin A antibodies were positive. A duodenal biopsy revealed lymphocyte infiltration, crypt hyperplasia, and villous atrophy compatible with celiac disease grade IIIb according to the Marsh classification. Following the diagnosis of celiac disease, human leukocyte antigen typing was performed, giving a definite diagnosis of celiac disease. She was started on a gluten-free diet. Due to failure to follow a gluten-free diet, episodes of carpal spasms appeared again. Unfortunately, at the age of 7 years she presents with delayed psychophysical development. Although hypocalcemia is a common finding in celiac disease, hypocalcemic carpal spasm is a rare initial manifestation of the disease. Therefore, the possibility of celiac disease should be considered in patients with repeated carpal spasms that seem unduly difficult to treat. This should be evaluated even in the absence of gastrointestinal symptoms since hypocalcemia and its manifestation may present as initial symptoms of celiac disease even in young children.

Bioactive Peptides Isolated from Casein Phosphopeptides Enhance Calcium and Magnesium Uptake in Caco-2 Cell Monolayers.

PubMed

Cao, Yong; Miao, Jianyin; Liu, Guo; Luo, Zhen; Xia, Zumeng; Liu, Fei; Yao, Mingfei; Cao, Xiaoqiong; Sun, Shengwei; Lin, Yanyin; Lan, Yaqi; Xiao, Hang

2017-03-22

The ability of casein phosphopeptides (CPPs) to bind and transport minerals has been previously studied. However, the single bioactive peptides responsible for the effects of CPPs have not been identified. This study was to purify calcium-binding peptides from CPPs and to determine their effects on calcium and magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1 to P5 were isolated and the amino acid sequences were determined using LC-MS/MS. Compared with the CPP-free control, all five monomeric peptides exhibited significant enhancing effects on the uptake of calcium and magnesium (P < 0.05). Interestingly, when calcium and magnesium were presented simultaneously with P5, magnesium was taken up with priority over calcium in the Caco-2 cell monolayers. For example, at 180 min, the amount of transferred magnesium and calcium was 78.4 ± 0.95 μg/well and 2.56 ± 0.64 μg/well, respectively, showing a more than 30-fold difference in the amount of transport caused by P5. These results provide novel insight into the mineral transport activity of phosphopeptides obtained from casein.

Effect of calcium(ion) uptake by rat adrenal mitochondria on pregnenolone formation and spectral properties of cytochrome P-450.

PubMed

Simpson, E R; Williams-Smith, D L

1975-10-09

The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenal glands. In the presence of succinate, addition of calcium chloride in the concentration range 20-150 muM caused an inhibition of pregnenolone formation in both decapsulated and capsular adrenal mitochondria. 11beta-hydroxylation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP+. Under these conditions, calcium chloride in the concentration range 120-875 muM caused a 2-4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone. The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium by adrenal mitochondria can stimulate pregnenolone formation by increasing the interaction of mitochondrial cytochrome P-450 with endogenous substrate.

Calcium transport across the inner mitochondrial membrane: molecular mechanisms and pharmacology

PubMed Central

Csordás, György; Várnai, Peter; Golenár, Tünde; Sheu, Shey-Shing; Hajnóczky, György

2011-01-01

Growing evidence supports that mitochondrial calcium uptake is important for cell metabolism, signaling and survival. However, both the molecular nature of the mitochondrial Ca2+ transport sites and the calcium signals they respond to remained elusive. Recent RNA interference studies have identified new candidate proteins for Ca2+ uptake across the inner mitochondrial membrane, including LETM1, MCU, MICU1 and NCLX. The sensitivity of these factors to several drugs has been tested and in parallel, some new inhibitors of mitochondrial Ca2+ uptake have been described. This paper provides an update on the pharmacological aspects of the molecular mechanisms of the inner mitochondrial membrane Ca2+ transport. PMID:22123069

Characterization and inhibition of nitrite uptake in shortnose sturgeon fingerlings

USGS Publications Warehouse

Fontenot, Q.C.; Isely, J.J.; Tomasso, J.R.

1999-01-01

Efforts are underway to culture the endangered shortnose sturgeon Acipenser brevirostrum for possible reintroduction. As part of a larger project to develop culture techniques for this species, the uptake of nitrite was evaluated in fingerlings (16.5 ?? 4.85 g; mean ?? SD). Plasma nitrite concentrations increased significantly with exposure time (0-5 d) and dose (0-4 mg nitrite-N/L). Shortnose sturgeon fingerlings were able to concentrate nitrite in their plasma to more than 63 times the environmental concentration. Chloride, as either sodium chloride or calcium chloride, partially inhibited nitrite uptake. However, calcium chloride was a better inhibitor. After previous exposure (2 d at 2.13 ?? 0.080 mg nitrite-N/L) plasma nitrite-N decreased from 165.5 to 36.7 mg/L during a 3-d simultaneous exposure to 2.13 ?? 0.080 mg nitrite-N/L and treatment with 40 mg chloride/L as calcium chloride. The addition of calcium chloride to the water appeared to be an effective means of preventing nitrite uptake and treating nitrite toxicity in hatchery-reared shortnose sturgeon fingerlings.

Initial stages of calcium uptake and mineral deposition in sea urchin embryos

PubMed Central

Vidavsky, Netta; Addadi, Sefi; Mahamid, Julia; Shimoni, Eyal; Ben-Ezra, David; Shpigel, Muki; Weiner, Steve; Addadi, Lia

2014-01-01

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5–1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20–30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule. PMID:24344263

Initial stages of calcium uptake and mineral deposition in sea urchin embryos.

PubMed

Vidavsky, Netta; Addadi, Sefi; Mahamid, Julia; Shimoni, Eyal; Ben-Ezra, David; Shpigel, Muki; Weiner, Steve; Addadi, Lia

2014-01-07

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.

Enhancing endosomal escape for nanoparticle mediated siRNA delivery

NASA Astrophysics Data System (ADS)

Ma, Da

2014-05-01

Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

PubMed

Kopp, Mathis; Rotan, Olga; Papadopoulos, Chrisovalantis; Schulze, Nina; Meyer, Hemmo; Epple, Matthias

2017-01-01

Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Diagnosis of Pediatric Hyperthyroidism: Technetium 99 Uptake Versus Thyroid Stimulating Immunoglobulins

PubMed Central

Misra, Madhusmita; Levitsky, Lynne L.

2015-01-01

Background: Treatment with antithyroid drugs is effective in conditions of increased thyroid hormone production (mostly Graves' Disease; GD), but not in subacute thyroiditis (SAT) or autoimmune thyroiditis (AIT). Positive thyroid stimulating immunoglobulins (TSI) make GD likely. However, not all children with GD have increased TSI. Uptake studies with 123I or 99Tc (99mTc) provide accurate and rapid diagnosis but are expensive and involve radiation exposure. Our objective was to compare TSI with 99mTc uptake for diagnosis of pediatric hyperthyroidism. Methods: We performed a retrospective chart review of hyperthyroid children who had both TSI estimation and 99mTc uptake assessment at presentation. Based on subsequent laboratory studies and follow-up, 37 had GD and 10 had non-GD thyroiditis. The TSI index was considered positive (TSI+) when it was above the upper limit of normal. 99mTc uptake was considered positive (Tc+) for any uptake >0.4% and negative (and low) (Tc-) for uptake ≤0.4%. Results: Forty-seven youth (83% females), aged 12.3±4.6 years, presented with a suppressed thyrotropin (TSH) and elevated free thyroxine and total triiodothyronine. All 37 patients with GD were Tc+ (100% sensitivity and specificity). The sensitivity of TSI for diagnosing GD was 84%, and the specificity was 100%. Six patients with GD were discordant with Tc+ but TSI–. Elevated TSI correlated with Tc+ (p=0.01) with a degree of agreement (kappa) of 0.69. Conclusion: 99mTc has excellent specificity and sensitivity in diagnosing GD. Given additional costs of 99mTc (two and a half times as much as TSI), it is reasonable to reserve 99mTc uptake assessment for hyperthyroidism of unclear etiology and negative TSI. PMID:25257665

Diagnosis of pediatric hyperthyroidism: technetium 99 uptake versus thyroid stimulating immunoglobulins.

PubMed

Baskaran, Charumathi; Misra, Madhusmita; Levitsky, Lynne L

2015-01-01

Treatment with antithyroid drugs is effective in conditions of increased thyroid hormone production (mostly Graves' Disease; GD), but not in subacute thyroiditis (SAT) or autoimmune thyroiditis (AIT). Positive thyroid stimulating immunoglobulins (TSI) make GD likely. However, not all children with GD have increased TSI. Uptake studies with (123)I or (99)Tc ((99m)Tc) provide accurate and rapid diagnosis but are expensive and involve radiation exposure. Our objective was to compare TSI with (99m)Tc uptake for diagnosis of pediatric hyperthyroidism. We performed a retrospective chart review of hyperthyroid children who had both TSI estimation and (99m)Tc uptake assessment at presentation. Based on subsequent laboratory studies and follow-up, 37 had GD and 10 had non-GD thyroiditis. The TSI index was considered positive (TSI+) when it was above the upper limit of normal. (99m)Tc uptake was considered positive (Tc+) for any uptake >0.4% and negative (and low) (Tc-) for uptake ≤0.4%. Forty-seven youth (83% females), aged 12.3±4.6 years, presented with a suppressed thyrotropin (TSH) and elevated free thyroxine and total triiodothyronine. All 37 patients with GD were Tc+ (100% sensitivity and specificity). The sensitivity of TSI for diagnosing GD was 84%, and the specificity was 100%. Six patients with GD were discordant with Tc+ but TSI-. Elevated TSI correlated with Tc+ (p=0.01) with a degree of agreement (kappa) of 0.69. (99m)Tc has excellent specificity and sensitivity in diagnosing GD. Given additional costs of (99m)Tc (two and a half times as much as TSI), it is reasonable to reserve (99m)Tc uptake assessment for hyperthyroidism of unclear etiology and negative TSI.

Elucidating the mechanisms of nickel compound uptake: A review of particulate and nano-nickel endocytosis and toxicity

PubMed Central

Muñoz, Alexandra; Costa, Max

2012-01-01

Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses - most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980’s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanism of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties. PMID:22206756

Elucidating the mechanisms of nickel compound uptake: A review of particulate and nano-nickel endocytosis and toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muñoz, Alexandra; Costa, Max, E-mail: Max.Costa@nyumc.org

Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses — most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanismmore » of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties.« less

The Transient Receptor Potential Ion Channel TRPV6 Is Expressed at Low Levels in Osteoblasts and Has Little Role in Osteoblast Calcium Uptake

PubMed Central

Little, Robert; Muimo, Richmond; Robson, Louise; Harris, Kate; Grabowski, Peter S.

2011-01-01

Background TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6 -/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. Results Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P = 0.77) or TE-85 (P = 0.69) osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9 × 10−5 relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0 × 10−2 and 2.38±0.28 × 10−4 the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24 × 10−5 relative to GAPDH, in contrast with 4.3±1.5 × 10−2 relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. Conclusions TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake. PMID:22163264

A sensor for calcium uptake

PubMed Central

Collins, Sean; Meyer, Tobias

2011-01-01

Mitochondria — the cell’s power plants — increase their energy production in response to calcium signals in the cytoplasm. A regulator of the elusive mitochondrial calcium channel has now been identified. PMID:20844529

Drug Hypersensitivity and Desensitizations: Mechanisms and New Approaches

PubMed Central

de las Vecillas Sánchez, Leticia; Alenazy, Leila A.; Garcia-Neuer, Marlene; Castells, Mariana C.

2017-01-01

Drug hypersensitivity reactions (HSRs) are increasing in the 21st Century with the ever expanding availability of new therapeutic agents. Patients with cancer, chronic inflammatory diseases, cystic fibrosis, or diabetes can become allergic to their first line therapy after repeated exposures or through cross reactivity with environmental allergens. Avoidance of the offending allergenic drug may impact disease management, quality of life, and life expectancy. Precision medicine provides new tools for the understanding and management of hypersensitivity reactions (HSRs), as well as a personalized treatment approach for IgE (Immunoglobuline E) and non-IgE mediated HSRs with drug desensitization (DS). DS induces a temporary hyporesponsive state by incremental escalation of sub-optimal doses of the offending drug. In vitro models have shown evidence that IgE desensitization is an antigen-specific process which blocks calcium flux, impacts antigen/IgE/FcεRI complex internalization and prevents the acute and late phase reactions as well as mast cell mediator release. Through a “bench to bedside” approach, in vitro desensitization models help elucidate the molecular pathways involved in DS, providing new insights to improved desensitization protocols for all patients. The aim of this review is to summarize up to date information on the drug HSRs, the IgE mediated mechanisms of desensitization, and their clinical applications. PMID:28632196

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

NASA Astrophysics Data System (ADS)

Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

GANP regulates recruitment of AID to immunoglobulin variable regions by modulating transcription and nucleosome occupancy

PubMed Central

Singh, Shailendra Kumar; Maeda, Kazuhiko; Eid, Mohammed Mansour Abbas; Almofty, Sarah Ameen; Ono, Masaya; Pham, Phuong; Goodman, Myron F.; Sakaguchi, Nobuo

2013-01-01

Somatic hypermutation in B cells is initiated by activation-induced cytidine deaminase-catalyzed C→U deamination at immunoglobulin variable regions. Here we investigate the role of the germinal centre-associated nuclear protein (GANP) in enhancing the access of activation-induced cytidine deaminase (AID) to immunoglobulin variable regions. We show that the nuclear export factor GANP is involved in chromatin modification at rearranged immunoglobulin variable loci, and its activity requires a histone acetyltransferase domain. GANP interacts with the transcription stalling protein Spt5 and facilitates RNA Pol-II recruitment to immunoglobulin variable regions. Germinal centre B cells from ganp-transgenic mice showed a higher AID occupancy at the immunoglobulin variable region, whereas B cells from conditional ganp-knockout mice exhibit a lower AID accessibility. These findings suggest that GANP-mediated chromatin modification promotes transcription complex recruitment and positioning at immunoglobulin variable loci to favour AID targeting. PMID:23652018

Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome.

PubMed

Kranias, Evangelia G; Hajjar, Roger J

2012-06-08

Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.

Importance of mitochondrial calcium uniporter in high glucose-induced endothelial cell dysfunction.

PubMed

Chen, Wei; Yang, Jie; Chen, Shuhua; Xiang, Hong; Liu, Hengdao; Lin, Dan; Zhao, Shaoli; Peng, Hui; Chen, Pan; Chen, Alex F; Lu, Hongwei

2017-11-01

Mitochondrial Ca 2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca 2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment. Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca 2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model. High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca 2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p < 0.05). These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively. Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.

CBL-CIPK network for calcium signaling in higher plants

NASA Astrophysics Data System (ADS)

Luan, Sheng

Plants sense their environment by signaling mechanisms involving calcium. Calcium signals are encoded by a complex set of parameters and decoded by a large number of proteins including the more recently discovered CBL-CIPK network. The calcium-binding CBL proteins specifi-cally interact with a family of protein kinases CIPKs and regulate the activity and subcellular localization of these kinases, leading to the modification of kinase substrates. This represents a paradigm shift as compared to a calcium signaling mechanism from yeast and animals. One example of CBL-CIPK signaling pathways is the low-potassium response of Arabidopsis roots. When grown in low-K medium, plants develop stronger K-uptake capacity adapting to the low-K condition. Recent studies show that the increased K-uptake is caused by activation of a specific K-channel by the CBL-CIPK network. A working model for this regulatory pathway will be discussed in the context of calcium coding and decoding processes.

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

PubMed Central

Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

2015-01-01

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane. PMID:26658290

Desalted Duck Egg White Peptides Promote Calcium Uptake and Modulate Bone Formation in the Retinoic Acid-Induced Bone Loss Rat and Caco-2 Cell Model.

PubMed

Hou, Tao; Liu, Yanshuang; Kolba, Nikolai; Guo, Danjun; He, Hui

2017-05-12

Desalted duck egg white peptides (DPs) have been proven to promote calcium uptake in Caco-2 cells and rats treated with a calcium-deficient diet. The retinoic acid-induced bone loss model was used to evaluate the effect of DPs on calcium absorption and bone formation. Three-month-old Wistar female rats were treated with 0.9% saline, DPs (800 mg/kg), or alendronate (5 mg/kg) for three weeks immediately after retinoic acid treatment (80 mg/kg) once daily for two weeks. The model group was significantly higher in serum bone alkaline phosphatase than the other three groups ( p < 0.05), but lower in calcium absorption rate, serum osteocalcin, bone weight index, bone calcium content, bone mineral density, and bone max load. After treatment with DPs or alendronate, the absorption rate increased and some serum and bone indices recovered. The morphology results indicated bone tissue form were ameliorated and numbers of osteoclasts decreased after supplementation with DPs or alendronate. The in vitro study showed that the transient receptor potential vanilloid 6 (TRPV6) calcium channel was the main transport pathway of both DPs and Val-Ser-Glu-Glu peptitde (VSEE), which was identified from DPs. Our results indicated that DPs could be a promising alternative to current therapeutic agents for bone loss because of the promotion of calcium uptake and regulation of bone formation.

Linking physiology and biomineralization processes to ecological inferences on the life history of fishes.

PubMed

Loewen, T N; Carriere, B; Reist, J D; Halden, N M; Anderson, W G

2016-12-01

Biomineral chemistry is frequently used to infer life history events and habitat use in fishes; however, significant gaps remain in our understanding of the underlying mechanisms. Here we have taken a multidisciplinary approach to review the current understanding of element incorporation into biomineralized structures in fishes. Biominerals are primarily composed of calcium-based derivatives such as calcium carbonate found in otoliths and calcium phosphates found in scales, fins and bones. By focusing on non-essential life elements (strontium and barium) and essential life elements (calcium, zinc and magnesium), we attempt to connect several fields of study to synergise how physiology may influence biomineralization and subsequent inference of life history. Data provided in this review indicate that the presence of non-essential elements in biominerals of fish is driven primarily by hypo- and hyper-calcemic environmental conditions. The uptake kinetics between environmental calcium and its competing mimics define what is ultimately incorporated in the biomineral structure. Conversely, circannual hormonally driven variations likely influence essential life elements like zinc that are known to associate with enzyme function. Environmental temperature and pH as well as uptake kinetics for strontium and barium isotopes demonstrate the role of mass fractionation in isotope selection for uptake into fish bony structures. In consideration of calcium mobilisation, the action of osteoclast-like cells on calcium phosphates of scales, fins and bones likely plays a role in fractionation along with transport kinetics. Additional investigations into calcium mobilisation are warranted to understand differing views of strontium, and barium isotope fractionation between calcium phosphates and calcium carbonate structures in fishes. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural changes in calcium-binding allergens: use of circular dichroism to study binding characteristics.

PubMed

Hebenstreit, D; Ferreira, F

2005-09-01

Several studies showed that calcium-binding proteins have a fixed place in the spectrum of allergenic substances. Often the binding of a calcium ion induces conformational changes and affects immunoglobulin E-binding to the allergen. Hence, the quantitative characterization of the binding to calcium is of importance to understand both the biologic and allergenic activity of these proteins. In the present study we describe a procedure for determining the stoichiometry and dissociation constant (K(D)) of calcium-binding allergens using circular dichroism (CD) techniques. For the experiments, we used recombinant Bet v 4, a two EF-hand allergen from birch pollen. Solutions of Bet v 4 were titrated with calcium and the change in molar ellipticity at 222 nm was monitored with a CD spectropolarimeter. The determination of the binding stoichiometry as well as of the K(D) for one EF-hand (4 microM) demonstrated the applicability of the method. CD-monitored calcium-titration of protein solutions represents a fast and easy method for determining the binding characteristics of calcium-binding allergens.

MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype

PubMed Central

Hong, Zhigang; Chen, Kuang-Hueih; DasGupta, Asish; Potus, Francois; Dunham-Snary, Kimberly; Bonnet, Sebastien; Tian, Lian; Fu, Jennifer; Breuils-Bonnet, Sandra; Provencher, Steeve; Wu, Danchen; Mewburn, Jeffrey; Ormiston, Mark L.

2017-01-01

Rationale: Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer-like phenotype is promoted by increased cytosolic calcium ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH’s cancer-like phenotype. Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore-forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid-mediated up-regulation, as well as through modulation of the upstream microRNAs (miRs) miR-138 and miR-25. In vivo, nebulized anti-miRs were administered to rats with monocrotaline-induced PAH. Measurements and Main Results: Impaired MCUC function, resulting from down-regulation of MCU and up-regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAH’s pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR-138) down-regulate MCU directly and also by decreasing MCU’s transcriptional regulator cAMP response element–binding protein 1. Nebulized anti-miRs against miR-25 and miR-138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model. Conclusions: These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted. PMID:27648837

Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins.

PubMed

Raman, Rajeev; Rajanikanth, V; Palaniappan, Raghavan U M; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P; Sharma, Yogendra; Chang, Yung-Fu

2010-12-29

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9(th) (Lig A9) and 10(th) repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²+ with dissociation constants of 2-4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. We demonstrate that the Lig are Ca²+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²+ binding.

Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins

PubMed Central

Palaniappan, Raghavan U. M.; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P.; Sharma, Yogendra; Chang, Yung-Fu

2010-01-01

Background Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. Principal Findings We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. Conclusions We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding. PMID:21206924

Update on the mechanism of action of antiepileptic drugs.

PubMed

Meldrum, B S

1996-01-01

Novel antiepileptic drugs (AEDs) are thought to act on voltage-sensitive ion channels, on inhibitory neurotransmission or on excitatory neurotransmission. Two successful examples of rational AED design that potentiate GABA-mediated inhibition are vigabatrin (VGB) by irreversible inhibition of GABA-transaminase, and tiagabine (TGB) by blocking GABA uptake. Lamotrigine (LTG) prolongs inactivation of voltage-dependent sodium channels. The anticonvulsant action of remacemide (RCM) is probably largely due to blockade of NMDA receptors and prolonged inactivation of sodium channels induced by its desglycinated metabolite. Felbamate (FBM) apparently blocks NMDA receptors, potentiates GABA-mediated responses, blocks L-type calcium channels, and possibly also prolongs sodium channel inactivation. Similarly, topiramate (TPM) has multiple probable sites of action, including sodium channels, GABA receptors, and glutamate (AMPA) receptors. Gabapentin (GBP) apparently has a completely novel type of action, probably involving potentiation of GABA-mediated inhibition and possibly also inactivation of sodium channels. The therapeutic advantages of the novel AEDs are as yet only partially explained by our present understanding of their mechanisms of action.

Relation between the location of elements in the periodic table and tumor-uptake rate.

PubMed

Ando, A; Ando, I; Hiraki, T; Hisada, K

1985-01-01

The bipositive ions and anions, with few exceptions, indicated a low tumor uptake rate. On the other hand, compounds of Hg, Au and Bi, which have a strong binding power to protein, showed a high tumor uptake rate. As Hg2+, Au+ and Bi3+ are soft acids according to the classification of Lewis acids, it was thought that these ions would bind strongly to soft bases (R-SH, R-S-) present in tumor tissue. For many hard acids such as 85Sr2+, 67Ga3+, 181Hf4+, and 95Nb5+, tumor uptake rates are shown as a function of ionic potentials (valency/ionic radii) of the metal ions. Considering the present data and previously reported results, it was presumed that hard acids of trivalence, quadrivalence and pentavalence would replace calcium in the calcium salts of hard bases (calcium salts of acid mucopolysaccharides, etc.). Ionic potentials of alkaline metals and Tl were small, but the tumor-uptake rate of these elements indicated various values. As Ge and Sb are bound by covalent bonds to chloride, GeCl4 and SbCl3 behaved differently from many metallic compounds in tumor tissue.

PubMed Central

Gallo, A.; Fusconi, M.; Pagliuca, G.; Greco, A.; De Virgilio, A.; De Vincentiis, M.

2017-01-01

SUMMARY Autoimmune diseases of major salivary glands include Sjögren's syndrome and a complex of disorders classified as immunoglobulin G4-related diseases. These pathologies are characterised by an autoimmune reaction mediated by T-helper lymphocytes that targets the ducts of exocrine glands in Sjögren's syndrome and glandular parenchyma in immunoglobulin G4-related diseases. Immunoglobulin G4-related diseases represent recently introduced multi-organ diseases that also involve the salivary glands. However, the morbid conditions once known as Mikulicz's disease and Kuttner's tumour were recently considered as two variants of immunoglobulin G4-related diseases affecting the major salivary glands ( immunoglobulin G4-related sialadenitis). This review briefly summarises the pathogenesis and clinical features of autoimmune diseases of the major salivary glands, focusing on the diagnostic and therapeutic role of sialendoscopy. PMID:28516978

Purification, immunotoxic effects, and cellular uptake of trichothecene mycotoxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witt, M.F.

1989-01-01

Studies were carried out to better understand how the trichothecenes alter immune function in animals and humans. Deoxynivalenol (DON) was purified for use in animal feeding studies. Dietary exposure to DON for 8 weeks altered the serum immunoglobulin profile in mice and decreased the splenic plaque-forming cell response to the antigen sheep red blood cells. The uptake of ({sup 3}H)T-2 toxin by a murine B-cell hybridoma was studied in order to learn more about the way in which trichothecenes interact with immune cells. A simple procedure was developed for the laboratory production and purification of gram quantities of crystalline DON.more » When Fusarium graminearum R6576 was grown on rice, concentrations of 600 to 700 ppm DON accumulated after 13 to 18 days of incubation. A DON derivative, 15-acetylDON, was also found at concentrations of 100 to 300 ppm after 7 to 10 days. DON was purified from crude culture extracts by water-saturated silica gel chromatography. Alpha-({sup 3}H)T-2 toxin of 99% chemical and radiochemical purity was prepared for use in uptake studies. Both the rate of uptake of ({sup 3}H)T-2 toxin by hybridomas and the time required for accumulation of ({sup 3}H)T-2 to reach equilibrium were proportional to the concentration of ({sup 3}H)T-2. ({sup 3}H)T-2 toxin accumulated by hybridomas was proportional to the concentration of ({sup 3}H)T-2 between 10{sup {minus}8} and 10{sup {minus}3} M. The rate of uptake of ({sup 3}H)jT-2 toxin by hybridomas was inhibited by the trichothecenes T-2 toxin, DON, verrucarin A, and roridin A, as well as the antibiotic anisomycin. The kinetics and concentration dependence of accumulation, along with the inhibition patterns, suggest that uptake of ({sup 3}H)T-2 toxin by hybridomas is mediated by binding of toxin to ribosomes.« less

Vincristine-sulphate-loaded liposome-templated calcium phosphate nanoshell as potential tumor-targeting delivery system.

PubMed

Thakkar, Hetal Paresh; Baser, Amit Kumar; Parmar, Mayur Prakashbhai; Patel, Ketul Harshadbhai; Ramachandra Murthy, Rayasa

2012-06-01

Vincristine-sulfate-loaded liposomes were prepared with an aim to improve stability, reduce drug leakage during systemic circulation, and increase intracellular uptake. Liposomes were prepared by the thin-film hydration method, followed by coating with calcium phosphate, using the sequential addition approach. Prepared formulations were characterized for size, zeta potential, drug-entrapment efficiency, morphology by transmission electron microscopy (TEM), in vitro drug-release profile, and in vitro cell cytotoxicity study. Effect of formulation variables, such as drug:lipid ratio as well as nature and volume of hydration media, were found to affect drug entrapment, and the concentration of calcium chloride in coating was found to affect size and coating efficiency. Size, zeta potential, and TEM images confirmed that the liposomes were effectively coated with calcium phosphate. The calcium phosphate nanoshell exhibited pH-dependent drug release, showing significantly lower release at pH 7.4, compared to the release at pH 4.5, which is the pH of the tumor interstitium. The in vitro cytotoxicity study done on the lung cancer cell line indicated that coated liposomes are more cytotoxic than plain liposomes and drug solution, indicating their potential for intracellular drug delivery. The cell-uptake study done on the lung cancer cell line indicated that calcium-phosphate-coated liposomes show higher cell uptake than uncoated liposomes.

Toxic β-Amyloid (Aβ) Alzheimer's Ion Channels: From Structure to Function and Design

NASA Astrophysics Data System (ADS)

Nussinov, Ruth

2012-02-01

Full-length amyloid beta peptides (Aβ1-40/42) form neuritic amyloid plaques in Alzheimer's disease (AD) patients and are implicated in AD pathology. Recent biophysical and cell biological studies suggest a direct mechanism of amyloid beta toxicity -- ion channel mediated loss of calcium homeostasis. Truncated amyloid beta fragments (Aβ11-42 and Aβ17-42), commonly termed as non-amyloidogenic are also found in amyloid plaques of Alzheimer's disease (AD) and in the preamyloid lesions of Down's syndrome (DS), a model system for early onset AD study. Very little is known about the structure and activity of these smaller peptides although they could be key AD and DS pathological agents. Using complementary techniques of explicit solvent molecular dynamics (MD) simulations, atomic force microscopy (AFM), channel conductance measurements, cell calcium uptake assays, neurite degeneration and cell death assays, we have shown that non-amyloidogenic Aβ9-42 and Aβ17-42 peptides form ion channels with loosely attached subunits and elicit single channel conductances. The subunits appear mobile suggesting insertion of small oligomers, followed by dynamic channel assembly and dissociation. These channels allow calcium uptake in APP-deficient cells and cause neurite degeneration in human cortical neurons. Channel conductance, calcium uptake and neurite degeneration are selectively inhibited by zinc, a blocker of amyloid ion channel activity. Thus truncated Aβ fragments could account for undefined roles played by full length Aβs and provide a novel mechanism of AD and DS pathology. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets are highly polymorphic, and spontaneously break into loosely interacting dynamic units (though still maintaining ion channel structures as imaged with AFM), that associate and dissociate leading to toxic ion flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting α-helices that robustly prevent ion leakage, rather than hydrogen-bonded β-strands. Moreover, in comparison with β-rich antimicrobial peptide (AMP) such as a protegrin-1 (PG-1), both Aβ and PG-1 are cytotoxic, and capable of forming fibrils and dynamic channels which consist of subunits with similar dimensions. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that PG-1 is amyloidogenic and amyloids may have an antimicrobial function.

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

PubMed

Caro, Andres A; Evans, Kerry L; Cederbaum, Arthur I

2009-01-31

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

A9C sensitive Cl− - accumulation in A. thaliana root cells during salt stress is controlled by internal and external calcium

PubMed Central

Saleh, Livia; Plieth, Christoph

2013-01-01

The involvement of chloride in salt stress symptoms and salt tolerance mechanisms in plants has been less investigated in the past. Therefore, we studied the salt-induced chloride influx in Arabidopsis expressing the GFP-based anion indicator Clomeleon. High salt concentrations induce two phases of chloride influx. The fast kinetic phase is likely caused by membrane depolarization, and is assumed to be mediated by channels. This is followed by a slower "saturation" phase, where chloride is accumulated in the cytoplasm. Both phases of chloride uptake are dependent on the presence of external calcium. In general: with high [Ca2+] less chloride is accumulated in the cytoplasm. Surprisingly, also the internal calcium availability has an impact on chloride transport. A complete block of the second phase of chloride influx is achieved by the anion channel blocker A9C and trivalent cations (La3+, Gd3+, and Al3+). Other channel blockers and diuretics were found to inhibit the process partially. The results suggest that several transporter species are involved here, including electroneutral cation-chloride-cotransporters, and a part of chloride possibly enters the cells through cation channels after salt application. PMID:23603974

Inulin and oligofructose and mineral metabolism: the evidence from animal trials.

PubMed

Scholz-Ahrens, Katharina E; Schrezenmeir, Jürgen

2007-11-01

Nondigestible oligosaccharides have been shown to increase the absorption of several minerals (calcium, magnesium, in some cases phosphorus) and trace elements (mainly copper, iron, zinc). Inulin-type fructans including oligofructose and fructooligosaccharides derived from sucrose by enzymatic transfructosylation are the best investigated food ingredients in this respect. The stimulation of absorption was more pronounced when the demand for calcium was high, i.e., in animals in the rapid growing stage and in animals with impaired calcium absorption because of either ovariectomy or gastrectomy. Even a small stimulation of calcium absorption increased the mineral accumulation in the skeleton because of its persisting effect over months. Inulin-type fructans stimulated mineral absorption and bone mineral accretion when combined with probiotic lactobacilli and in the presence of antibiotics. Direct comparison of different inulin-type fructans revealed a more pronounced effect by inulin or a mixture of long-chain inulin and oligofructose than by oligofructose alone. Mechanisms on how inulin-type fructans mediate this effect include acidification of the intestinal lumen by short-chain fatty acids increasing solubility of minerals in the gut, enlargement of the absorption surface, increased expression of calcium-binding proteins mainly in the large intestine, modulated expression of bone-relevant cytokines, suppression of bone resorption, increased bioavailability of phytoestrogens, and, via stimulation of beneficial commensal microorganisms, increase of calcium uptake by enterocytes. Under certain conditions, inulin-type fructans may improve mineral absorption by their impact on the amelioration of gut health including stabilization of the intestinal flora and reduction of inflammation. The abundance of reports indicate that inulin-type fructans are promising substances that could help to improve the supply with available calcium in human nutrition and by this contribute to bone health.

Calcium and the heat-shock response in the human monocytic line U-937.

PubMed

Kantengwa, S; Capponi, A M; Bonventre, J V; Polla, B S

1990-07-01

In the human monocytic line U-937, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases cytosolic free calcium concentration ([Ca2+]i). 1,25-(OH)2D3 also enhances the synthesis of heat-shock proteins (HSPs) when U-937 cells are exposed to elevated temperatures. To establish whether these two effects of 1,25-(OH)2D3 are related, we examined the effects of calcium on the heat-shock (HS) response, as well as the influence of 1,25-(OH)2D3 on this system. The equilibrium dissociation constant (Kd) of the fluorescent probe used to measure [Ca2+]i, fura-2, at 37 and 45 degrees C was found to be 191 and 234 nM, respectively. Exposure of U-937 cells to 45 degrees C did not increase [Ca2+]i under conditions in which active efflux of the dye was prevented by the organic anion transport inhibitor probenecid (1 mM). In cells preincubated in calcium-free medium, with subsequent addition of 4 mM EGTA before HS, or exposed to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), the increase in HSPs synthesis was not affected. Cell viability, assessed by [3H]thymidine uptake, was not different between cells exposed to HS in calcium-containing or calcium-free media. Moreover, the effects of 1,25-(OH)2D3 on the HS response were also observed in a calcium-depleted medium, indicating that the effects of 1,25-(OH)2D3 on HSP synthesis were not mediated by [Ca2+]i.

Distinct Akt phosphorylation states are required for insulin regulated Glut4 and Glut1-mediated glucose uptake.

PubMed

Beg, Muheeb; Abdullah, Nazish; Thowfeik, Fathima Shazna; Altorki, Nasser K; McGraw, Timothy E

2017-06-07

Insulin, downstream of Akt activation, promotes glucose uptake into fat and muscle cells to lower postprandial blood glucose, an enforced change in cellular metabolism to maintain glucose homeostasis. This effect is mediated by the Glut4 glucose transporter. Growth factors also enhance glucose uptake to fuel an anabolic metabolism required for tissue growth and repair. This activity is predominantly mediated by the Glut1. Akt is activated by phosphorylation of its kinase and hydrophobic motif (HM) domains. We show that insulin-stimulated Glut4-mediated glucose uptake requires PDPK1 phosphorylation of the kinase domain but not mTORC2 phosphorylation of the HM domain. Nonetheless, an intact HM domain is required for Glut4-mediated glucose uptake. Whereas, Glut1-mediated glucose uptake also requires mTORC2 phosphorylation of the HM domain, demonstrating both phosphorylation-dependent and independent roles of the HM domain in regulating glucose uptake. Thus, mTORC2 links Akt to the distinct physiologic programs related to Glut4 and Glut1-mediated glucose uptake.

99Tcm-labelled polyclonal human immunoglobulin G scintigraphy before and after intra-articular knee injection of triamcinolone hexacetonide in patients with rheumatoid arthritis.

PubMed

de Bois, M H; Arndt, J W; Tak, P P; Kluin, P M; van der Velde, E A; Pauwels, E K; Breedveld, F C

1993-10-01

The ability of 99Tcm-labelled polyclonal human immunoglobulin G (99Tcm-IgG) scintigraphy to monitor intra-individual variation in arthritis activity was studied in seven patients with rheumatoid arthritis (RA). These patients were treated with an intra-articular injection of 20 mg triamcinolone hexacetonide. The results of semiquantitative 99Tcm-IgG scintigraphy were compared with the degree of joint swelling and the histological changes observed in synovial biopsies before and 14 days after the injection. In all seven patients the local treatment resulted in a decreased arthritis activity of the treated knee as measured clinically or histologically. This decrease was parallelled, in all patients except one, by a lower uptake of 99Tcm-IgG after the injection when compared to uptake prior to treatment. This study shows that 99Tcm-IgG scintigraphy is able to reflect intra-individual variations in arthritis activity in patients with RA.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

PubMed

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Role of mitochondrial calcium uptake homeostasis in resting state fMRI brain networks.

PubMed

Kannurpatti, Sridhar S; Sanganahalli, Basavaraju G; Herman, Peter; Hyder, Fahmeed

2015-11-01

Mitochondrial Ca(2+) uptake influences both brain energy metabolism and neural signaling. Given that brain mitochondrial organelles are distributed in relation to vascular density, which varies considerably across brain regions, we hypothesized different physiological impacts of mitochondrial Ca(2+) uptake across brain regions. We tested the hypothesis by monitoring brain "intrinsic activity" derived from the resting state functional MRI (fMRI) blood oxygen level dependent (BOLD) fluctuations in different functional networks spanning the somatosensory cortex, caudate putamen, hippocampus and thalamus, in normal and perturbed mitochondrial Ca(2+) uptake states. In anesthetized rats at 11.7 T, mitochondrial Ca(2+) uptake was inhibited or enhanced respectively by treatments with Ru360 or kaempferol. Surprisingly, mitochondrial Ca(2+) uptake inhibition by Ru360 and enhancement by kaempferol led to similar dose-dependent decreases in brain-wide intrinsic activities in both the frequency domain (spectral amplitude) and temporal domain (resting state functional connectivity; RSFC). The fact that there were similar dose-dependent decreases in the frequency and temporal domains of the resting state fMRI-BOLD fluctuations during mitochondrial Ca(2+) uptake inhibition or enhancement indicated that mitochondrial Ca(2+) uptake and its homeostasis may strongly influence the brain's functional organization at rest. Interestingly, the resting state fMRI-derived intrinsic activities in the caudate putamen and thalamic regions saturated much faster with increasing dosage of either drug treatment than the drug-induced trends observed in cortical and hippocampal regions. Regional differences in how the spectral amplitude and RSFC changed with treatment indicate distinct mitochondrion-mediated spontaneous neuronal activity coupling within the various RSFC networks determined by resting state fMRI. Copyright © 2015 John Wiley & Sons, Ltd.

Biotic Control of Surface pH and Evidence of Light-Induced H+ Pumping and Ca2+-H+ Exchange in a Tropical Crustose Coralline Alga.

PubMed

Hofmann, Laurie C; Koch, Marguerite; de Beer, Dirk

2016-01-01

Presently, an incomplete mechanistic understanding of tropical reef macroalgae photosynthesis and calcification restricts predictions of how these important autotrophs will respond to global change. Therefore, we investigated the mechanistic link between inorganic carbon uptake pathways, photosynthesis and calcification in a tropical crustose coralline alga (CCA) using microsensors. We measured pH, oxygen (O2), and calcium (Ca2+) dynamics and fluxes at the thallus surface under ambient (8.1) and low (7.8) seawater pH (pHSW) and across a range of irradiances. Acetazolamide (AZ) was used to inhibit extracellular carbonic anhydrase (CAext), which mediates hydrolysis of HCO3-, and 4,4' diisothiocyanatostilbene-2,2'-disulphonate (DIDS) that blocks direct HCO3- uptake by anion exchange transport. Both inhibited photosynthesis, suggesting both diffusive uptake of CO2 via HCO3- hydrolysis to CO2 and direct HCO3- ion transport are important in this CCA. Surface pH was raised approximately 0.3 units at saturating irradiance, but less when CAext was inhibited. Surface pH was lower at pHSW 7.8 than pHSW 8.1 in the dark, but not in the light. The Ca2+ fluxes were large, complex and temporally variable, but revealed net Ca2+ uptake under all conditions. The temporal variability in Ca2+ dynamics was potentially related to localized dissolution during epithallial cell sloughing, a strategy of CCA to remove epiphytes. Simultaneous Ca2+ and pH dynamics suggest the presence of Ca2+/H+ exchange. Rapid light-induced H+ surface dynamics that continued after inhibition of photosynthesis revealed the presence of a light-mediated, but photosynthesis-independent, proton pump. Thus, the study indicates metabolic control of surface pH can occur in CCA through photosynthesis and light-inducible H+ pumps. Our results suggest that complex light-induced ion pumps play an important role in biological processes related to inorganic carbon uptake and calcification in CCA.

Biotic Control of Surface pH and Evidence of Light-Induced H+ Pumping and Ca2+-H+ Exchange in a Tropical Crustose Coralline Alga

PubMed Central

Hofmann, Laurie C.; Koch, Marguerite; de Beer, Dirk

2016-01-01

Presently, an incomplete mechanistic understanding of tropical reef macroalgae photosynthesis and calcification restricts predictions of how these important autotrophs will respond to global change. Therefore, we investigated the mechanistic link between inorganic carbon uptake pathways, photosynthesis and calcification in a tropical crustose coralline alga (CCA) using microsensors. We measured pH, oxygen (O2), and calcium (Ca2+) dynamics and fluxes at the thallus surface under ambient (8.1) and low (7.8) seawater pH (pHSW) and across a range of irradiances. Acetazolamide (AZ) was used to inhibit extracellular carbonic anhydrase (CAext), which mediates hydrolysis of HCO3-, and 4,4′ diisothiocyanatostilbene-2,2′-disulphonate (DIDS) that blocks direct HCO3- uptake by anion exchange transport. Both inhibited photosynthesis, suggesting both diffusive uptake of CO2 via HCO3- hydrolysis to CO2 and direct HCO3- ion transport are important in this CCA. Surface pH was raised approximately 0.3 units at saturating irradiance, but less when CAext was inhibited. Surface pH was lower at pHSW 7.8 than pHSW 8.1 in the dark, but not in the light. The Ca2+ fluxes were large, complex and temporally variable, but revealed net Ca2+ uptake under all conditions. The temporal variability in Ca2+ dynamics was potentially related to localized dissolution during epithallial cell sloughing, a strategy of CCA to remove epiphytes. Simultaneous Ca2+ and pH dynamics suggest the presence of Ca2+/H+ exchange. Rapid light-induced H+ surface dynamics that continued after inhibition of photosynthesis revealed the presence of a light-mediated, but photosynthesis-independent, proton pump. Thus, the study indicates metabolic control of surface pH can occur in CCA through photosynthesis and light-inducible H+ pumps. Our results suggest that complex light-induced ion pumps play an important role in biological processes related to inorganic carbon uptake and calcification in CCA. PMID:27459463

Interactions between calcium precipitation and the polyphosphate-accumulating bacteria metabolism.

PubMed

Barat, R; Montoya, T; Borrás, L; Ferrer, J; Seco, A

2008-07-01

A sequencing batch reactor that is operated for biological phosphorus removal has been operated under different influent calcium concentrations to study the precipitation process and the possible effects of phosphorus precipitation in the biological phosphorus removal process. Four experiments were carried out under different influent calcium concentrations ranging from 10 to 90 g Ca m(-3). The experimental results and the equilibrium study, which are based on the saturation index calculation, confirm that the process controlling the calcium behaviour is the calcium phosphate precipitation. This precipitation takes place at two stages: initially, precipitation of the amorphous calcium phosphate, and later crystallization of hydroxyapatite. Also the accumulation of phosphorus precipitated was observed when the influent calcium concentration was increased. In all the experiments, the influent wastewater ratio P/COD was kept constant. It has been observed that, at high calcium concentration, the ratio between phosphate release and acetate uptake (P(rel)/Ac(uptake)) decreases. Changes in the polyphosphate-accumulating organism (PAO) population and in the glycogen-accumulating organism (GAO) population during the experimental period were ruled out by means of fluorescence in situ hybridization. These results could suggest that PAO are able to change their metabolic pathways based on external conditions, such as influent calcium concentration. The accumulation of phosphorus precipitated as calcium phosphate at high influent calcium concentration throughout the experimental period confirmed that phosphate precipitation is a process that can affect the PAO metabolism.

Immunoglobulin-Mediated Agglutination of and Biofilm Formation by Escherichia coli K-12 Require the Type 1 Pilus Fiber

PubMed Central

Orndorff, Paul E.; Devapali, Aditya; Palestrant, Sarah; Wyse, Aaron; Everett, Mary Lou; Bollinger, R. Randal; Parker, William

2004-01-01

The binding of human secretory immunoglobulin A (SIgA), the primary immunoglobulin in the gut, to Escherichia coli is thought to be dependent on type 1 pili. Type 1 pili are filamentous bacterial surface attachment organelles comprised principally of a single protein, the product of the fimA gene. A minor component of the pilus fiber (the product of the fimH gene, termed the adhesin) mediates attachment to a variety of host cell molecules in a mannose inhibitable interaction that has been extensively described. We found that the aggregation of E. coli K-12 by human secretory IgA (SIgA) was dependent on the presence of the pilus fiber, even in the absence of the mannose specific adhesin or in the presence of 25 mM α-CH3Man. The presence of pilus without adhesin also facilitated SIgA-mediated biofilm formation on polystyrene, although biofilm formation was stronger in the presence of the adhesin. IgM also mediated aggregation and biofilm formation in a manner dependent on pili with or without adhesin. These findings indicate that the pilus fiber, even in the absence of the adhesin, may play a role in biologically important processes. Under conditions in which E. coli was agglutinated by SIgA, the binding of SIgA to E. coli was not increased by the presence of the pili, with or without adhesin. This observation suggests that the pili, with or without adhesin, affect factors such as cell surface rigidity or electrostatic repulsion, which can affect agglutination but which do not necessarily determine the level of bound immunoglobulin. PMID:15039312

Dynamics of cytokines and immunoglobulins serum profiles in primary and secondary Cryptosporidium parvum infection: usefulness of Luminex® xMAP technology.

PubMed

Codices, Vera; Martins, Catarina; Novo, Carlos; de Sousa, Bruno; Lopes, Ângela; Borrego, Miguel; Matos, Olga

2013-01-01

Infection by Cryptosporidium parvum triggers a complex array of innate and adaptive cell mediated immune response, playing an important role in controlling the infection. To date, there are no studies applying the Luminex® xMAP technology to determine profiles of cytokines and immunoglobulins in the context of an infection by C. parvum. In this study, we analyzed these immune mediators in the serum of immunocompetent mice inoculated with C. parvum oocysts, using Luminex, to understand how the immune system responds to an infection by this parasite. Animal sera were also analyzed by ELISA to determine the expressed immunoglobulin isotype profile, and compare the obtained trend with data obtained by Luminex. Specific-pathogen-free BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Peripheral blood was aseptically collected from sacrificed mice on several time points, and immune mediators were evaluated in serum samples. Infection was confirmed by the presence of C. parvum DNA in feces by a nested-PCR assay (60-kDa glycoprotein). Luminex results showed predominance in the secretion of IgG1 and IgG2a, confirmed by ELISA, which also showed that IgG1 is the major immunoglobulin isotype produced during the infection. The analysis of cytokines suggests a preferential Th(1) over the Th(2) response, with increased production of TNF-α, IFN-γ and GM-CSF. This work contributed to a better understanding of the immune response to the infection by C. parvum, as well as demonstrated the advantage of Luminex® xMAP technology to study immune mediators, using small sample volumes. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural basis for PECAM-1 homophilic binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paddock, C.; Zhou, D.; Lertkiatmongkol, P.

2015-12-23

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1–mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1–mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientationmore » of the PECAM-1–PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a β-sandwich topology formed by 2 sheets of antiparallel β strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å 2. These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions.« less

The spontaneous and evoked release of spermine from rat brain in vitro.

PubMed Central

Harman, R. J.; Shaw, G. G.

1981-01-01

1 The efflux of previously accumulated [3H]-spermine from brain slices was measured using a continuous perfusion system. The spontaneous efflux was biphasic, consisting of an initial rapid efflux followed by a much slower release. 2 The slices were depolarized by the addition to the medium of high potassium concentrations, ouabain or veratrine. 3 At concentrations greater than 30 mM, potassium evoked a striking increase in the release of [3H]-spermine. Following uptake in the presence of 5.7 x 10(-9)M [3H]-spermine, K+-evoked release was dependent on the presence of calcium ions. Release of spermine after uptake at 5.6 x 10(-8)M or 5.0 x 10(-7)M was not calcium-dependent. 4 The calcium-dependent, K+-stimulated release of spermine was inhibited in the presence of diphenylhydantoin (5 x 10(-5)M) or ruthenium red (10(-5)M). 5 Following uptake of 5.7 x 10(-9)M [3H]-spermine in a sodium-free medium, the calcium-dependent, K+-stimulated release was significantly inhibited. 5 Ouabain (10(-4)M) caused a large but calcium-independent increase in the efflux of [3H]-spermine. 7 Veratrine-induced release was less substantial but was increased in a calcium-free medium. Release evoked by veratrine was abolished in the absence of sodium. 8 These results are discussed with respect to a possible 'neurotransmitter' or 'neuromodulator' role for spermine. PMID:6169383

Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal.

PubMed

Maeda, A; Kurosaki, M; Ono, M; Takai, T; Kurosaki, T

1998-04-20

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

T-dependent activation of resting B cells mediated by concanavalin A.

PubMed

Ratcliffe, M J; Julius, M H

1984-03-01

In cultures containing long-term cultured lines of antigen-specific helper T (Th) cells, normal unprimed B cells and concanavalin A (Con A), induction of B cells to immunoglobulin secretion and DNA synthesis was observed. The plaque-forming cell (PFC) response was large (frequently greater than 75 000 PFC/10(6) input B cells) demonstrating the polyspecific nature of the response. Con A-mediated maturation and induction to DNA synthesis of responding B cells was completely Th cell dependent and inhibited with methyl-alpha-D-mannoside. Both resting and blasted B cells, separated by Percoll density centrifugation, were induced to DNA synthesis and immunoglobulin secretion. Responses were completely unrestricted by the B cell major histocompatibility complex, even at the level of the resting B cell. The polyclonal nature of the response taken together with the Con A-mediated bypassing of T cell specificity and restricting haplotype indicates that this response is analogous to lectin-mediated cytotoxicity.

Inhibition of IgE-induced mast cell activation by ethyl tertiary-butyl ether, a bioethanol-derived fuel oxygenate.

PubMed

Yamaki, Kouya; Yoshino, Shin

2009-09-01

The effect of ethyl tertiary-butyl ether (ETBE), which is widely used as a fuel oxygenate commonly produced from bioethanol, on immunoglobulin (Ig)E-dependent mast cell activation was investigated. The rat mast cell line RBL2H3 sensitised with monoclonal anti-ovalbumin IgE was challenged with ovalbumin in the presence or absence of ETBE, tert-butanol (TBA), which is the main metabolite of ETBE in humans, and ethanol. Degranulation of RBL2H3 was examined by the release of beta-hexosaminidase. To understand the mechanisms responsible for regulating mast cell function, the effects of ETBE, TBA and ethanol on the levels of intracellular calcium, phosphorylation of Akt (as a marker of phosphatidylinositol 3-kinase) and global tyrosine phosphorylation were also measured as indicators of mast cell activation. In the presence of ETBE, TBA or ethanol, IgE-induced release of beta-hexosaminidase was decreased. These compounds also attenuated the IgE-mediated increase in the levels of intracellular Ca(2+), phosphorylation of Akt and global tyrosine phosphorylation in RBL2H3 cells. ETBE, TBA and ethanol inhibited mast cell degranulation by inhibiting the increase in intracellular calcium ion concentration and activation of phosphatidylinositol 3-kinase and protein tyrosine kinase activation, suggesting that exposure to ETBE might affect immune responses, particularly in allergic diseases.

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

PubMed Central

Felgner, P L; Gadek, T R; Holm, M; Roman, R; Chan, H W; Wenz, M; Northrop, J P; Ringold, G M; Danielsen, M

1987-01-01

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique. Images PMID:2823261

Proofs concerning the existence, in the blood of hypertensive patients, of some serum factors influencing the vascular smooth muscle and the myocardium physiology.

PubMed

Mocanu, M; Botea, S; Dragomir, C T

1991-01-01

Starting from the existence of some autoimmune diseases (i.e. bronchial asthma or miastenia gravis) we asked ourselves if some plasmatic factors do exist, influencing the receptor--mediator relations in cardiovascular system during some illnesses having unknown etiology, as arterial hypertension. For this reason, in this work was tested the hypothesis that, in some chronic cardiovascular diseases would exist factors circulating and affecting the functions of the cellular membranes of the arterial wall, particularly of the smooth muscle cells and myocardial cells. Our results show a significant modification of the calcium fluxes and of some neuromediators uptake at the hypertensive patients.

Calcium silicate hydrates: Solid and liquid phase composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lothenbach, Barbara, E-mail: Barbara.lothenbach@empa.ch; Nonat, André

This paper presents a review on the relationship between the composition, the structure and the solution in which calcium silicate hydrate (C–S–H) is equilibrated. The silica chain length in C–S–H increases with the silicon concentration and the calcium content in the interlayer space with the calcium concentrations. Sodium and potassium are taken up in the interlayer space, preferentially at low calcium concentrations and thus by low Ca/Si C–S–H. Aluminium uptake in C–S–H increases strongly at higher aluminium concentrations in the solution. At low Ca/Si, aluminium substitutes silica in the bridging position, at Ca/Si > 1 aluminium is bound in TAH.more » Recently developed thermodynamic models are closely related to the structure of C–S–H and tobermorite, and able to model not only the solubility and the chemical composition of the C–S–H, but also to predict the mean silica chain length and the uptake of aluminium.« less

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

PubMed Central

1979-01-01

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity. PMID:512629

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling.

PubMed

Rojas, Fabiola; Gonzalez, David; Cortes, Nicole; Ampuero, Estibaliz; Hernández, Diego E; Fritz, Elsa; Abarzua, Sebastián; Martinez, Alexis; Elorza, Alvaro A; Alvarez, Alejandra; Court, Felipe; van Zundert, Brigitte

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1(G93A) (ACM-hSOD1(G93A)) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1(G93A), but not ACM-hSOD1(WT), increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1(G93A)-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1(G86R) or TDP43(A315T). We further find that co-application of ACM-SOD1(G93A) with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1(G93A) induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

The Calcium Solution: Developing Potato Cultivars With Enhanced Tuber Storage and Internal Quality by Genetic Improvement of Tuber Calcium Accumulation Ability Enetic Improvement of Potato for Tuber Calcium Uptake

USDA-ARS?s Scientific Manuscript database

Tuber internal quality is a major limiting factor for the U.S. potato industry. Breeders invest time and money in producing advanced selections which, in the end, often fail because of tuber internal defects, tuber bruising, or storage quality issues. In-season fertilization with calcium is known to...

Effects of calcium channel blockers on the kinetics of voltage-dependent changes in synaptosomal calcium concentrations.

PubMed

Thomas, M M; Puligandla, P S; Dunn, S M

1994-01-28

Synaptosomal preparations from rat cerebral cortex have been used in stopped-flow fluorescence studies to measure rapid changes in intrasynaptosomal calcium concentrations upon depolarization. Synaptosomes were loaded with the fluorescent calcium chelating dye, Fura-2, by incubation with the membrane permeant acetoxymethyl ester derivative. Depolarization by elevated external K+ concentration resulted in a rapid increase in cytoplasmic Ca2+ as measured by a quench in Fura-2 fluorescence when excited at 390 nm. The fluorescence change could be reasonably fit by a single exponential process with an apparent rate of 10-15 s-1 and the magnitude of the response was voltage-dependent, increasing with increasing external K+ over the range of 5-30 mM. The observed quench was blocked by micromolar concentrations of the inorganic calcium channel blockers, Cd2+, Co2+ and La3+. Nimodipine, a dihydropyridine which blocks L-type calcium channels, inhibited only 10-15% of the flux response while nitrendipine had no consistent effect. omega-Conotoxin GVIA, a blocker of N-type channels in many species, had only a small inhibitory effect at high (1-10 microM) concentrations. The response was, however, inhibited by pre-incubation of the synaptosomes with venom of the funnel web spider. Agelenopsis aperta (0.1-300 micrograms/ml). Inhibition was observed with both a purified polyamine fraction (FTX) from the venom (IC50 = 4 nl/ml) and a purified peptide toxin, omega-AgaIVA (IC50 = 30 nM). These results indicate that voltage-dependent Ca2+ uptake by mammalian nerve terminals is mediated primarily by channels that are insensitive to dihydropyridines and omega-conotoxin GVIA but are sensitive to components of funnel web spider venom.

Calcium translocations during the moulting cycle of the semiterrestrial isopod Ligia hawaiiensis (Oniscidea, Crustacea).

PubMed

Ziegler, Andreas; Hagedorn, Monica; Ahearn, Gregory A; Carefoot, Thomas H

2007-01-01

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO(3) deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. (45)Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.

Mechanism of Aluminum Inhibition of Net 45Ca2+ Uptake by Amaranthus Protoplasts 1

PubMed Central

Rengel, Zdenko; Elliott, Daphne C.

1992-01-01

Calcium ions serve as a second messenger in signal transduction and metabolic regulation. Effects of Al on calcium homeostasis remain to be elucidated. Short-term net 45Ca2+ uptake by Amaranthus tricolor protoplasts was monitored from uptake media prepared to test the influence of pH, Al, and various inhibitors. Accumulation of 45Ca2+ increased during the first 3 to 6 minutes and then leveled off or declined. Al and Ca2+ channel blockers (verapamil and bepridil) decreased net 45Ca2+ uptake. This decrease was more pronounced when Al and bepridil were both present in uptake media, but Al did not aggravate verapamil-induced reduction of net 45Ca2+ uptake. Erythrosin B and calmidazolium each increased net 45Ca2+ uptake, probably by interfering with Ca2+ efflux. This effect was undetectable in the presence of Al. Mycophenolic acid decreased net 45Ca2+ uptake; guanosine alleviated this effect. Al-induced reduction of net 45Ca2+ uptake was not aggravated by mycophenolic acid. Net 45Ca2+ uptake was generally less at pH 4.5 than at 5.5 for all treatments. It is concluded that Al ions affect net 45Ca2+ uptake by binding to the verapamil-specific channel site that is different from the bepridil-specific one, as well as by interfering with the action of guanosine 5′-triphosphate-binding proteins. PMID:16668688

Enhancement of nitrate uptake and growth of barley seedlings by calcium under saline conditions

NASA Technical Reports Server (NTRS)

Ward, M. R.; Aslam, M.; Huffaker, R. C.

1986-01-01

The effect of Ca2+ on NO3- assimilation in young barley (Hordeum vulgare L. var CM 72) seedlings in the presence and absence of NaCl was studied. Calcium increased the activity of the NO3- transporter under saline conditions, but had little effect under nonsaline conditions. Calcium decreased the induction period for the NO3- transporter under both saline and nonsaline conditions but had little effect on its apparent Km for NO3- both in the presence and absence of NaCl. The enhancement of NO3- transport by Ca2+ under saline conditions was dependent on the presence of Ca2+ in the uptake solution along with the salt, since Ca2+ had no effect when supplied before or after salinity stress. Although Mn2+ and Mg2+ enhanced NO3- uptake under saline conditions, neither was as effective as Ca2+. In longer studies, increasing the Ca2+ concentration in saline nutrient solutions resulted in increases in NO3- assimilation and seedling growth.

A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

PubMed

Cai, Xixi; Yang, Qian; Lin, Jiaping; Fu, Nanyan; Wang, Shaoyun

2017-03-29

Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

Calcium Activates Nedd4 E3 Ubiquitin Ligases by Releasing the C2 Domain-mediated Auto-inhibition*

PubMed Central

Wang, Jian; Peng, Qisheng; Lin, Qiong; Childress, Chandra; Carey, David; Yang, Wannian

2010-01-01

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-β. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination. PMID:20172859

Selected Phytochemicals and Culinary Plant Extracts Inhibit Fructose Uptake in Caco-2 Cells.

PubMed

Lee, Yurim; Lim, Yeni; Kwon, Oran

2015-09-18

This study compared the ability of nine culinary plant extracts containing a wide array of phytochemicals to inhibit fructose uptake and then explored the involvement of intestinal fructose transporters and phytochemicals for selected samples. The chemical signature was characterized by high performance liquid chromatography with mass spectrometry. Inhibition of [(14)C]-fructose uptake was tested by using human intestinal Caco-2 cells. Then, the relative contribution of the two apical-facing intestinal fructose transporters, GLUT2 and GLUT5, and the signature components for fructose uptake inhibition was confirmed in naive, phloretin-treated and forskolin-treated Caco-2 cells. HPLC/MS analysis of the chemical signature revealed that guava leaf contained quercetin and catechin, and turmeric contained curcumin, bisdemethoxycurcumin and dimethoxycurcumin. Similar inhibition of fructose uptake (by ~50%) was observed with guava leaf and turmeric in Caco-2 cells, but with a higher contribution of GLUT2 for turmeric and that of GLUT5 for guava leaf. The data suggested that, in turmeric, demethoxycurcumin specifically contributed to GLUT2-mediated fructose uptake inhibition, and curcumin did the same to GLUT5-mediated fructose uptake inhibition, but GLUT2 inhibition was more potent. By contrast, in guava leaf, catechin specifically contributed to GLUT5-mediated fructose uptake inhibition, and quercetin affected both GLUT5- and GLUT2-mediated fructose uptake inhibition, resulting in the higher contribution of GLUT5. These results suggest that demethoxycurcumin is an important contributor to GLUT2-mediated fructose uptake inhibition for turmeric extract, and catechin is the same to GLUT5-mediated fructose uptake inhibition for guava leaf extract. Quercetin, curcumin and bisdemethoxycurcumin contributed to both GLUT5- and GLUT2-mediated fructose uptake inhibition, but the contribution to GLUT5 inhibition was higher than the contribution to GLUT2 inhibition.

PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Xuhui; Department of Laboratory Medicine, Tongji Hospital and Tongji Medical College of Huazhong University of Science and Technology, Wuhan; Yao Honghong

2009-10-15

The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, therebymore » underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.« less

AID targeting: old mysteries and new challenges.

PubMed

Chandra, Vivek; Bortnick, Alexandra; Murre, Cornelis

2015-09-01

Activation-induced cytidine deaminase (AID) mediates cytosine deamination and underlies two central processes in antibody diversification: somatic hypermutation and class-switch recombination. AID deamination is not exclusive to immunoglobulin loci; it can instigate DNA lesions in non-immunoglobulin genes and thus stringent checks are in place to constrain and restrict its activity. Recent findings have provided new insights into the mechanisms that target AID activity to specific genomic regions, revealing an involvement for noncoding RNAs associated with polymerase pausing and with enhancer transcription as well as genomic architecture. We review these findings and integrate them into a model for multilevel regulation of AID expression and targeting in immunoglobulin and non-immunoglobulin loci. Within this framework we discuss gaps in understanding, and outline important areas of further research. Copyright © 2015 Elsevier Ltd. All rights reserved.

AID Targeting: Old Mysteries and New Challenges

PubMed Central

Chandra, Vivek; Bortnick, Alexandra; Murre, Cornelis

2015-01-01

Activation-induced cytidine deaminase (AID) mediates cytosine deamination and underlies two central processes in antibody diversification: somatic hypermutation and class-switch recombination. AID deamination is not exclusive to immunoglobulin loci; it can instigate DNA lesions in non-immunoglobulin genes and thus, stringent checks are in place to constrain and restrict its activity. Recent findings have provided new insights into the mechanisms that target AID activity to specific genomic regions, revealing an involvement for non-coding RNAs associated with polymerase pausing and with enhancer transcription as well as genomic architecture. We review these findings and integrate them into a model for multi-level regulation of AID expression and targeting in immunoglobulin and non-immunoglobulin loci. Within this framework we discuss gaps in understanding, and outline important areas of further research. PMID:26254147

Functional identification of pathogenic autoantibody responses in patients with multiple sclerosis

PubMed Central

Elliott, Christina; Lindner, Maren; Arthur, Ariel; Brennan, Kathryn; Jarius, Sven; Hussey, John; Chan, Andrew; Stroet, Anke; Olsson, Tomas; Willison, Hugh; Barnett, Susan C.; Meinl, Edgar

2012-01-01

Pathological and clinical studies implicate antibody-dependent mechanisms in the immunopathogenesis of multiple sclerosis. We tested this hypothesis directly by investigating the ability of patient-derived immunoglobulins to mediate demyelination and axonal injury in vitro. Using a myelinating culture system, we developed a sensitive and reproducible bioassay to detect and quantify these effects and applied this to investigate the pathogenic potential of immunoglobulin G preparations obtained from patients with multiple sclerosis (n = 37), other neurological diseases (n = 10) and healthy control donors (n = 13). This identified complement-dependent demyelinating immunoglobulin G responses in approximately 30% of patients with multiple sclerosis, which in two cases was accompanied by significant complement-dependent antibody mediated axonal loss. No pathogenic immunoglobulin G responses were detected in patients with other neurological disease or healthy controls, indicating that the presence of these demyelinating/axopathic autoantibodies is specific for a subset of patients with multiple sclerosis. Immunofluorescence microscopy revealed immunoglobulin G preparations with demyelinating activity contained antibodies that specifically decorated the surface of myelinating oligodendrocytes and their contiguous myelin sheaths. No other binding was observed indicating that the response is restricted to autoantigens expressed by terminally differentiated myelinating oligodendrocytes. In conclusion, our study identifies axopathic and/or demyelinating autoantibody responses in a subset of patients with multiple sclerosis. This observation underlines the mechanistic heterogeneity of multiple sclerosis and provides a rational explanation why some patients benefit from antibody depleting treatments. PMID:22561643

Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles.

PubMed

McKenzie, Zofi; Kendall, Michaela; Mackay, Rose-Marie; Whitwell, Harry; Elgy, Christine; Ding, Ping; Mahajan, Sumeet; Morgan, Cliff; Griffiths, Mark; Clark, Howard; Madsen, Jens

2015-01-01

The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A(-/-) mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.

Mitochondriotropic and Cardioprotective Effects of Triphenylphosphonium-Conjugated Derivatives of the Diterpenoid Isosteviol

PubMed Central

Strobykina, Irina; Semenov, Victor V.; Semenova, Marina; Martelli, Alma; Citi, Valentina; Breschi, Maria C.; Kataev, Vladimir E.; Calderone, Vincenzo

2017-01-01

Mitochondria play a crucial role in the cell fate; in particular, reducing the accumulation of calcium in the mitochondrial matrix offers cardioprotection. This affect is achieved by a mild depolarization of the mitochondrial membrane potential, which prevents the assembly and opening of the mitochondrial permeability transition pore. For this reason, mitochondria are an attractive target for pharmacological interventions that prevent ischaemia/reperfusion injury. Isosteviol is a diterpenoid created from the acid hydrolysis of Stevia rebaudiana Bertoni (fam. Asteraceae) glycosides that has shown protective effects against ischaemia/reperfusion injury, which are likely mediated through the activation of mitochondrial adenosine tri-phosphate (ATP)-sensitive potassium (mitoKATP) channels. Some triphenylphosphonium (triPP)-conjugated derivatives of isosteviol have been developed, and to evaluate the possible pharmacological benefits that result from these synthetic modifications, in this study, the mitochondriotropic properties of isosteviol and several triPP-conjugates were investigated in rat cardiac mitochondria and in the rat heart cell line H9c2. This study’s main findings highlight the ability of isosteviol to depolarize the mitochondrial membrane potential and reduce calcium uptake by the mitochondria, which are typical functions of mitochondrial potassium channel openings. Moreover, triPP-conjugated derivatives showed a similar behavior to isosteviol but at lower concentrations, indicative of their improved uptake into the mitochondrial matrix. Finally, the cardioprotective property of a selected triPP-conjugated derivative was demonstrated in an in vivo model of acute myocardial infarct. PMID:28954424

Calcium/Calmodulin-Mediated Gravitropic Response in Plants

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.

2002-01-01

The goal of this project was to gain a fundamental understanding of how calcium/calmodulin-mediated signaling is involved in gravity signal transduction in plants. During the period of support, significant progress was made in elucidating the role of calmodulin and its target proteins in gravitropism. This laboratory has made breakthroughs by cloning and characterizing genes that are involved in calcium/calmodulin-mediated signaling. Some of these genes show altered expression under hypergravity and simulated microgravity conditions. A major advance was made in our attempts to understand gravity signal transduction by cloning and characterizing a catalase which requires calcium/calmodulin for its activation. Our results suggest that calcium/calmodulin have dual roles in regulating the level of hydrogen peroxide (H202), a signal molecule that plays a major role in gravitropism. It is well established that auxin plays a major role in gravitropism. Our results indicate that there is a 'cross-talk' between calcium/calmodulin-mediated signaling and auxin-mediated signal transduction. Auxin-regulated SAUR proteins that are involved in gravitropism bind to calmodulin in a calcium-dependent manner. A novel chimeric calcium/calmodulin-dependent protein kinase was cloned and characterized and its role in gravity signal transduction was investigated. These studies have provided some answers to the fundamental questions about how signal molecules such as calcium, H202, and hormones such as auxin bring about the ultimate gravitropic response and the integral role of calmodulin in gravity signal transduction. This NASA-funded study has led to some spinoffs that have applications in solving agricultural problems. The Washington State University Research Foundation has obtained several patents related to this work.

xCT expression reduces the early cell cycle requirement for calcium signaling

PubMed Central

Lastro, Michele; Kourtidis, Antonis; Farley, Kate; Conklin, Douglas S.

2009-01-01

Calcium has long been recognized as an important regulator of cell cycle transitions although the mechanisms are largely unknown. A functional genomic screen has identified genes involved in the regulation of early cell cycle progression by calcium. These genes when overexpressed confer the ability to bypass the G1/S arrest induced by Ca2+- channel antagonists in mouse fibroblasts. Overexpression of the cystine-glutamate exchanger, xCT, had the greatest ability to evade calcium antagonist-induced cell cycle arrest. xCT carries out the rate limiting step of glutathione synthesis in many cell types and is responsible for the uptake of cystine in most human cancer cell lines. Functional analysis indicates that the cystine uptake activity of xCT overcomes the G1/S arrest induced by Ca2+- channel antagonists by bypassing the requirement for calcium signaling. Since cells overexpressing xCT were found to have increased levels and activity of the AP-1 transcription factor in G1, redox stimulation of AP-1 activity accounts for the observed growth of these cells in the presence of calcium channel antagonists. These results suggest that reduced calcium signaling impairs AP-1 activation and that xCT expression may directly affect cell proliferation. PMID:18054200

Effect of dephytinization on bioavailability of iron, calcium and zinc from infant cereals assessed in the Caco-2 cell model

PubMed Central

Frontela, Carmen; Scarino, Maria Laura; Ferruzza, Simonetta; Ros, Gaspar; Martínez, Carmen

2009-01-01

AIM: To test the effect of the dephytinization of three different commercial infant cereals on iron, calcium, and zinc bioavailability by estimating the uptake, retention, and transport by Caco-2 cells. METHODS: Both dephytinized (by adding an exogenous phytase) and non-dephytinized infant cereals were digested using an in vitro digestion protocol adapted to the gastrointestinal conditions of infants younger than 6 mo. Mineral cell retention, transport, and uptake from infant cereals were measured using the soluble fraction of the simulated digestion and the Caco-2 cells. RESULTS: Dephytinization of infant cereals significantly increased (P < 0.05) the cell uptake efficiency (from 0.66%-6.05% to 3.93%-13%), retention (from 6.04%-16.68% to 14.75%-20.14%) and transport efficiency (from 0.14%-2.21% to 1.47%-6.02%), of iron, and the uptake efficiency (from 5.0%-35.4% to 7.3%-41.6%) and retention (from 4.05%-20.53% to 14.45%-61.3%) of zinc, whereas calcium only cell uptake showed a significant increase (P < 0.05) after removing phytate from most of the samples analyzed. A positive relationship (P < 0.05) between mineral solubility and the cell uptake and transport efficiencies was observed. CONCLUSION: Removing phytate from infant cereals had a beneficial effect on iron and zinc bioavailability when infant cereals were reconstituted with water. Since in developing countries cereal-based complementary foods for infants are usually consumed mixed with water, exogenous phytase additions could improve the nutritional value of this weaning food. PMID:19399930

CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells.

PubMed

Schofield, L; McConville, M J; Hansen, D; Campbell, A S; Fraser-Reid, B; Grusby, M J; Tachado, S D

1999-01-08

Immunoglobulin G (IgG) responses require major histocompatibility complex (MHC)-restricted recognition of peptide fragments by conventional CD4(+) helper T cells. Immunoglobulin G responses to glycosylphosphatidylinositol (GPI)- anchored protein antigens, however, were found to be regulated in part through CD1d-restricted recognition of the GPI moiety by thymus-dependent, interleukin-4-producing CD4(+), natural killer cell antigen 1.1 [(NK1.1)+] helper T cells. The CD1-NKT cell pathway regulated immunogobulin G responses to the GPI-anchored surface antigens of Plasmodium and Trypanosoma and may be a general mechanism for rapid, MHC-unrestricted antibody responses to diverse pathogens.

Calcium/calmodulin-mediated signal network in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2003-01-01

Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. Calmodulin, the predominant calcium receptor, is one of the best-characterized calcium sensors in eukaryotes. In recent years, completion of the Arabidopsis genome project and advances in functional genomics have helped to identify and characterize numerous calmodulin-binding proteins in plants. There are some similarities in Ca(2+)/calmodulin-mediated signaling in plants and animals. However, plants possess multiple calmodulin genes and many calmodulin target proteins, including unique protein kinases and transcription factors. Some of these proteins are likely to act as "hubs" during calcium signal transduction. Hence, a better understanding of the function of these calmodulin target proteins should help in deciphering the Ca(2+)/calmodulin-mediated signal network and its role in plant growth, development and response to environmental stimuli.

Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the aryl hydrocarbon receptor (AHR)-mediated impairment of immunoglobulin secretion in human primary B cells.

PubMed

Zhou, Jiajun; Zhang, Qiang; Henriquez, Joseph E; Crawford, Robert B; Kaminski, Norbert E

2018-05-31

The aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing, cell cycle regulation and cell development. In humans, the activation of AHR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AHR-ligand, impairs the secretion of immunoglobulin M (IgM) to suppress humoral immunity. However, the mechanisms bridging the activation of AHR and the impairment of IgM secretion by human primary B cells remain poorly understood. Recent transcriptomic analysis revealed upregulation of lymphocyte-specific protein tyrosine kinase (LCK) in AHR activated human primary B cells. LCK is a well-characterized tyrosine kinase that phosphorylates critical signaling proteins involved in activation and cytokine production in T cells. Conversely, the role of LCK in human primary B cells is not well understood. In the current studies, we have verified the transcriptomic finding by detecting AHR-mediated upregulation of LCK protein in human primary B cells. We also confirmed the role of AHR in the upregulation of LCK by using a specific AHR antagonist, which abolished the AHR-mediated increase of LCK. Furthermore, we have confirmed the role of LCK in the AHR-mediated suppression of IgM by using LCK specific inhibitors, which restored IgM secretion by human B cells in the presence of TCDD. Collectively, the current studies demonstrate a novel role of LCK in IgM secretion and provide new insights into the mechanism for AHR-mediated impairment of immunoglobulin secretion by human primary B cells.

Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium

PubMed Central

1989-01-01

The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol- induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o- aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the elevation of [Ca]i and alterations in Ki, Nai, and cell volume spontaneously returned to near baseline levels. In addition to quantitating the activation of various ion flux pathways in the rat parotid acinar cell, these results demonstrate that the activation of ion transport systems responsible for fluid secretion in this tissue is closely linked to the elevation of [Ca]i. PMID:2467962

The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

PubMed Central

Liao, Yajin; Dong, Yuan; Cheng, Jinbo

2017-01-01

The mitochondrial calcium uniporter (MCU)—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP); however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders. PMID:28208618

Physiological characterisation of a pH- and calcium-dependent sodium uptake mechanism in the freshwater crustacean, Daphnia magna.

PubMed

Glover, Chris N; Wood, Chris M

2005-03-01

Daphnia are highly sensitive to sodium metabolism disruption caused by aquatic acidification and ionoregulatory toxicants, due to their finely balanced ion homeostasis. Nine different water chemistries of varying pH (4, 6 and 8) and calcium concentration (0, 0.5 and 1 mmol l(-1)) were used to delineate the mechanism of sodium influx in Daphnia magna. Lowering water pH severely inhibited sodium influx when calcium concentration was high, but transport kinetic analysis revealed a stimulated sodium influx capacity (J(max)) when calcium was absent. At low pH increasing water calcium levels decreased J(max) and raised K(m) (decreased sodium influx affinity), while at high pH the opposite pattern was observed (elevated J(max) and reduced K(m)). These effects on sodium influx were mirrored by changes in whole body sodium levels. Further examination of the effect of calcium on sodium influx showed a severe inhibition of sodium uptake by 100 micromol l(-1) calcium gluconate at both low (50 micromol l(-1)) and high (1000 micromol l(-1)) sodium concentrations. At high sodium concentrations, stimulated sodium influx was noted with elevated calcium levels. These results, in addition to data showing amiloride inhibition of sodium influx (K(i)=180 micromol l(-1)), suggest a mechanism of sodium influx in Daphnia magna that involves the electrogenic 2Na(+)/1H(+) exchanger.

Effects of extracellular calcium on calcium transport during hyperthermia of tumor cells.

PubMed

Anghileri, L J; Marcha, C; Crone-Escanyé, M C; Robert, J

1985-08-01

The effects of different concentrations of extracellular ion calcium on the transport of calcium by tumor cells have been studied by means of the uptake of radiocalcium. Tumor cells incubated at 45 degrees C take up 4-10 times the amount of radioactivity incorporated by cells incubated at 37 degrees C. The difference is still greater (up to 100 times) for the intracellular incorporation as assessed by elimination of the membrane-bound calcium by EGTA treatment. The possible mechanisms involved in this differential behavior are discussed.

Low-intensity pulsed ultrasound (LIPUS) stimulates mineralization of MC3T3-E1 cells through calcium and phosphate uptake.

PubMed

Tassinary, João Alberto Fioravante; Lunardelli, Adroaldo; Basso, Bruno de Souza; Dias, Henrique Bregolin; Catarina, Anderson Velasque; Stülp, Simone; Haute, Gabriela Viegas; Martha, Bianca Andrade; Melo, Denizar Alberto da Silva; Nunes, Fernanda Bordignon; Donadio, Márcio Vinícius Fagundes; Oliveira, Jarbas Rodrigues de

2018-03-01

The present study aimed to evaluate the effect of low-intensity pulsed ultrasound (LIPUS) on pre-osteoblast mineralization using in vitro bioassays. Pre-osteoblastic MC3T3-E1 cells were exposed to LIPUS at 1 MHz frequency, 0.2 W/cm 2 intensity and 20% duty cycle for 30 min. The analyses were carried out up to 336 h (14 days) after exposure. The concentration of collagen, phosphate, alkaline phosphatase, calcium and transforming growth factor beta 1 (TGF-β1) in cell supernatant and the presence of calcium deposits in the cells were analyzed. Our results showed that LIPUS promotes mineralized nodules formation. Collagen, phosphate, and calcium levels were decreased in cell supernatant at 192 h after LIPUS exposure. However, alkaline phosphatase and TGF-β1 concentrations remained unchanged. Therapeutic pulsed ultrasound is capable of stimulating differentiation and mineralization of pre-osteoblastic MC3T3-E1 cells by calcium and phosphate uptake with consequent hydroxyapatite formation. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

PubMed

Fechner, H; Suckau, L; Kurreck, J; Sipo, I; Wang, X; Pinkert, S; Loschen, S; Rekittke, J; Weger, S; Dekkers, D; Vetter, R; Erdmann, V A; Schultheiss, H-P; Paul, M; Lamers, J; Poller, W

2007-02-01

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes

PubMed Central

Li, Dongdong; Hérault, Karine; Oheim, Martin; Ropert, Nicole

2009-01-01

The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca2+) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca2+ homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca2+ entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca2+ for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca2+ from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence. PMID:20007370

P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

PubMed Central

Uchitel, O D; Protti, D A; Sanchez, V; Cherksey, B D; Sugimori, M; Llinás, R

1992-01-01

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel. Images PMID:1348859

P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

PubMed

Uchitel, O D; Protti, D A; Sanchez, V; Cherksey, B D; Sugimori, M; Llinás, R

1992-04-15

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.

Cobalt-55 positron emission tomography in traumatic brain injury: a pilot study.

PubMed Central

Jansen, H M; van der Naalt, J; van Zomeren, A H; Paans, A M; Veenma-van der Duin, L; Hew, J M; Pruim, J; Minderhoud, J M; Korf, J

1996-01-01

Traumatic brain injury is usually assessed with the Glasgow coma scale (GCS), CT, or MRI. After such injury, the injured brain tissue is characterised by calcium mediated neuronal damage and inflammation. Positron emission tomography with the isotope cobalt-55 (Co-PET) as a calcium tracer enables imaging of affected tissue in traumatic brain injury. The aim was to determine whether additional information can be gained by Co-PET in the diagnosis of moderate traumatic brain injury and to assess any prognostic value of Co-PET. Five patients with recent moderately severe traumatic brain injury were studied. CT was performed on the day of admission, EEG within one week, and MRI and Co-PET within four weeks of injury. Clinical assessment included neurological examination, GCS, neuropsychological testing, and Glasgow outcome scale (GOS) after one year. Co-PET showed focal uptake that extended beyond the morphological abnormalities shown by MRI and CT, in brain regions that were actually diagnosed with EEG. Thus Co-PET is potentially useful for diagnostic localisation of both structural and functional abnormalities in moderate traumatic brain injury. Images PMID:8708661

BSA nanoparticle loaded atorvastatin calcium--a new facet for an old drug.

PubMed

Sripriyalakshmi, S; Anjali, C H; George, Priya Doss C; Rajith, B; Ravindran, Aswathy

2014-01-01

Currently, the discovery of effective chemotherapeutic agents poses a major challenge to the field of cancer biology. The present study focuses on enhancing the therapeutic and anti cancer properties of atorvastatin calcium loaded BSA (ATV-BSA) nanoparticles in vitro. BSA-ATV nanoparticles were prepared using desolvation technique. The process parameters were optimized based on the amount of desolvating agent, stabilization conditions as well as the concentration of the cross linker. The anti cancer properties of the protein coated ATV nanoparticles were tested on MiaPaCa-2 cell lines. In vitro release behavior of the drug from the carrier suggests that about 85% of the drug gets released after 72 hrs. Our studies show that ATV-BSA nanoparticles showed specific targeting and enhanced cytotoxicity to MiaPaCa-2 cells when compared to the bare ATV. We hereby propose that the possible mechanism of cellular uptake of albumin bound ATV could be through caveolin mediated endocytosis. Hence our studies open up new facet for an existing cholesterol drug as a potent anti-cancer agent.

Endocytic mechanisms and osteoinductive profile of hydroxyapatite nanoparticles in human umbilical cord Wharton’s jelly-derived mesenchymal stem cells

PubMed Central

Zhang, Juan; Wang, Chen

2018-01-01

Background As a potentially bioactive material, the widespread application of nanosized hydroxyapatite (nano-HAP) in the field of bone regeneration has increased the risk of human exposure. However, our understanding of the interaction between nano-HAP and stem cells implicated in bone repair remains incomplete. Methods Here, we characterized the adhesion and cellular internalization of HAP nanoparticles (HANPs) with different sizes (20 nm np20 and 80 nm np80) and highlighted the involved pathway in their uptake using human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs). In addition, the effects of HANPs on cell viability, apoptosis response, osteogenic differentiation, and underlying related mechanisms were explored. Results It was shown that both types of HANPs readily adhered to the cellular membrane and were transported into the cells compared to micro-sized HAP particles (m-HAP; 12 μm). Interestingly, the endocytic routes of np20 and np80 differed, although they exhibited similar kinetics of adhesion and uptake. Our study revealed involvement of clathrin- and caveolin-mediated endocytosis as well as macropinocytosis in the np20 uptake. However, for np80, clathrin-mediated endocytosis and some as-yet-unidentified important uptake routes play central roles in their internalization. HANPs displayed a higher preference to accumulate in the cytoplasm compared to m-HAP, and HANPs were not detected in the nucleolus. Exposure to np20 for 24 h caused a decrease in cell viability, while cells completely recovered with an exposure time of 72 h. Furthermore, HANPs did not influence apoptosis and necrosis of hWJ-MSCs. Strikingly, HANPs enhanced mRNA levels of osteoblast-related genes and stimulated calcium mineral deposition, and this directly correlated with the activation in c-Jun N-terminal kinases and p38 pathways. Conclusion Our data provide additional insight about the interactions of HANPs with MSCs and suggest their application potential in hard tissue regeneration. PMID:29559775

Childhood-Onset Multifocal Motor Neuropathy With Immunoglobulin M Antibodies to Gangliosides GM1 and GM2: A Case Report and Review of the Literature.

PubMed

Ishigaki, Hidetoshi; Hiraide, Takuya; Miyagi, Yoshifumi; Hayashi, Taiju; Matsubayashi, Tomoko; Shimoda, Ayumi; Kusunoki, Susumu; Fukuda, Tokiko

2016-09-01

Multifocal motor neuropathy is a rare immune-mediated neuropathy characterized by progressive asymmetric weakness and atrophy without sensory abnormalities. Although disease onset is usually in adulthood, a few childhood-onset cases have been reported. Here, we report the case of an 8-year-old boy with multifocal motor neuropathy who presented with a slowly progressive left and distal upper limb weakness without sensory loss. The initial high-dose intravenous immunoglobulin treatment significantly improved left upper limb muscle weakness. Continued monthly intravenous immunoglobulin treatment gradually improved muscle strength for several months initially. While the muscle strength decreased slightly after 8 months of therapy, it was better than that before intravenous immunoglobulin treatment. One year and eight months after the initiation of treatment, serum testing for IgM antibodies to gangliosides, GM1 and GM2, was negative. This is the first pediatric report of the serum IgM autoantibodies positive to GM1 and GM2. The clinical course is similar to that of partial intravenous immunoglobulin responders among patients with adulthood-onset multifocal motor neuropathy. Since the symptoms plateaued after the initial intravenous immunoglobulin therapy, prognosis appears to be determined by the patient's initial response to intravenous immunoglobulin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Cadmium biodynamics in the oligochaete Lumbriculus variegatus and its implications for trophic transfer

USGS Publications Warehouse

Xie, Lingtian; Lambert, D.; Martin, C.; Cain, D.J.; Luoma, S.N.; Buchwalter, D.

2008-01-01

It has become increasingly apparent that diet can be a major source of trace metal bioaccumulation in aquatic organisms. In this study, we examined cadmium uptake, efflux, and subcellular compartmentalization dynamics in the freshwater oligochaete Lumbriculus variegatus. L. variegatus is an important component of freshwater food webs in Europe and North America and is potentially useful as a standard food source for laboratory-based trophic transfer studies. Cadmium accumulation and depuration were each followed for 10 days. Rate constants of uptake (ku) and efflux (ke) were estimated and subcellular Cd compartmentalization was followed over the course of uptake and efflux. The partitioning of Cd into operationally-defined subcellular compartments was relatively consistent throughout the 20-day experiment, with the majority of Cd accumulating in the cytosol. No major changes in Cd compartmentalization were observed over uptake or depuration, but there appeared to be some exchange between heat-stable and heat-labile cytosolic protein fractions. Cadmium accumulation from solution was strongly affected by ambient calcium concentrations, suggesting competition between Cd and Ca for uptake sites. Finally, we demonstrate the ability to manipulate the whole body calcium content of L. variegatus as a potential tool for examining calcium influences on dietary Cd dynamics. The potential for this species to be an important conduit of Cd to higher trophic levels is discussed, along with its potential as a standardized food source in metal trophic transfer studies. ?? 2007 Elsevier B.V. All rights reserved.

Oestrogen receptor specificity in oestradiol-mediated effects on B lymphopoiesis and immunoglobulin production in male mice

PubMed Central

Erlandsson, M C; Jonsson, C A; Islander, U; Ohlsson, C; Carlsten, H

2003-01-01

Oestrogen treatment down-regulates B lymphopoiesis in the bone marrow of mice. Meanwhile it up-regulates immunoglobulin production. To understand better the oestrogen action on bone marrow male mice lacking oestrogen receptor α (ERα; ERKO mice), lacking ERβ (BERKO mice), lacking both receptors (DERKO mice) or wild-type (wt) littermates were castrated and treated for 2·5 weeks with 30 μg/kg 17β-oestradiol (E2) or vehicle oil as controls. The B lymphopoiesis in the bone marrow was examined by flow cytometry and mature B-cell function was studied using an ELISPOT assay enumerating the B cells in bone marrow and spleen that were actively producing immunoglobulins. In wt mice the frequency of B-lymphopoietic (B220+) cells in the bone marrow decreased from 15% to 5% upon E2 treatment. In ERKO and BERKO mice significant reduction was seen but not of the same magnitude. In DERKO mice no reduction of B lymphopoiesis was seen. In addition, our results show that E2 mediated reduction of different steps in B lymphopoiesis require only ERα or both receptors. In wt and BERKO mice E2 treatment resulted in significantly increased levels of B cells actively producing immunoglobulin, while in ERKO and DERKO mice no such change was seen. Similar results were found in both bone marrow and spleen. In conclusion our results clearly show that both ERα and ERβ are required for complete down-regulation of B lymphopoiesis while only ERα is needed to up-regulate immunoglobulin production in both bone marrow and spleen. PMID:12603601

Chrysin suppresses mast cell-mediated allergic inflammation: Involvement of calcium, caspase-1 and nuclear factor-{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Yunju; Lee, Soyoung; Kim, Sang-Hyun, E-mail: shkim72@knu.ac.kr

A great number of people are suffering from allergic inflammatory diseases such as asthma, atopic dermatitis, and sinusitis. Therefore discovery of drugs for the treatment of these diseases is an important subject in human health. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid contained in propolis, blue passion flower, and fruits. Several studies reported that chrysin has beneficial effects including anti-tumor and anti-oxidant activities. The aim of the present study was to elucidate whether chrysin modulates the allergic inflammatory reaction and to study its possible mechanisms of action using mast cell-based in vitro and in vivo models. Chrysin inhibited immediate-type systemic hypersensitivitymore » and serum histamine release. Chrysin attenuated immunoglobulin E-mediated local anaphylaxis. These inhibitory effects of chrysin on the systemic and local allergic reaction were more potent than cromolyn, a known anti-allergic drug. Chrysin reduced histamine release from mast cells. The inhibitory effect of chrysin on the histamine release was mediated by the modulation of intracellular calcium. In addition, chrysin decreased gene expression of pro-inflammatory cytokines such as, tumor necrosis factor-{alpha}, IL (interleukin)-1{beta}, IL-4, and IL-6 in mast cells. The inhibitory effect of chrysin on the pro-inflammatory cytokine was nuclear factor-{kappa}B and caspase-1 dependent. Our findings provide evidence that chrysin inhibits mast cell-derived allergic inflammatory reactions by blocking histamine release and pro-inflammatory cytokine expression, and suggest the mechanisms of action. Furthermore, in vivo and in vitro anti-allergic inflammatory effect of chrysin suggests a possible therapeutic application of this agent in allergic inflammatory diseases. - Research Highlights: > Discovery of drugs for the allergic inflammation is important in human health. > Chrysin is a natural flavonoid contained in propolis, blue passion flower, and fruits. > Chrysin inhibited systemic and local hypersensitivity, and serum histamine release. > Chrysin decreased inflammatory cytokines through the inhibition of NF-{kappa}B and caspase-1. > Chrysin might be a candidate for the treatment of allergic inflammatory diseases.« less

Porphyrin-laser photodynamic induction of focal brain necrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stroop, W.G.; Battles, E.J.; Townsend, J.J.

A noninvasive photodynamic method has been developed to produce focal brain necrosis using porphyrin activated in vivo with laser light. After peripheral injection of the photosensitive porphyrin derivative, Photofrin I, mice were irradiated on the posterior lateral aspect of the head through the intact depilated scalp with 632 nm argon-dye laser light. Animals were studied at one, two and seven days after irradiation. Blood-brain barrier damage was detected by the intravenous injection of Evans blue, horseradish peroxidase and heterologous immunoglobulins. At one and two days after irradiation, the lesions were characterized by extravasation of immunoglobulin and Evans blue, and bymore » edema, ischemia and infiltration by monocytes. On the seventh day after irradiation, the lesion was smaller than it had been two days after irradiation, and had reactive changes at its edges and coagulative necrosis at its center. Extravasation of Evans blue and immunoglobulin was markedly reduced by the seventh day after irradiation, but uptake of horseradish peroxidase by macrophages located at the periphery of the lesion was evident.« less

Diselenolane-mediated cellular uptake.

PubMed

Chuard, Nicolas; Poblador-Bahamonde, Amalia I; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi; Matile, Stefan

2018-02-21

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

ACUTE EFFECT OF ETHANOL ON HEPATIC RETICULAR G6Pase AND Ca2+ POOL

PubMed Central

Jacobs-Harper, Amy; Crumbly, Ashlee; Romani, Andrea

2012-01-01

Background Hydrolysis of glucose 6-phosphate via glucose 6-phosphatase enlarges the reticular Ca2+ pool of the hepatocyte. Exposure of liver cells to ethanol impairs reticular Ca2+ homeostasis. The present study investigated the effect of acute ethanol administration on glucose 6-phosphate supported Ca2+ accumulation in liver cells. Methods Total microsomes were isolated from rat livers acutely perfused with varying doses of ethanol (0.01%, 0.1%, or 1% v/v) for 8 minutes. Calcium uptake was assessed by 45Ca redistribution. Inorganic phosphate (Pi) formation was measured as an indicator of glucose 6-phosphatase hydrolytic activity. Results Glucose 6-phosphate-supported Ca2+ uptake decreased in a manner directly proportional to the dose of ethanol infused in the liver whereas Ca2+ uptake via SERCA pumps was decreased by ~25% only at the highest dose of alcohol administered. The reduced accumulation of Ca2+ within the microsomes resulted in a smaller IP3-induced Ca2+ release. Kinetic assessment of IP3 and passive Ca2+ release indicated a faster mobilization in microsomes from ethanol-treated livers, suggesting alcohol-induced alteration of Ca2+ releasing mechanisms. Pre-treatment of livers with chloromethiazole or dithio-threitol, but not 4-methyl-pyrazole prevented the inhibitory effect of ethanol on glucose 6-phosphatase activity and Ca2+ homeostasis. Conclusions Liver glucose 6-phosphatase activity and IP3-mediated Ca2+ release are rapidly inhibited following acute (8 min) exposure to ethanol, thus compromising the ability of the endoplasmic reticulum to dynamically modulate Ca2+ homeostasis in the hepatocyte. The protective effect of chloromethiazole and di-thio-threitol suggests that the inhibitory effect of ethanol is mediated through its metabolism via reticular cyP4502E1 and consequent free radicals formation. PMID:22958133

A Rare Case of Cerebellar Ataxia Due to Voltage-Gated Calcium Channel and Glutamic Acid Decarboxylase Autoantibodies.

PubMed

Annunziata, Giuseppe; Lobo, Pamela; Carbuccia, Cristian

2017-11-27

BACKGROUND Autoimmune cerebellar ataxia can be paraneoplastic in nature or can occasionally present without evidence of an ongoing malignancy. The detection of specific autoantibodies has been statistically linked to different etiologies. CASE REPORT A 55-year-old African-American woman with hypertension and a past history of morbid obesity and uncontrolled diabetes status post gastric bypass four years prior to the visit (with significantly improved body mass index and hemoglobin A1c controlled at the time of the clinical encounter) presented to the office complaining of gradual onset of unsteadiness and recurrent falls for the past three years, as well as difficulties coordinating routine daily activities. The neurologic exam showed moderate dysarthria and ataxic gait with bilateral dysmetria and positive Romberg test. Routine laboratory test results were only remarkable for a mild elevation of erythrocyte sedimentation rate, and most laboratory and imaging tests for common causes of ataxia failed to demonstrate an etiology. Upon further workup, evidence of anti-voltage-gated calcium channel and anti-glutamic acid decarboxylase antibody was demonstrated. She was then treated with intravenous immunoglobulins with remarkable clinical improvement. CONCLUSIONS We present a case of antibody-mediated ataxia not associated with malignancy. While ataxia is rarely related to autoantibodies, in such cases it is critical to understand the etiology of this disabling condition in order to treat it correctly. Clinicians should be aware of the possible association with specific autoantibodies and the necessity to rule out an occult malignancy in such cases.

Osteogenic differentiation capacity of human mesenchymal stromal cells in response to extracellular calcium with special regard to connexin 43.

PubMed

Wagner, Alena-Svenja; Glenske, Kristina; Wolf, Verena; Fietz, Daniela; Mazurek, Sybille; Hanke, Thomas; Moritz, Andreas; Arnhold, Stefan; Wenisch, Sabine

2017-01-01

The effects of extracellular calcium on osteogenic differentiation capacity of human bone-derived mesenchymal stromal cells with special regard to connexin 43 (cx43) have been investigated by means of cell culture experiments. Mesenchymal stromal cells isolated from human cancellous bone were cultured on tissue culture plates at different calcium ion (Ca 2+ ) concentrations (1.8mmoll -1 , 10mmoll -1 , 20mmoll -1 ). Cell responses were evaluated by quantitative RT-PCR, immunofluorescence staining, and Lucifer Yellow fluorescence uptake experiments. It could be shown that increasing Ca 2+ concentrations correlate with increasing cx43 and bone sialoprotein mRNA levels as well as with enhanced cx43 fluorescence signaling and matrix mineralization of the cultures as shown by von Kossa staining. Hemichannel gating - assessed by Lucifer Yellow uptake - increases with increasing extracellular Ca 2+ concentrations suggesting that regulatory effects at the hemichannel level are calcium-dependent. Copyright © 2016 Elsevier GmbH. All rights reserved.

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

NASA Technical Reports Server (NTRS)

Tischner, R.; Ward, M. R.; Huffaker, R. C.

1989-01-01

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Hydrocortisone selectively inhibits IgE-dependent arachidonic acid release from rat peritoneal mast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiman, A.S.; Crews, F.T.

Purified rat mst cells were used to study the effects of antiinflammatory steroids on the release of (1-14C)-arachidonic acid ((1-14C)AA) and metabolites. Mast cell were incubated overnight with glucocorticoids, (1-14C)AA incorporated into cellular phospholipids and the release of (1-14C)AA, and metabolites determined using a variety of secretagogues. Release of (1-14C)AA and metabolites by concanavalin A, the antigen ovalbumin and anti-immunoglobulin E antibody was markedly reduced by glucocorticoid treatment. Neither the total incorporation of (1-14C)AA nor the distribution into phospholipids was altered by hydrocortisone pretreatment. Glucocorticoid pretreatment did not alter (1-14C)AA release stimulated by somatostatin, compound 48/80, or the calcium ionophore,more » A23187. These data indicate that antiinflammatory steroids selectively inhibit immunoglobulin dependent release of arachidonic acid from rat mast cells. These findings question the role of lipomodulin and macrocortin as general phospholipase inhibitors and suggest that they may be restricted to immunoglobulin stimuli.« less

Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

PubMed

Di Croce, D; Trinks, P W; Grifo, M B; Takara, D; Sánchez, G A

2015-11-01

The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

Development of a rapid biolistic assay to determine changes in relative levels of intracellular calcium in leaves following tetracycline uptake by pinto bean plants.

PubMed

Farkas, Michael H; Mojica, Elmer-Rico E; Patel, Minesh; Aga, Diana S; Berry, James O

2009-08-01

Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced into agricultural lands through the application of manure as fertilizer. These compounds are phytotoxic to certain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation. While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. We describe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium in leaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellular calcium levels, but current methodology requires stable transformation. Many plant species, including pinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determine the role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently express the cameleon protein. This method can easily be adapted to other plant systems. Our findings provide evidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by this antibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaves were further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry, confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.

Social Cognitive Mediators of Sociodemographic Differences in Colorectal Cancer Screening Uptake

PubMed Central

Lo, Siu Hing; Waller, Jo; Vrinten, Charlotte; Kobayashi, Lindsay; von Wagner, Christian

2015-01-01

Background. This study examined if and how sociodemographic differences in colorectal cancer (CRC) screening uptake can be explained by social cognitive factors. Methods. Face-to-face interviews were conducted with individuals aged 60–70 years (n = 1309) living in England as part of a population-based omnibus survey. Results. There were differences in screening uptake by SES, marital status, ethnicity, and age but not by gender. Perceived barriers (stand. b = −0.40, p < 0.001), social norms (stand. b = 0.33, p < 0.001), and screening knowledge (stand. b = 0.17, p < 0.001) had independent associations with uptake. SES differences in uptake were mediated through knowledge, social norms, and perceived barriers. Ethnic differences were mediated through knowledge. Differences in uptake by marital status were primarily mediated through social norms and to a lesser extent through knowledge. Age differences were largely unmediated, except for a small mediated effect via social norms. Conclusions. Sociodemographic differences in CRC screening uptake were largely mediated through social cognitive factors. Impact. Our findings suggest that multifaceted interventions might be needed to reduce socioeconomic inequalities. Ethnic differences might be reduced through improved screening knowledge. Normative interventions could emphasise screening as an activity endorsed by important others outside the immediate family to appeal to a wider audience. PMID:26504782

Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

PubMed

Nakanishi, Tomonori; Xu, Xiaoyan; Wynn, Carmen; Yamada, Toshiko; Pan, Fan; Erickson, Laurie; Teo, Haeman; Nakagawa, Terry; Masunaga, Taro; Abe, Jumpei; Akamatsu, Masahiko; Tamura, Kouichi; Jiang, Hongsi

2015-08-01

Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores. The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

Calcium-Mediated Signaling during Sandalwood Somatic Embryogenesis. Role for Exogenous Calcium as Second Messenger1

PubMed Central

Anil, Veena S.; Rao, K. Sankara

2000-01-01

The possible involvement of Ca2+-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. 45Ca2+-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,4-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca2+]cyt of PEMs, increasing from a resting concentration of 30 to 50 nm to 650 to 800 nm. Chelation of exogenous Ca2+ with ethyleneglycol-bis(aminoethyl ether)-N,N′-tetraacetic acid arrests such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca2+-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell clumps. Culture treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving sandalwood Ca2+-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca2+ during sandalwood somatic embryogenesis. PMID:10938349

Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger.

PubMed

Anil, V S; Rao, K S

2000-08-01

The possible involvement of Ca(2+)-mediated signaling in the induction/regulation of somatic embryogenesis from pro-embryogenic cells of sandalwood (Santalum album) has been investigated. (45)Ca(2+)-uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca(2+)](cyt) of pro-embryogenic cells in response to culture conditions conducive for embryo development. Sandalwood pro-embryogenic cell masses (PEMs) are obtained in the callus proliferation medium that contains the auxin 2,4-dichlorophenoxyacetic acid. Subculture of PEMs into the embryo differentiation medium, which lacks 2,4-dichlorophenoxyacetic acid and has higher osmoticum, results in a 4-fold higher (45)Ca(2+) incorporation into the symplast. Fura-2 ratiometric analysis corroboratively shows a 10- to 16-fold increase in the [Ca(2+)](cyt) of PEMs, increasing from a resting concentration of 30 to 50 nM to 650 to 800 nM. Chelation of exogenous Ca(2+) with ethyleneglycol-bis(aminoethyl ether)-N,N'-tetraacetic acid arrests such an elevation in [Ca(2+)](cyt). Exogenous Ca(2+) when chelated or deprived also arrests embryo development and inhibits the accumulation of a sandalwood Ca(2+)-dependent protein kinase. However, such culture conditions do not cause cell death as the PEMs continue to proliferate to form larger cell clumps. Culture treatment with N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide reduced embryogenic frequency by 85%, indicating that blockage of Ca(2+)-mediated signaling pathway(s) involving sandalwood Ca(2+)-dependent protein kinase and/or calmodulin causes the inhibition of embryogenesis. The observations presented are evidence to suggest a second messenger role for exogenous Ca(2+) during sandalwood somatic embryogenesis.

Effects of seawater alkalinity on calcium and acid-base regulation in juvenile European lobster (Homarus gammarus) during a moult cycle.

PubMed

Middlemiss, Karen L; Urbina, Mauricio A; Wilson, Rod W

2016-03-01

Fluxes of NH4(+) (acid) and HCO3(-) (base), and whole body calcium content were measured in European lobster (Homarus gammarus) during intermoult (megalopae stage), and during the first 24h for postmoult juveniles under control (~2000 μeq/L) and low seawater alkalinity (~830 μeq/L). Immediately after moulting, animals lost 45% of the total body calcium via the shed exoskeleton (exuvia), and only 11% was retained in the uncalcified body. At 24h postmoult, exoskeleton calcium increased to ~46% of the intermoult stage. Ammonia excretion was not affected by seawater alkalinity. After moulting, bicarbonate excretion was immediately reversed from excretion to uptake (~4-6 fold higher rates than intermoult) over the whole 24h postmoult period, peaking at 3-6h. These data suggest that exoskeleton calcification is not completed by 24h postmoult. Low seawater alkalinity reduced postmoult bicarbonate uptake by 29% on average. Net acid-base flux (equivalent to net base uptake) followed the same pattern as HCO3(-) fluxes, and was 22% lower in low alkalinity seawater over the whole 24h postmoult period. The common occurrence of low alkalinity in intensive aquaculture systems may slow postmoult calcification in juvenile H. gammarus, increasing the risk of mortalities through cannibalism. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic Improvement of Potato for Tuber Calcium Uptake

USDA-ARS?s Scientific Manuscript database

Tuber internal quality is a major limiting factor for the U.S. potato industry. Breeders invest time and money in producing advanced selections which, in the end, often fail because of tuber internal defects, tuber bruising, or storage quality issues. In-season fertilization with calcium is known to...

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

PubMed

Anzinger, Joshua J; Chang, Janet; Xu, Qing; Buono, Chiara; Li, Yifu; Leyva, Francisco J; Park, Bum-Chan; Greene, Lois E; Kruth, Howard S

2010-10-01

To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Saponarin activates AMPK in a calcium-dependent manner and suppresses gluconeogenesis and increases glucose uptake via phosphorylation of CRTC2 and HDAC5.

PubMed

Seo, Woo-Duck; Lee, Ji Hae; Jia, Yaoyao; Wu, Chunyan; Lee, Sung-Joon

2015-11-15

This study investigated the molecular mechanism of saponarin, a flavone glucoside, in the regulation of insulin sensitivity. Saponarin suppressed the rate of gluconeogenesis and increased cellular glucose uptake in HepG2 and TE671 cells by regulating AMPK. Using an in vitro kinase assay, we showed that saponarin did not directly interact with the AMPK protein. Instead, saponarin increased intracellular calcium levels and induced AMPK phosphorylation, which was diminished by co-stimulation with STO-609, an inhibitor of CAMKKβ. Transcription of hepatic gluconeogenesis genes was upregulated by nuclear translocation of CRTC2 and HDAC5, coactivators of CREB and FoxO1 transcription factors, respectively. This nuclear translocation was inhibited by increased phosphorylation of CRTC2 and HDAC5 by saponarin-induced AMPK in HepG2 cells and suppression of CREB and FoxO1 transactivation activities in cells stimulated by saponarin. The results from a chromatin immunoprecipitation assay confirmed the reduced binding of CRTC2 on the PEPCK and G6Pase promoters. In TE671 cells, AMPK phosphorylated HDAC5, which suppressed nuclear penetration and upregulated GLUT4 transcription, leading to enhanced glucose uptake. Collectively, these results suggest that saponarin activates AMPK in a calcium-dependent manner, thus regulating gluconeogenesis and glucose uptake. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.).

PubMed

Mirza, Neelofar; Taj, Gohar; Arora, Sandeep; Kumar, Anil

2014-10-25

Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca(2+)/H(+) antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca(2+) ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca(2+) ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet. Copyright © 2014 Elsevier B.V. All rights reserved.

Modification of nanostructured calcium carbonate for efficient gene delivery.

PubMed

Zhao, Dong; Wang, Chao-Qun; Zhuo, Ren-Xi; Cheng, Si-Xue

2014-06-01

In this study, a facile method to modify nanostructured calcium carbonate (CaCO3) gene delivery systems by adding calcium phosphate (CaP) component was developed. CaCO3/CaP/DNA nanoparticles were prepared by the co-precipitation of Ca(2+) ions with plasmid DNA in the presence of carbonate and phosphate ions. For comparison, CaCO3/DNA nanoparticles and CaP/DNA co-precipitates were also prepared. The effects of carbonate ion/phosphate ion (CO3(2-)/PO4(3-)) ratio on the particle size and gene delivery efficiency were investigated. With an appropriate CO3(2-)/PO4(3-) ratio, the co-existence of carbonate and phosphate ions could control the size of co-precipitates effectively, and CaCO3/CaP/DNA nanoparticles with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly improved as compared with CaCO3/DNA nanoparticles and CaP/DNA co-precipitates. The confocal microscopy study indicated that the cellular uptake and nuclear localization of CaCO3/CaP/DNA nanoparticles were significantly enhanced as compared with unmodified CaCO3/DNA nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake.

PubMed

Ramasamy, Umadevi; Anwer, M Sawkat; Schonhoff, Christopher M

2013-10-01

The Na(+) taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.

Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake

PubMed Central

Ramasamy, Umadevi; Anwer, M. Sawkat

2013-01-01

The Na+ taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO. PMID:23886862

Persistent Graves' hyperthyroidism despite rapid negative conversion of thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results: a case report.

PubMed

Ohara, Nobumasa; Kaneko, Masanori; Kitazawa, Masaru; Uemura, Yasuyuki; Minagawa, Shinichi; Miyakoshi, Masashi; Kaneko, Kenzo; Kamoi, Kyuzi

2017-02-06

Graves' disease is an autoimmune thyroid disorder characterized by hyperthyroidism, and patients exhibit thyroid-stimulating hormone receptor antibody. The major methods of measuring circulating thyroid-stimulating hormone receptor antibody include the thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Although the diagnostic accuracy of these assays has been improved, a minority of patients with Graves' disease test negative even on second-generation and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulins. We report a rare case of a thyroid-stimulating hormone-binding inhibitory immunoglobulin-positive patient with Graves' disease who showed rapid lowering of thyroid-stimulating hormone-binding inhibitory immunoglobulin levels following administration of the anti-thyroid drug thiamazole, but still experienced Graves' hyperthyroidism. A 45-year-old Japanese man presented with severe hyperthyroidism (serum free triiodothyronine >25.0 pg/mL; reference range 1.7 to 3.7 pg/mL) and tested weakly positive for thyroid-stimulating hormone-binding inhibitory immunoglobulins on second-generation tests (2.1 IU/L; reference range <1.0 IU/L). Within 9 months of treatment with oral thiamazole (30 mg/day), his thyroid-stimulating hormone-binding inhibitory immunoglobulin titers had normalized, but he experienced sustained hyperthyroidism for more than 8 years, requiring 15 mg/day of thiamazole to correct. During that period, he tested negative on all first-generation, second-generation, and third-generation thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, but thyroid scintigraphy revealed diffuse and increased uptake, and thyroid ultrasound and color flow Doppler imaging showed typical findings of Graves' hyperthyroidism. The possible explanations for serial changes in the thyroid-stimulating hormone-binding inhibitory immunoglobulin results in our patient include the presence of thyroid-stimulating hormone receptor antibody, which is bioactive but less reactive on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays, or the effect of reduced levels of circulating thyroid-stimulating hormone receptor antibody upon improvement of thyroid autoimmunity with thiamazole treatment. Physicians should keep in mind that patients with Graves' disease may show thyroid-stimulating hormone-binding inhibitory immunoglobulin assay results that do not reflect the severity of Graves' disease or indicate the outcome of the disease, and that active Graves' disease may persist even after negative results on thyroid-stimulating hormone-binding inhibitory immunoglobulin assays. Timely performance of thyroid function tests in combination with sensitive imaging tests, including thyroid ultrasound and scintigraphy, are necessary to evaluate the severity of Graves' disease and treatment efficacy.

Influence of calcium on lipid mixing mediated by influenza hemagglutinin.

PubMed

Zhukovsky, Mikhail A; Markovic, Ingrid; Bailey, Austin L

2007-09-01

We studied the influence of calcium on lipid mixing mediated by influenza hemagglutinin (HA). Lipid mixing between HA-expressing cells and liposomes containing disialoganglioside, influenza virus receptor, was studied at 37 degrees C and neutral pH after a low-pH pulse at 4 degrees C. With DSPC/cholesterol liposomes, calcium present after raising the temperature significantly promoted lipid mixing only when it was triggered by a short low-pH application. In case of DOPC/cholesterol liposomes, calcium promotion was observed regardless of the duration of the low-pH pulse. Calcium present during a short, but not long, low-pH application to HA-expressing cells with bound DSPC/cholesterol liposomes at 4 degrees C inhibited subsequent lipid mixing. We hypothesize that calcium influences lipid mixing because it binds to a vestigial esterase domain of hemagglutinin or causes expulsion of the fusion peptide from an electronegative cavity. We suggest that calcium promotes the transition from early and reversible conformation(s) of low pH-activated HA towards an irreversible conformation that underlies both HA-mediated lipid mixing and HA inactivation.

Feasibility of CO₂/SO₂ uptake enhancement of calcined limestone modified with rice husk ash during pressurized carbonation.

PubMed

Chen, Huichao; Zhao, Changsui; Ren, Qiangqiang

2012-01-01

The calcination/carbonation cycle using calcium-based sorbents appears to be a viable method for carbon dioxide (CO₂) capture from combustion gases. Recent attempts to improve the CO₂/SO₂ uptake of a calcium-based sorbent modified by using rice husk ash (RHA) in the hydration process have succeeded in enhancing its effectiveness. The optimal mole ratio of RHA to calcined limestone (M(Si/Ca)) was adjusted to 0.2. The cyclic CO₂ capture characteristics and the SO₂ uptake activity of the modified sorbent were evaluated in a calcination/pressurized carbonation reactor system. Scanning electron microscope (SEM) images and X-ray diffraction (XRD) spectrum of the sorbent were also taken to supplement the study. The results showed that the carbonation conversion was greatly increased for the sorbent with M(Si/Ca) ratio of 0.2. For this sorbent formulation the optimal operating conditions were 700-750 °C and 0.5-0.7 MPa. CO₂ absorption was not proportional to CO₂ concentration in the carbonation atmosphere, but was directly related to reaction time. The CO₂ uptake decreased in the presence of SO₂. SO₂ uptake increased, and the total calcium utilization was maintained over multiple cycles. Analysis has shown that the silicate component is evenly or well distributed, and this serves as a framework to prevent sintering, thus preserving the available microstructure for reaction. The sorbent also displayed high activity to SO₂ absorption and could be used to capture CO₂ and SO₂ simultaneously. Copyright © 2011 Elsevier Ltd. All rights reserved.

Calcium Signaling Is Required for Erythroid Enucleation

PubMed Central

Russell, Sarah M.; Humbert, Patrick O.

2016-01-01

Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate. Together these functional studies highlight a critical role for calcium signaling in the regulation of erythroid enucleation. PMID:26731108

Local recurrence as immunoglobulin G4 (IgG4)-related disease 10 years after radiotherapy to ocular adnexal extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue.

PubMed

Matsuo, Toshihiko; Ichimura, Kouichi; Yoshino, Tadashi

2011-01-01

In 2000, a 48-year-old woman developed a left orbital mass with lacrimal gland involvement and then, in 2003, a right orbital mass with lacrimal gland involvement, both of which were diagnosed as extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). She underwent 30 Gy external beam radiation to bilateral orbital lesions. The lymphoma cells in both lesions did not share the same clonality, as shown by amplification by polymerase chain reaction of the immunoglobulin heavy chain gene. Immunoglobulin light chain analysis by immunohistochemistry and messenger RNA in situ hybridization showed λ chain monotype in the left orbital lesion but κ chain monotype in the right orbital lesion. She developed recurrent left orbital mass with high uptake on fluorodeoxyglucose positron emission tomography fused with computed tomography in 2010, and excisional biopsy disclosed the formation of follicles and infiltration with immunoglobulin G4 (IgG4)-positive plasma cells mainly in interfollicular areas. The immunoglobulin light chain analysis showed the λ chain and κ chain bitype. With the immunohistopathological diagnosis of IgG4-related disease, the serum IgG4 level was found to show elevation at 376 mg/dL, and the patient chose observation. This is the first reported case of development of IgG4-related disease after bilataral orbital MALT lymphoma with external beam radiotherapy.

Soil-calcium depletion linked to acid rain and forest growth in the eastern United States

USGS Publications Warehouse

Lawrence, Gregory B.; Huntington, T.G.

1999-01-01

Since the discovery of acid rain in the 1970's, scientists have been concerned that deposition of acids could cause depletion of calcium in forest soils. Research in the 1980's showed that the amount of calcium in forest soils is controlled by several factors that are difficult to measure. Further research in the 1990's, including several studies by the U.S. Geological Survey, has shown that (1) calcium in forest soils has decreased at locations in the northeastern and southeastern U.S., and (2) acid rain and forest growth (uptake of calcium from the soil by roots) are both factors contributing to calcium depletion.

IgM-mediated opsonization and cytotoxicity in the shark.

PubMed

McKinney, E C; Flajnik, M F

1997-02-01

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice

PubMed Central

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-01-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury. PMID:12067297

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice.

PubMed

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-06-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury.

Effects of Opsonization and Gamma Interferon on Growth of Brucella Melitensis 16M in Mouse Peritoneal Macrophages In Vitro

DTIC Science & Technology

2000-01-01

bacterial CFU (Fig. 3). Macrophages cultured without brucel - lae or cultured with brucellae but without IFN-7 made ɘ.3 nmol of nitrite/200 JJLI well...receptors used for the uptake of nonopsonized brucel - lae are unknown. Synergy between the receptor for the Fc domain of immunoglobulin G (FcR) and

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling

PubMed Central

Rojas, Fabiola; Gonzalez, David; Cortes, Nicole; Ampuero, Estibaliz; Hernández, Diego E.; Fritz, Elsa; Abarzua, Sebastián; Martinez, Alexis; Elorza, Alvaro A.; Alvarez, Alejandra; Court, Felipe; van Zundert, Brigitte

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1G93A (ACM-hSOD1G93A) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1G93A, but not ACM-hSOD1WT, increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1G93A-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1G86R or TDP43A315T. We further find that co-application of ACM-SOD1G93A with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1G93A induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death. PMID:26106294

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murayama, T.; Ui, M.

1985-06-25

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separatemore » effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.« less

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Upper Extremity Multifocal Neuropathy in a 10-Year-Old Boy Associated With NS6S Disaccharide Antibodies.

PubMed

Edelman, Frederick; Naddaf, Elie; Waclawik, Andrew J

2015-06-01

We present a 10-year-old boy with a predominantly motor multifocal neuropathy with demyelinating and axonal changes with sensory involvement, affecting only one upper extremity. Laboratory studies revealed an elevated titer of immunoglobulin M (IgM) antibodies against the NS6S antigen. He responded to treatment with high dose intravenous immunoglobulins. Focal or multifocal immune-mediated neuropathies are not common in children and may be underdiagnosed. © The Author(s) 2014.

Calcium signaling in taste cells: regulation required.

PubMed

Medler, Kathryn F

2010-11-01

Peripheral taste receptor cells depend on distinct calcium signals to generate appropriate cellular responses that relay taste information to the central nervous system. Some taste cells have conventional chemical synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release from stores to formulate an output signal through a hemichannel. Despite the importance of calcium signaling in taste cells, little is known about how these signals are regulated. This review summarizes recent studies that have identified 2 calcium clearance mechanisms expressed in taste cells, including mitochondrial calcium uptake and sodium/calcium exchangers (NCXs). These studies identified a unique constitutive calcium influx that contributes to maintaining appropriate calcium homeostasis in taste cells and the role of the mitochondria and exchangers in this process. The additional role of NCXs in the regulation of evoked calcium responses is also discussed. Clearly, calcium signaling is a dynamic process in taste cells and appears to be more complex than has previously been appreciated.

Effect of pH and calcium on short-term NO3- fluxes in roots of barley seedlings

NASA Technical Reports Server (NTRS)

Aslam, M.; Travis, R. L.; Huffaker, R. C.

1995-01-01

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2(-)-induced seedlings contain little internal NO3- initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293-1301). NO3-, uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3(-)-induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3- and NO2(-)-induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2(-)-induced roots at either pH 3 or 6. However, in NO3(-)-induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodman, Kerry M.; Yamagata, Masahito; Jin, Xiangshu

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (viatransinteractions) and Sdk clusteringmore » in isolated cells (viacisinteractions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition betweencisandtransinteractions provides a novel mechanism to sharpen the specificity of cell-cell interactions.« less

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

PubMed Central

Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

2016-01-01

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

PubMed

Kraehling, Jan R; Chidlow, John H; Rajagopal, Chitra; Sugiyama, Michael G; Fowler, Joseph W; Lee, Monica Y; Zhang, Xinbo; Ramírez, Cristina M; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L; Fernández-Hernando, Carlos; Sessa, William C

2016-11-21

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

GLUT4 Is Not Necessary for Overload-Induced Glucose Uptake or Hypertrophic Growth in Mouse Skeletal Muscle

PubMed Central

McMillin, Shawna L.; Schmidt, Denise L.; Kahn, Barbara B.

2017-01-01

GLUT4 is necessary for acute insulin- and contraction-induced skeletal muscle glucose uptake, but its role in chronic muscle loading (overload)-induced glucose uptake is unknown. Our goal was to determine whether GLUT4 is required for overload-induced glucose uptake. Overload was induced in mouse plantaris muscle by unilateral synergist ablation. After 5 days, muscle weights and ex vivo [3H]-2-deoxy-d-glucose uptake were assessed. Overload-induced muscle glucose uptake and hypertrophic growth were not impaired in muscle-specific GLUT4 knockout mice, demonstrating that GLUT4 is not necessary for these processes. To assess which transporters mediate overload-induced glucose uptake, chemical inhibitors were used. The facilitative GLUT inhibitor cytochalasin B, but not the sodium-dependent glucose cotransport inhibitor phloridzin, prevented overload-induced uptake demonstrating that GLUTs mediate this effect. To assess which GLUT, hexose competition experiments were performed. Overload-induced [3H]-2-deoxy-d-glucose uptake was not inhibited by d-fructose, demonstrating that the fructose-transporting GLUT2, GLUT5, GLUT8, and GLUT12 do not mediate this effect. To assess additional GLUTs, immunoblots were performed. Overload increased GLUT1, GLUT3, GLUT6, and GLUT10 protein levels twofold to fivefold. Collectively, these results demonstrate that GLUT4 is not necessary for overload-induced muscle glucose uptake or hypertrophic growth and suggest that GLUT1, GLUT3, GLUT6, and/or GLUT10 mediate overload-induced glucose uptake. PMID:28279980

GLUT4 Is Not Necessary for Overload-Induced Glucose Uptake or Hypertrophic Growth in Mouse Skeletal Muscle.

PubMed

McMillin, Shawna L; Schmidt, Denise L; Kahn, Barbara B; Witczak, Carol A

2017-06-01

GLUT4 is necessary for acute insulin- and contraction-induced skeletal muscle glucose uptake, but its role in chronic muscle loading (overload)-induced glucose uptake is unknown. Our goal was to determine whether GLUT4 is required for overload-induced glucose uptake. Overload was induced in mouse plantaris muscle by unilateral synergist ablation. After 5 days, muscle weights and ex vivo [ 3 H]-2-deoxy-d-glucose uptake were assessed. Overload-induced muscle glucose uptake and hypertrophic growth were not impaired in muscle-specific GLUT4 knockout mice, demonstrating that GLUT4 is not necessary for these processes. To assess which transporters mediate overload-induced glucose uptake, chemical inhibitors were used. The facilitative GLUT inhibitor cytochalasin B, but not the sodium-dependent glucose cotransport inhibitor phloridzin, prevented overload-induced uptake demonstrating that GLUTs mediate this effect. To assess which GLUT, hexose competition experiments were performed. Overload-induced [ 3 H]-2-deoxy-d-glucose uptake was not inhibited by d-fructose, demonstrating that the fructose-transporting GLUT2, GLUT5, GLUT8, and GLUT12 do not mediate this effect. To assess additional GLUTs, immunoblots were performed. Overload increased GLUT1, GLUT3, GLUT6, and GLUT10 protein levels twofold to fivefold. Collectively, these results demonstrate that GLUT4 is not necessary for overload-induced muscle glucose uptake or hypertrophic growth and suggest that GLUT1, GLUT3, GLUT6, and/or GLUT10 mediate overload-induced glucose uptake. © 2017 by the American Diabetes Association.

Lysophospholipids stimulate prostate cancer cell migration via TRPV2 channel activation.

PubMed

Monet, Michaël; Gkika, Dimitra; Lehen'kyi, V'yacheslav; Pourtier, Albin; Vanden Abeele, Fabien; Bidaux, Gabriel; Juvin, Véronique; Rassendren, François; Humez, Sandrine; Prevarsakaya, Natalia

2009-03-01

The physiological role, the mechanisms of activation, as well as the endogenous regulators for the non-selective cationic channel TRPV2 are not known so far. In the present work we report that endogenous lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) induce a calcium influx via TRPV2 channel. This activation is dependent on the length of the side-chain and the nature of the lysophospholipid head-group. TRPV2-mediated calcium uptake stimulated by LPC and LPI occurred via Gq/Go-protein and phosphatidylinositol-3,4 kinase (PI3,4K) signalling. We have shown that the mechanism of TRPV2 activation induced by LPC and LPI is due to the TRPV2 channel translocation to the plasma membrane. The activation of TRPV2 channel by LPC and LPI leads to an increase in the cell migration of the prostate cancer cell line PC3. We have demonstrated that TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cancer cell migration. Thus, for the first time, we have identified one of the natural regulators of TRPV2 channel, one of the mechanisms of TRPV2 activation and regulation, as well as its pathophysiological role in cancer.

Carrier-mediated uptake of nobiletin, a citrus polymethoxyflavonoid, in human intestinal Caco-2 cells.

PubMed

Kimura, Osamu; Ohta, Chiho; Koga, Nobuyuki; Haraguchi, Koichi; Kato, Yoshihisa; Endo, Tetsuya

2014-07-01

The mechanism of intestinal absorption of nobiletin (NBL) was investigated using Caco-2 cells. The uptake of NBL from the apical membranes of Caco-2 cells was rapid and temperature-dependent and the presence of metabolic inhibitors, NaN3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone, did not cause a decrease in NBL uptake. The relationship between the initial uptake of NBL and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The Km and uptake clearance (Vmax/Km) values for NBL were 50.6 and 168.1μl/mg protein/min, respectively. This clearance value was about 9-fold greater than that of the non-saturable uptake clearance (Kd: 18.5μl/mg protein/min). The presence of structurally similar compounds, such as quercetin and luteolin, competitively inhibited NBL uptake. These results suggest that uptake of NBL from the apical membranes of Caco-2 cells is mainly mediated by an energy-independent facilitated diffusion process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Intravenous immunoglobulin in kidney transplantation.

PubMed

Tedla, Fasika M; Roche-Recinos, Andrea; Brar, Amarpali

2015-12-01

Antibody-mediated injury of renal allografts has assumed increasing importance with the availability of potent immunosuppressants directed against T-lymphocytes. Intravenous immunoglobulin (IVIG) has been used for prevention and treatment of antibody-mediated rejection. The review summarizes recent advances that shed light on mechanisms of action of IVIG and outlines current roles of IVIG in kidney transplantation. Observational studies support the use of IVIG for desensitization and treatment of acute rejection. Most studies are small and uncontrolled, but a matched case-control study reported a better survival with incompatible live-donor kidney transplant after desensitization using IVIG-containing regimens compared with dialysis or waiting for compatible transplant. Recent data indicate that variations in glycosylation and amino acid sequence cause the crystallizable fragment of immunoglobulin G to assume specific conformations that have high affinity for canonical crystallizable fragment receptors (FcR) or a newly discovered class of FcRs, labelled type II FcRs. Signaling through type II FcRs appears to trigger anti-inflammatory pathways. Recent discoveries expand our understanding of the mechanism of action of IVIG. Future research is expected to clarify the relevance of these findings to humans and could lead to the development of novel immunomodulatory agents.

Ion release and in vitro enamel fluoride uptake associated with pit and fissure sealants containing microencapsulated remineralizing agents.

PubMed

Burbank, Brant D; Cooper, Ryan L; Kava, Alyssa; Hartjes, Jennifer M; McHale, William A; Latta, Mark A; Gross, Stephen M

2017-04-01

To determine if pit-and-fissure sealants with microencapsulated remineralizing agents with sustained release of fluoride, calcium and phosphate ions could promote enamel fluoride uptake by demineralized tooth structure. Sealants that contained 5 w/w% microcapsules with aqueous solutions of 5M Ca(NO3)2 or 0.8M NaF or 6.0M K2HPO4 or a mixture of all three were prepared. Ion release profiles were measured as a function of time. Enamel fluoride uptake by demineralized tooth structure was determined. Sustained release of fluoride, calcium and phosphate ions from a sealant was demonstrated. Fluoride uptake by demineralized enamel was significantly increased compared to a control sealant manufactured without microcapsules (P< 0.01). Bovine enamel that contained 2.2±2.1 µg F/g of enamel prior to exposure to a sealant without microcapsules had 2.3±0.5 after 90 days. Enamel exposed to sealant with 5w/% NaF microcapsules went from 3.5±3.5 µg F/g of enamel prior to exposure to 148±76 after 90 days. Enamel exposed to sealant with 2 w/w% NaF, 2 w/w% Ca(NO3)2 and 1 w/w% K2HPO4 microcapsules went from 1.7±0.7 µg F/g of enamel prior to exposure to 190±137 after 90 days. Sealants with encapsulated remineralizing agents were capable of releasing biologically available fluoride, calcium, and phosphate ions. Incorporation of these microcapsules in pit and fissure sealants is a promising method for remineralization determined by enamel fluoride uptake measurements.

Protective Effects of Calcium on Cadmium Accumulation in Co-Cultured Silver Carp (Hypophthalmichthys molitrix) and Triangle Sail Mussel (Hyriopsis cumingii).

PubMed

Li, Deliang; Pi, Jie; Wang, Jianping; Zhu, Pengfei; Lei, Liuping; Zhang, Ting; Liu, Deming

2016-12-01

Discovering cost effective strategies to reduce cadmium (Cd) uptake is of great concern for consumer food safety in the aquaculture industry. This study investigated the protective effects of calcium (Ca) on Cd uptake in co-cultured silver carp (Hypophthalmichthys molitrix) and triangle sail mussel (Hyriopsis cumingii). The results show that Ca-depending on its applied concentration-caused a significant decrease in the Cd uptake into muscle (by 48 %-72 %), gills (by 51 %-57 %), liver (by 52 %-81 %) and kidney (by 54 %-81 %) of silver carp (p < 0.001), as well as foot (by 8 %-32 %) and visceral mass (by 40 %-47 %) of triangle sail mussels (p < 0.05). The results indicate that Ca treatment is an effective means of reducing Cd accumulation in aquaculture. Since Ca is often used to increase the quality of pearls produced by triangle sail mussel, the quality of co-cultured edible fish might improve as a consequence of the potentially reduced Cd uptake.

An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells.

PubMed Central

Borle, A B

1990-01-01

An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total cell calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca2+ compartmentalization, but the methods suffer from the possibility of Ca2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45Ca uptake or desaturation curves have been used to study the distribution of Ca2+ among various kinetic pools in living cells and their rate of Ca2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45Ca uptake can detect instantaneous changes in calcium influx, while 45Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. Permeabilized cells have been successfully used to gauge the relative role of intracellular organelles in controlling [Ca2+]i. The measurement of the cytosolic ionized calcium ([Ca2+]i) is undoubtedly the most important and, physiologically, the most relevant method available. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca2(+)-sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study. Each probe has specific assets and drawbacks. The study of plasma membrane vesicles derived from baso-lateral or apical plasmalemma can also bring important information on the (Ca2(+)-Mg2+) ATPase-dependent calcium pump and on the kinetics and stoichiometry of the Na(+)-Ca2+ antiporter. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles. PMID:2190818

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat.

PubMed

Takahashi, Yu; Nishimura, Tomohiro; Maruyama, Tetsuo; Tomi, Masatoshi; Nakashima, Emi

2017-04-01

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and L-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and L-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10 mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [ 14 C]α-methylaminoisobutyric acid ([ 14 C]MeAIB) uptake, while 5 mM L-arginine specifically and completely inhibited [ 3 H]glycine uptake via human SNAT4, as well as [ 14 C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [ 14 C]MeAIB was almost completely inhibited by 20 mM unlabeled MeAIB. L-Arginine (5 mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10 mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and L-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

Kefir improves bone mass and microarchitecture in an ovariectomized rat model of postmenopausal osteoporosis.

PubMed

Chen, H-L; Tung, Y-T; Chuang, C-H; Tu, M-Y; Tsai, T-C; Chang, S-Y; Chen, C-M

2015-02-01

Kefir treatment in ovariectomized (OVX) rats could significantly decrease the levels of bone turnover markers and prevent OVX-induced bone loss, deterioration of trabecular microarchitecture, and biomechanical dysfunction that may be due to increase intracellular calcium uptake through the TRPV6 calcium channel. Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to an increased fracture risk. The incidence of osteoporosis increases with age and occurs most frequently in postmenopausal women due to estrogen deficiency, as the balance between bone resorption and bone formation shifts towards increased levels of bone resorption. Among various methods of prevention and treatment for osteoporosis, an increase in calcium intake is the most commonly recommended preventive measure. Kefir is a fermented milk product made with kefir grains that degrade milk proteins into various peptides with health-promoting effects, including immunomodulating-, antithrombotic-, antimicrobial-, and calcium-absorption-enhancing bioactivities. The aim of this study is to investigate the effect of kefir on osteoporosis prophylaxis in an ovariectomized rat model. A total of 56 16-week-old female Sprague-Dawley (SD) rats were divided into 7 experimental groups: sham (normal), OVX/Mock, OVX/1X kefir (164 mg/kg BW/day), OVX/2X kefir (328 mg/kg BW/day), OVX/4X kefir (656 mg/kg BW/day), OVX/ALN (2.5 mg/kg BW/day), and OVX/REBONE (800 mg/kg BW/day). After 12-week treatment with kefir, the bone physiology in the OVX rat model was investigated. Accordingly, the aim of this study was to investigate the possible transport mechanism involved in calcium absorption using the Caco-2 human cell line. A 12-week treatment with kefir on the OVX-induced osteoporosis model reduced the levels of C-terminal telopeptides of type I collagen (CTx), bone turnover markers, and trabecular separation (Tb. Sp.). Additionally, treatment with kefir increased trabecular bone mineral density (BMD), bone volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and the biomechanical properties (hardness and modulus) of the distal femur with a dose-dependent efficacy. In addition, in in vitro assay, we found that kefir increased intracellular calcium uptake in Caco-2 cell through TRPV6 calcium channels and not through L-type voltage-operated calcium channels. The protective effect of kefir in the OVX rat model may occur through increasing intracellular calcium uptake through the TRPV6 calcium channel.

Collagen Peptides from Crucian Skin Improve Calcium Bioavailability and Structural Characterization by HPLC-ESI-MS/MS.

PubMed

Hou, Tao; Liu, Yanshuang; Guo, Danjun; Li, Bo; He, Hui

2017-10-11

The effects of collagen peptides (CPs), which are derived from crucian skin, were investigated in a retinoic acid-induced bone loss model. The level of serum bone alkaline phosphatase (BALP) in the model group (117.65 ± 4.66 units/L) was significantly higher than those of the other three groups (P < 0.05). After treatment with 600 and 1200 mg of CPs/kg, the level of BALP decreased to 85.26 ± 7.35 and 97.03 ± 7.21 units/L, respectively. After treatment with 600 mg of CPs/kg, the bone calcium content significantly increased by 22% (femur) and 12.38% (tibia) compared to those of the model group. In addition, the bone mineral density in the 600 mg of CPs/kg group was significantly higher (femur, 0.37 ± 0.02 g/cm 2 ; tibia, 0.33 ± 0.02 g/cm 2 ) than in the model group (femur, 0.26 ± 0.01 g/cm 2 ; tibia, 0.23 ± 0.02 g/cm 2 ). The morphology results indicated bone structure improved after the treatment with CPs. Structural characterization demonstrated that Glu, Lys, and Arg play important roles in binding calcium and promoting calcium uptake. Our results indicated that CPs could promote calcium uptake and regulate bone formation.

Calcium channel currents in bovine adrenal chromaffin cells and their modulation by anaesthetic agents.

PubMed Central

Charlesworth, P; Pocock, G; Richards, C D

1994-01-01

1. The calcium channel currents of bovine adrenal chromaffin cells were characterized using a variety of voltage pulse protocols and selective channel blockers before examination of their modulation by anaesthetic agents. 2. All the anaesthetics studied (halothane, methoxyflurane, etomidate and methohexitone) inhibited the calcium channel currents in a concentration-dependent manner and increased the rate of current decay. 3. The anaesthetics did not shift the current-voltage relation nor did they change the voltage for half-maximal channel activation derived from analysis of the voltage dependence of the tail currents. None of the anaesthetics appeared to alter the time constant of tail current decay. 4. To complement earlier studies of the inhibitory actions of anaesthetics on K(+)-evoked catecholamine secretion and the associated Ca2+ uptake, the IC50 values for etomidate and methohexitone were determined using a biochemical assay. The IC50 values for anaesthetic inhibition of calcium channel currents corresponded closely with those for inhibition of K(+)-evoked calcium uptake and catecholamine secretion. 5. The inhibitory effect of the volatile anaesthetics and etomidate is best explained by dual action: a reduction in the probability of channel opening coupled with an increase in the rate of channel inactivation. Methohexitone appeared to inhibit the currents by a use-dependent slow block. PMID:7707224

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet.

PubMed

Langer, Julia; Gerkau, Niklas J; Derouiche, Amin; Kleinhans, Christian; Moshrefi-Ravasdjani, Behrouz; Fredrich, Michaela; Kafitz, Karl W; Seifert, Gerald; Steinhäuser, Christian; Rose, Christine R

2017-02-01

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308. © 2016 Wiley Periodicals, Inc.

Use of Mutated Self-Cleaving 2A Peptides as a Molecular Rheostat to Direct Simultaneous Formation of Membrane and Secreted Anti-HIV Immunoglobulins

PubMed Central

Yu, Kenneth K.; Aguilar, Kiefer; Tsai, Jonathan; Galimidi, Rachel; Gnanapragasam, Priyanthi; Yang, Lili; Baltimore, David

2012-01-01

In nature, B cells produce surface immunoglobulin and secreted antibody from the same immunoglobulin gene via alternative splicing of the pre-messenger RNA. Here we present a novel system for genetically programming B cells to direct the simultaneous formation of membrane-bound and secreted immunoglobulins that we term a “Molecular Rheostat”, based on the use of mutated “self-cleaving” 2A peptides. The Molecular Rheostat is designed so that the ratio of secreted to membrane-bound immunoglobulins can be controlled by selecting appropriate mutations in the 2A peptide. Lentiviral transgenesis of Molecular Rheostat constructs into B cell lines enables the simultaneous expression of functional b12-based IgM-like BCRs that signal to the cells and mediate the secretion of b12 IgG broadly neutralizing antibodies that can bind and neutralize HIV-1 pseudovirus. We show that these b12-based Molecular Rheostat constructs promote the maturation of EU12 B cells in an in vitro model of B lymphopoiesis. The Molecular Rheostat offers a novel tool for genetically manipulating B cell specificity for B-cell based gene therapy. PMID:23209743

Evidence of saturable uptake mechanisms at maternal and fetal sides of the perfused human placenta by rapid paired-tracer dilution: studies with calcium and choline.

PubMed

Sweiry, J H; Page, K R; Dacke, C G; Abramovich, D R; Yudilevich, D L

1986-12-01

Rapid uptake and efflux of 45Ca2+ and [3H]choline at the maternal and fetal interfaces of the syncytiotrophoblast in the dually-perfused human placenta was investigated by application of the single circulation paired-tracer dilution method (Yudilevich, Eaton, Short & Leichtweiss 1979). Cotyledons were perfused with Krebs-bicarbonate containing dextran (30 g/l; MW = 60-70,000) at 20 and 6 ml/min on maternal and fetal sides, respectively. The paired-tracer (test substrate and extracellular marker) technique consisted of an intra-arterial injection of a tracer bolus, followed by venous sampling over 5-6 min. There was a rapid (sec) uptake of 45Ca2+, followed by backflux (efflux into the ipsilateral circulation) which, over 5-6 min, was 59-100% on the fetal side. It was more variable but generally lower on the maternal interface. At 0.1 mM calcium, 45Ca2+ maximal uptake (Umax) was about 53% on the fetal side but on the maternal side it was variable and averaged 17%. At 2.4 mM calcium fetal side Umax was reduced to 40%. However, on the maternal side the effect was not consistent. Unidirectional influx (nmol/min per g) appeared to be not different on the two sides of the placenta. For [3H]choline (in choline-free perfusates) Umax was about 50% and 30% on fetal and maternal sides, respectively; tracer backflux was variable on the maternal side and averaged 50% on the fetal side. [3H]Choline uptake was highly inhibited by either 1.0 mM choline or the specific competitive inhibitor, hemicholinium-3 (0.1 mM). Specific transplacental transfer of 45Ca2+ (i.e. in excess of the extracellular marker) was not significant in either direction. For [3H]choline there was an apparent small excess (about 4%) preferential towards the fetal circulation. These findings in the human placenta are similar to those demonstrated previously in the guinea-pig placenta which suggested the existence of specific transport systems for choline and calcium on both sides of the syncytiotrophoblast.

Chemico-Genetic Identification of Drebrin as a Regulator of Calcium Responses

PubMed Central

Mercer, Jason C.; Qi, Qian; Mottram, Laurie F.; Law, Mankit; Bruce, Danny; Iyer, Archana; Morales, J. Luis; Yamazaki, Hiroyuki; Shirao, Tomoaki; Peterson, Blake R.; August, Avery

2009-01-01

Store-operated calcium channels are plasma membrane Ca2+ channels that are activated by depletion of intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca2+ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca2+ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and tore-operated channel mediated calcium influx. PMID:19948240

The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

PubMed

Nolz, Jeffrey C; Gomez, Timothy S; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B; Shimizu, Yoji; Burkhardt, Janis K; Freedman, Bruce D; Billadeau, Daniel D

2006-01-10

The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Isolated contact urticaria caused by immunoglobulin E-mediated fish allergy.

PubMed

Onesimo, Roberta; Giorgio, Valentina; Pill, Stefania; Monaco, Serena; Sopo, Stefano Miceli

2012-01-01

Fish is a common cause of food allergy. The reactions usually occur after its ingestion. In most immunoglobulin E-mediated reactions, the allergens are gastroresistant and heat-stable proteins of low molecularweight (parvalbumin). On the other hand, isolated contact urticaria following the handling of raw fish but without symptoms after its ingestion was found among cooks and professional fish handlers. In these cases, the fish allergens are gastrosensitive and thermolabile, as demonstrated by the decrease in the diameter of the wheal in the skin-prick test using cooked fish. To the best of our knowledge isolated fish contact urticaria in children has not been previously reported. We analyze the features of three pediatric cases of contact urticaria from cod (one of them was sensitized to parvalbumin), with tolerance after ingestion of this fish on oral food challenge.

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis

PubMed Central

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-01-01

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF–EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications. PMID:28629179

Epidermal Growth Factor Enhances Cellular Uptake of Polystyrene Nanoparticles by Clathrin-Mediated Endocytosis.

PubMed

Phuc, Le Thi Minh; Taniguchi, Akiyoshi

2017-06-19

The interaction between nanoparticles and cells has been studied extensively, but most research has focused on the effect of various nanoparticle characteristics, such as size, morphology, and surface charge, on the cellular uptake of nanoparticles. In contrast, there have been very few studies to assess the influence of cellular factors, such as growth factor responses, on the cellular uptake efficiency of nanoparticles. The aim of this study was to clarify the effects of epidermal growth factor (EGF) on the uptake efficiency of polystyrene nanoparticles (PS NPs) by A431 cells, a human carcinoma epithelial cell line. The results showed that EGF enhanced the uptake efficiency of A431 cells for PS NPs. In addition, inhibition and localization studies of PS NPs and EGF receptors (EGFRs) indicated that cellular uptake of PS NPs is related to the binding of EGF-EGFR complex and PS NPs. Different pathways are used to enter the cells depending on the presence or absence of EGF. In the presence of EGF, cellular uptake of PS NPs is via clathrin-mediated endocytosis, whereas, in the absence of EGF, uptake of PS NPs does not involve clathrin-mediated endocytosis. Our findings indicate that EGF enhances cellular uptake of PS NPs by clathrin-mediated endocytosis. This result could be important for developing safe nanoparticles and their safe use in medical applications.

Saccharomyces boulardii Stimulates Intestinal Immunoglobulin A Immune Response to Clostridium difficile Toxin A in Mice

PubMed Central

Qamar, Amir; Aboudola, Samer; Warny, Michel; Michetti, Pierre; Pothoulakis, Charalabos; LaMont, J. Thomas; Kelly, Ciarán P.

2001-01-01

Saccharomyces boulardii is a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrent Clostridium difficile colitis. The administration of C. difficile toxoid A by gavage to S. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P = 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P < 0.001). Enhancing host intestinal immune responses may be an important mechanism for S. boulardii-mediated protection against diarrheal illnesses. PMID:11254650

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation.

PubMed

Reis, Carlos R; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L

2017-01-17

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand-death receptor (TRAIL-DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL-DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL-DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

PubMed Central

Reis, Carlos R.; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L.

2017-01-01

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors. PMID:28049841

Microsomal Ca2+ flux modulation as an indicator of heavy metal toxicity.

PubMed

Pentyala, Srinivas; Ruggeri, Jeanine; Veerraju, Amulya; Yu, Zhangzhang; Bhatia, Anjori; Desaiah, Durisala; Vig, Parminder

2010-07-01

Inositol 1,4,5-trisphosphatee (IP3), an intracellular messenger, releases Ca2+ from microsomes. Ca2+ plays a major role in regulating various cellular events like neural transmission and regulation of hormones and growth factors. Aluminum (Al), lead (Pb) and mercury (Hg) were reported to alter Ca(2+)-regulated events thereby causing neurotoxicity. Hence, an attempt was made characterize IP3 mediated Ca2+ release from rat brain microsomes under the influence of Al, Pb and Hg. Different concentrations of metals were tested over a designated time scale and their effects on IP3 mediated Ca2+ release from microsomes were monitored using Fura-2 technique. All the three metals inhibited IP3 mediated Ca2+ release, Pb being more potent. The order of potency of these three metals was Pb>Hg>Al. Except for Al, both Hg and Pb independently released Ca2+ from microsomes. Re-uptake of Ca2+ into microsomes was inhibited by all the three metals, Pb being more potent. Microsomal Ca(2+)-ATPase activity was also inhibited by all the three metals. These results suggest that neurotoxicity exerted by Al, Pb and Hg may be due to the interference of these metals with IP3 mediated calcium release and also interfering with the microsomal Ca2+ sequestration mechanism. Differential effects of heavy metal induced changes in Ca2+ flux can be used as an index of relative toxicity.

Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin.

PubMed

Johnson, Adam S; García, Dana M

2007-12-19

Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.

Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin

PubMed Central

Johnson, Adam S; García, Dana M

2007-01-01

Background Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. Results Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. Conclusion A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC. PMID:18093324

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ginkel, Paul R. van; Yan, Michael B.; Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cellsmore » to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP{sub 3} pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca{sup 2+}-dependent pro-apoptotic pathways inhibit cancer cell growth.« less

Copper retention, calcium release and ultrastructural evidence indicate specific Cuprolinic Blue uptake and peculiar modifications in mineralizing aortic valves.

PubMed

Ortolani, F; Tubaro, F; Petrelli, L; Gandaglia, A; Spina, M; Marchini, M

2002-01-01

Previously, reactions with copper phthalocyanines at 0.05 M critical electrolyte concentration were found to cause demineralization in calcifying porcine aortic valves after subdermal implantation in rat, as well as simultaneous visualization of peculiar phthalocyanine-positive layers around cells and cell-derived matrix vesicles. In the present investigation, an appraisal was made of the mechanism and specificity of reactions with Cuprolinic Blue by comparing quantitatively calcium release and copper retention by calcified aortic valves reacted with this phthalocyanine under different critical electrolyte concentration conditions, and the corresponding ultrastructural patterns. It was found that (i) decalcifying properties are inversely proportional to salt molarity; (ii) reactivity to Cuprolinic Blue is critical electrolyte concentration-dependent, since the greatest copper retention occurred in 0.05 M critical electrolyte concentration Cuprolinic Blue-reacted samples, the only ones that also exhibited phthalocyanine-positive layers; (iii) the appearance of phthalocyanine-positive layers depends on Cuprolinic Blue uptake, revealing pericellular clustering of calcium-binding, anionic molecules; and (iv) minor Cuprolinic Blue uptake occurs by residual proteoglycans which still remain in the extracellular matrix after 6-week-long subdermal implantation. The present results indicate that this method is appropriate for the study of mineralized tissues and illustrate peculiar tissue modifications occurring at least in the experimental conditions used here.

[Painless thyroiditis].

PubMed

Okamura, Ken; Fujikawa, Megumi; Bandai, Sachiko

2006-12-01

Painless thyroiditis is characterized by painless low-uptake thyrotoxicosis (thyrotoxicosis without hyperthyroidism). Destructive damage of the thyroid has been thought to be the mechanism for self-limited thyrotoxicosis. However, hydrolysis of thyroglobulin must be responsible for the release of excessive thyroid hormone. Low-uptake of iodine and excessive release of thyroid hormone suggest the uncoupling of hormone synthesis and hormone secretion in the thyroid gland. Suppressed serum TSH level, various cytokines or growth factors including TGFbeta1, and thyroglobulin itself may be responsible for the suppressed hormone synthesis. The mechanism for persistent hormone release despite suppressed hormone synthesis should be clarified. Quantitative TSH binding inhibitor immunoglobulin assay is helpful for the differential diagnosis of painless thyroiditis and Graves' hyperthyroidism.

Effects of calcium at toxic concentrations of cadmium in plants.

PubMed

Huang, Danlian; Gong, Xiaomin; Liu, Yunguo; Zeng, Guangming; Lai, Cui; Bashir, Hassan; Zhou, Lu; Wang, Dafei; Xu, Piao; Cheng, Min; Wan, Jia

2017-05-01

This review provides new insight that calcium plays important roles in plant growth, heavy metal accumulation and translocation, photosynthesis, oxidative damage and signal transduction under cadmium stress. Increasing heavy metal pollution problems have raised word-wide concerns. Cadmium (Cd), being a highly toxic metal, poses potential risks both to ecosystems and human health. Compared with conventional technologies, phytoremediation, being cost-efficient, highly stable and environment-friendly, is believed to be a promising green technology for Cd decontamination. However, Cd can be easily taken up by plants and may cause severe phytotoxicity to plants, thus limiting the efficiency of phytoremediation. Various researches are being done to investigate the effects of exogenous substances on the mitigation of Cd toxicity to plants. Calcium (Ca) is an essential plant macronutrient that involved in various plant physiological processes, such as plant growth and development, cell division, cytoplasmic streaming, photosynthesis and intracellular signaling transduction. Due to the chemical similarity between Ca and Cd, Ca may mediate Cd-induced physiological or metabolic changes in plants. Recent studies have shown that Ca could be used as an exogenous substance to protect plants against Cd stress by the alleviation of growth inhibition, regulation of metal uptake and translocation, improvement of photosynthesis, mitigation of oxidative damages and the control of signal transduction in the plants. The effects of Ca on toxic concentrations of Cd in plants are reviewed. This review also provides new insight that plants with enhanced Ca level have improved resistance to Cd stress.

Diospyros kaki calyx inhibits immediate-type hypersensitivity via the reduction of mast cell activation.

PubMed

Kim, Min-Jong; Park, Hae Ran; Shin, Tae-Yong; Kim, Sang-Hyun

2017-12-01

Diospyros kaki L. (Ebenaceae) fruit is widely distributed in Asia and is known to exert anti-inflammatory and antithrombotic effects. We evaluated the inhibitory effect of aqueous extract of D. kaki calyx (AEDKC) on mast cell-mediated immediate-type hypersensitivity and underlying mechanism of action. For in vivo, ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) and immunoglobulin (Ig) E-mediated passive cutaneous anaphylaxis (PCA) models were used. In the ASA, AEDKC (1-100 mg/kg) was orally administered 3 times during 14 days. In the PCA, AEDKC was orally treated 1 h before the antigen challenge. The control drug dexamethasone was used to compare the effectiveness of AEDKC. For in vitro, IgE-stimulated RBL-2H3 cells and primary cultured peritoneal mast cells were used to determine the role of AEDKC (0.01-1 mg/mL). Oral administration of AEDKC dose dependently suppressed rectal temperature decrease and increases in serum histamine, total IgE, OVA-specific IgE, and interleukin (IL)-4 in the ASA. In the PCA, AEDKC reduced Evans blue pigmentation. Compared to dexamethasone (10 mg/kg), AEDKC (100 mg/kg) showed similar inhibitory effects in vivo. AEDKC concentration dependently suppressed the release of histamine and β-hexosaminidase through the reduction of intracellular calcium in mast cells. In addition, AEDKC decreased the expression and secretion of tumour necrosis factor-α and IL-4 by the reduction of nuclear factor-κB. The inhibitory potential of AEDKC (1 mg/mL) was similar with dexamethasone (10 μM) in vitro. We suggest that AEDKC may be a potential candidate for the treatment of mast cell-mediated allergic diseases.

Diselenolane-mediated cellular uptake† †Electronic supplementary information (ESI) available: Detailed procedures and results for all reported experiments. See DOI: 10.1039/c7sc05151d

PubMed Central

Chuard, Nicolas; Poblador-Bahamonde, Amalia I.; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi

2018-01-01

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides. PMID:29675232

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

PubMed

Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle

PubMed Central

Sandström, Marie E; Zhang, Shi-Jin; Bruton, Joseph; Silva, José P; Reid, Michael B; Westerblad, Håkan; Katz, Abram

2006-01-01

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by ∼50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a ∼25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation ∼10-fold, and NAC blocked ∼50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle. PMID:16777943

Tansley Review No. 104, Calcium Physiology and Terrestrial Ecosystem Processes

Treesearch

S.B. McLaughlin; R. Wimmer

1999-01-01

Calcium occupies a unique position among plant nutrients both chemically and functionally. Its chemical properties allow it to exist in a wide range of binding states and to serve in both structural and messenger roles. Despite its importance in many plant processes, Ca mobility is low, making Ca uptake and distribution rate a limiting process for many key plant...

Effect of broiler litter ash and flue gas desulfurization gypsum on yield, calcium and phosphorus uptake by peanut

USDA-ARS?s Scientific Manuscript database

Peanut (Arachis hyogaea) is an important oil seed crop that is grown as a principle source of edible oil and vegetable protein. Over 1.6 million acres of peanuts were planted in the United States during 2012. Peanuts require large amounts of Calcium (Ca) and Phosphorus (P). In 2010, over 10 milli...

CALCIUM ABSORPTION IN MAN: BASED ON LARGE VOLUME LIQUID SCINTILLATION COUNTER STUDIES.

PubMed

LUTWAK, L; SHAPIRO, J R

1964-05-29

A technique has been developed for the in vivo measurement of absorption of calcium in man after oral administration of 1 to 5 microcuries of calcium-47 and continuous counting of the radiation in the subject's arm with a large volume liquid scintillation counter. The maximum value for the arm counting technique is proportional to the absorption of tracer as measured by direct stool analysis. The rate of uptake by the arm is lower in subjects with either the malabsorption syndrome or hypoparathyroidism. The administration of vitamin D increases both the absorption rate and the maximum amount of calcium absorbed.

Crystal structure of the epithelial calcium channel TRPV6.

PubMed

Saotome, Kei; Singh, Appu K; Yelshanskaya, Maria V; Sobolevsky, Alexander I

2016-06-23

Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca(2+)-selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca(2+) uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca(2+) permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular 'skirt' involved in allosteric modulation. In the K(+) channel-like transmembrane domain, Ca(2+) selectivity is determined by direct coordination of Ca(2+) by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca(2+) permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca(2+) uptake and its role in pathophysiology.

Calcium-mediated actin reset (CaAR) mediates acute cell adaptations.

PubMed

Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland

2016-12-06

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

THE REGULATORY ROLE OF DIVALENT CATIONS IN HUMAN GRANULOCYTE CHEMOTAXIS

PubMed Central

Gallin, John I.; Rosenthal, Alan S.

1974-01-01

Optimal human granulocyte chemotaxis has been shown to require both calcium and magnesium. Exposure of granulocytes to three different chemotactic factors (C5a, kallikrein, and dialyzable transfer factor) yielded a rapid calcium release, depressed calcium uptake, and was associated with a shift of calcium out of the cytoplasm and into a granule fraction. Colchicine, sodium azide, and cytochalasin B, in concentrations that inhibited chemotaxis, also inhibited calcium release while low concentrations of cytochalasin B, which enhanced chemotaxis, also enhanced calcium release. Microtubule assembly was visualized both in cells suspended in C5a without a chemotactic gradient and in cells actively migrating through a Micropore filter. The data suggest microtubule assembly is regulated, at least, in part, by the level of cytoplasmic calcium. It is proposed that asymmetric assembly of microtubules may be instrumental in imparting the net vector of motion during chemotaxis. PMID:4855032

Dimebon Inhibits Calcium-Induced Swelling of Rat Brain Mitochondria But Does Not Alter Calcium Retention or Cytochrome C Release

PubMed Central

Naga, Kranthi Kumari

2012-01-01

Dimebon was originally introduced as an antihistamine and subsequently investigated as a possible therapeutic for a variety of disorders, including Alzheimer's disease. One putative mechanism underlying the neuroprotective properties of Dimebon is inhibition of mitochondrial permeability transition, based on the observation that Dimebon inhibited the swelling of rat liver mitochondria induced by calcium and other agents that induce permeability transition. Because liver and brain mitochondria differ substantially in their properties and response to conditions associated with opening of the permeability transition pore, we sought to determine whether Dimebon inhibited permeability transition in brain mitochondria. Dimebon reduced calcium-induced mitochondrial swelling but did not enhance the calcium retention capacity or impair calcium-induced cytochrome C release from non-synaptic mitochondria isolated from rat brain cerebral cortex. These findings indicate that Dimebon does not inhibit mitochondrial permeability transition, induced by excessive calcium uptake, in brain mitochondria. PMID:20625939

Dimebon inhibits calcium-induced swelling of rat brain mitochondria but does not alter calcium retention or cytochrome C release.

PubMed

Naga, Kranthi Kumari; Geddes, James W

2011-03-01

Dimebon was originally introduced as an antihistamine and subsequently investigated as a possible therapeutic for a variety of disorders, including Alzheimer's disease. One putative mechanism underlying the neuroprotective properties of Dimebon is inhibition of mitochondrial permeability transition, based on the observation that Dimebon inhibited the swelling of rat liver mitochondria induced by calcium and other agents that induce permeability transition. Because liver and brain mitochondria differ substantially in their properties and response to conditions associated with opening of the permeability transition pore, we sought to determine whether Dimebon inhibited permeability transition in brain mitochondria. Dimebon reduced calcium-induced mitochondrial swelling but did not enhance the calcium retention capacity or impair calcium-induced cytochrome C release from non-synaptic mitochondria isolated from rat brain cerebral cortex. These findings indicate that Dimebon does not inhibit mitochondrial permeability transition, induced by excessive calcium uptake, in brain mitochondria.

Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels.

PubMed

Ase, Ariel R; Honson, Nicolette S; Zaghdane, Helmi; Pfeifer, Tom A; Séguéla, Philippe

2015-04-01

P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Selective dopamine receptor 4 activation mediates the hippocampal neuronal calcium response via IP3 and ryanodine receptors.

PubMed

Wang, Ya-Li; Wang, Jian-Gang; Guo, Fang-Li; Gao, Xia-Huan; Zhao, Dan-Dan; Zhang, Lin; Wang, Jian-Zhi; Lu, Cheng-Biao

2017-09-01

Intracellular calcium is a key factor in most cellular processes, including cell growth, differentiation, proliferation and neurotransmitter release. Dopamine (DA) mediates synaptic transmission by regulating the intracellular calcium content. It is not clear, however, which specific subunit of the DA receptor contributes to DA modulation of intracellular calcium content changes. Through the traditional technique of Fura-2 calcium imaging, this study demonstrated that the DA can induce transient calcium in cultured hippocampal neurons and that this response can be mimicked by a selective dopamine receptor 4 (DR4) agonist PD168077 (PD). PD-induced calcium transience can be blocked by a calcium chelator, such as BAPTA-AM, or by pre-treatment of neurons with thapsigargin, a IP 3 receptor antagonist, or a micromolar concentration of ryanodine, a ryanodine receptor (RyR) antagonist. However PD-induced calcium transience cannot be blocked by pre-treatment of neurons with a free-calcium medium or a cocktail of NMDA receptor, L-type calcium channel and alpha7 nicotinic acetylcholine receptor blockers. These results indicate that the calcium response induced by DR4 activation is mainly through activation of IP 3 receptor in internal stores, which is likely to contribute to the DA modulation of synaptic transmission and cognitive function. Copyright © 2017. Published by Elsevier B.V.

The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

PubMed Central

Nolz, Jeffrey C.; Gomez, Timothy S.; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B.; Shimizu, Yoji; Burkhardt, Janis K.; Freedman, Bruce D.; Billadeau, Daniel D.

2007-01-01

Summary Background The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. Results By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and β-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCγ1 activation and IP3-mediated store release. Conclusions These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation. PMID:16401421

TRPV6 calcium channel translocates to the plasma membrane via Orai1-mediated mechanism and controls cancer cell survival

PubMed Central

Raphaël, Maylis; Lehen’kyi, V’yacheslav; Vandenberghe, Matthieu; Beck, Benjamin; Khalimonchyk, Sergiy; Vanden Abeele, Fabien; Farsetti, Leonardo; Germain, Emmanuelle; Bokhobza, Alexandre; Mihalache, Adriana; Gosset, Pierre; Romanin, Christoph; Clézardin, Philippe; Skryma, Roman; Prevarskaya, Natalia

2014-01-01

Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca2+/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca2+/Annexin I/S100A11 pathway. PMID:25172921

IMMUNOGLOBULINS IN DEFENSE, PATHOGENESIS AND THERAPY OF FUNGAL DISEASES

PubMed Central

Casadevall, Arturo; Pirofski, Liise-anne

2012-01-01

Summary Only two decades ago antibodies to fungi were thought to have little or no role in protection against fungal diseases. However, subsequent research has provided convincing evidence that certain antibodies can modify the course of fungal infection to the benefit or detriment of the host. Hybridoma technology was the breakthrough that enabled the characterization of antibodies to fungi, illuminating some of the requirements for antibody efficacy. As discussed in this review, fungal specific antibodies mediate protection through direct actions on fungal cells and through classical mechanisms such as phagocytosis and complement activation. Although mechanisms of antibody-mediated protection are often species-specific, numerous fungal antigens can be targeted to generate vaccines and therapeutic immunoglobulins. Furthermore, the study of antibody function against medically important fungi has provided fresh immunological insights into the complexity of humoral immunity that are likely to apply to other pathogens. PMID:22607798

Probing the limit of magnesium uptake by β-tricalcium phosphate in biphasic mixtures formed from calcium deficient apatites

NASA Astrophysics Data System (ADS)

Kumar, P. Nandha; Mishra, Sandeep K.; Kannan, S.

2015-11-01

A series of magnesium doped non-stoichiometric calcium deficient apatites were synthesized through an aqueous precipitation route. The resultant structural changes during heat treatment were investigated by X-ray diffraction, Raman and FT-IR spectroscopy and Rietveld refinement. The results confirmed the formation of biphasic mixtures comprising Ca10(PO4)6(OH)2 and β-Ca3(PO4)2 after heat treatment at 1000 °C with the preferential occupancy of Mg2+ at the crystal lattice of β-Ca3(PO4)2. The concentration of Mg2+ uptake in β-Ca3(PO4)2 is limited till reaching the stoichiometric ratio of (Ca+Mg)/P=1.67 and beyond this stoichiometric value [(Ca+Mg)/P>1.67], Mg2+ precipitates as Mg(OH)2 and thereafter gets converted to MgO during heat treatment. Any kind of Mg2+ uptake in the crystal lattice of Ca10(PO4)6(OH)2 is discarded from the investigation.

The influence of methionine-5-enkephalin on calcium uptake by the bovine aortic media.

PubMed

Kokkas, B; Kotoula, M; Kouyoumtzis, A; Kouvelas, D; Papadopoulos, K; Paradelis, A G

1990-04-01

The influence of methionine-5-enkephalin (M-5-E), an endogenous opioid receptor agonist, on calcium uptake by bovine aortic media was investigated in vitro. 45Ca was used and radioactivity was counted in a beta scintillation counter. M-5-E increases Ca2+ uptake by the preparation. This action is inhibited by naloxone and that is proof that an opioid receptor is stimulated. A comparative study showed that phenylephrine, an alpha-adrenoceptor agonist, exhibits the same action as M-5-E, whereas morphine's action is negligible. Phenylephrine contracts the deendotheliazed ring of the bovine aorta, whereas M-5-E fails to do so. It is concluded that an opioid receptor was identified at the bovine aortic smooth muscle. This receptor is stimulated by M-5-E resulting in an increase of the extracellular Ca2+ entrance. Although no relationship was observed between the receptor and the contraction mechanism, a possible role of M-5-E in the maintenance of the vascular tone cannot be excluded.

Prostaglandin E2 activates channel-mediated calcium entry in human erythrocytes: an indication for a blood clot formation supporting process.

PubMed

Kaestner, Lars; Tabellion, Wiebke; Lipp, Peter; Bernhardt, Ingolf

2004-12-01

Prostaglandin E(2) (PGE(2)) is released from platelets when they are activated. Using fluorescence imaging and the patch-clamp technique, we provide evidence that PGE(2) at physiological concentrations (10(-10) M) activates calcium rises mediated by calcium influx through a non-selective cation-channel in human red blood cells. The extent of calcium increase varied between cells with a total of 45% of the cells responding. It is well known that calcium increases elicited the calcium-activated potassium channel (Gardos channel) in the red cell membrane. Previously, it was shown that the Gardos channel activation results in potassium efflux and shrinkage of the cells. Therefore, we conclude that the PGE(2) responses of red blood cells described here reveal a direct and active participation of erythrocytes in blood clot formation.

Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

PubMed Central

Ghosal, Abhisek; Sekar, Thillai V.

2014-01-01

Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

Lipoprotein lipase-dependent binding and uptake of low density lipoproteins by THP-1 monocytes and macrophages: possible involvement of lipid rafts.

PubMed

Makoveichuk, Elena; Castel, Susanna; Vilaró, Senen; Olivecrona, Gunilla

2004-11-08

Lipoprotein lipase (LPL) is produced by cells in the artery wall and can mediate binding of lipoproteins to cell surface heparan sulfate proteoglycans (HSPG), resulting in endocytosis (the bridging function). Active, dimeric LPL may dissociate to inactive monomers, the main form found in plasma. We have studied binding/internalization of human low density lipoprotein (LDL), mediated by bovine LPL, using THP-1 monocytes and macrophages. Uptake of (125)I-LDL was similar in monocytes and macrophages and was not affected by the LDL-receptor family antagonist receptor-associated protein (RAP) or by the phagocytosis inhibitor cytochalasin D. In contrast, uptake depended on HSPG and on membrane cholesterol. Incubation in the presence of dexamethasone increased the endogenous production of LPL by the cells and also increased LPL-mediated binding of LDL to the cell surfaces. Monomeric LPL was bound to the cells mostly in a heparin-resistant fashion. We conclude that the uptake of LDL mediated by LPL dimers is receptor-independent and involves cholesterol-enriched membrane areas (lipid rafts). Dimeric and monomeric LPL differ in their ability to mediate binding/uptake of LDL, probably due to different mechanisms for binding/internalization.

The Clinical Significance of Serum Amyloid A and the Effects of Corticosteroids on Its Production by HEPG2 Cells

DTIC Science & Technology

1992-07-01

SAA was a more sensitive measure of inflammation in CF patients, particularly ir CFS patients, and correlated with pulmonary function tests . A...1990). CRP is a heat labile, non-immunoglobulin serum substance which requires calcium for precipitation of Fra!tion C. It is a O- globulin , and is... SLE ), and may be partially responsible for some of the sequelae or complications of this disease (Linke and Stolle, 1984). It has been suggested that

Drosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves

NASA Astrophysics Data System (ADS)

Restrepo, Simon; Basler, Konrad

2016-08-01

Calcium signalling is a highly versatile cellular communication system that modulates basic functions such as cell contractility, essential steps of animal development such as fertilization and higher-order processes such as memory. We probed the function of calcium signalling in Drosophila wing imaginal discs through a combination of ex vivo and in vivo imaging and genetic analysis. Here we discover that wing discs display slow, long-range intercellular calcium waves (ICWs) when mechanically stressed in vivo or cultured ex vivo. These slow imaginal disc intercellular calcium waves (SIDICs) are mediated by the inositol-3-phosphate receptor, the endoplasmic reticulum (ER) calcium pump SERCA and the key gap junction component Inx2. The knockdown of genes required for SIDIC formation and propagation negatively affects wing disc recovery after mechanical injury. Our results reveal a role for ICWs in wing disc homoeostasis and highlight the utility of the wing disc as a model for calcium signalling studies.

TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via Different Routes is Calcium-Dependent.

PubMed

Tan, Ling-Yan; Huang, Bin; Xu, Shen; Wei, Zhong-Bo; Yang, Liu-Yan; Miao, Ai-Jun

2016-07-19

Calcium plays versatile roles in aquatic ecosystems. In this study, we investigated its effects on the uptake of polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) by the water flea (cladoceran) Daphnia magna. Particle distribution in these daphnids was also visualized using synchrotron radiation-based micro X-ray fluorescence spectroscopy, transmission electron microscopy, and scanning electron microscopy. At low ambient Ca concentrations in the experimental medium ([Ca]dis), PAA-TiO2-NPs were well dispersed and distributed throughout the daphnid; the particle concentration was highest in the abdominal zone and the gut, as a result of endocytosis and passive drinking of the nanoparticles, respectively. Further, Ca induced PAA-TiO2-NP uptake as a result of the increased Ca influx. At a high [Ca]dis, the PAA-TiO2-NPs formed micrometer-sized aggregates that were ingested by D. magna and concentrated only in its gut, independent of the Ca influx. Our results demonstrated the multiple effects of Ca on nanoparticle bioaccumulation. Specifically, well-dispersed nanoparticles were taken up by D. magna through endocytosis and passive drinking whereas the uptake of micrometer-sized aggregates relied on active ingestion.

Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

PubMed

Eom, Dae Seok; Inoue, Shinya; Patterson, Larissa B; Gordon, Tiffany N; Slingwine, Rebecca; Kondo, Shigeru; Watanabe, Masakatsu; Parichy, David M

2012-01-01

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

Melanophore Migration and Survival during Zebrafish Adult Pigment Stripe Development Require the Immunoglobulin Superfamily Adhesion Molecule Igsf11

PubMed Central

Patterson, Larissa B.; Gordon, Tiffany N.; Slingwine, Rebecca; Kondo, Shigeru; Watanabe, Masakatsu; Parichy, David M.

2012-01-01

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation. PMID:22916035

The AID enzyme induces class switch recombination in fibroblasts.

PubMed

Okazaki, Il-mi; Kinoshita, Kazuo; Muramatsu, Masamichi; Yoshikawa, Kiyotsugu; Honjo, Tasuku

2002-03-21

The switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA is mediated by class switch recombination (CSR). CSR changes the immunoglobulin heavy chain constant region (CH) gene from Cmu to one of the other CH genes. Somatic hypermutation introduces massive numbers of point mutations in the immunoglobulin variable (V) region gene, giving rise to immunoglobulin with higher affinity. Activation-induced cytidine deaminase (AID), a putative RNA-editing cytidine deaminase, is expressed strictly in activated B cells and is indispensable in both CSR and somatic hypermutation. But the exact function of AID is unknown. Here we show that ectopic expression of AID induces CSR in an artificial switch construct in fibroblasts at a level comparable to that in stimulated B cells. Sequences around recombination junctions in the artificial substrate have features similar to endogenous CSR junctions. Furthermore, AID-induced CSR in fibroblasts is dependent on transcription of the target S region, as shown in endogenous CSR in B cells. The results show that AID is the only B-cell-specific factor required for initiation of the CSR reaction in the activated locus.

The Role of the Polymeric Immunoglobulin Receptor and Secretory Immunoglobulins during Mucosal Infection and Immunity.

PubMed

Turula, Holly; Wobus, Christiane E

2018-05-03

The gastrointestinal tract houses millions of microbes, and thus has evolved several host defense mechanisms to keep them at bay, and prevent their entry into the host. One such mucosal surface defense is the secretion of secretory immunoglobulins (SIg). Secretion of SIg depends on the polymeric immunoglobulin receptor (pIgR), which transports polymeric Ig (IgA or IgM) from the basolateral surface of the epithelium to the apical side. Upon reaching the luminal side, a portion of pIgR, called secretory component (SC) is cleaved off to release Ig, forming SIg. Through antigen-specific and non-specific binding, SIg can modulate microbial communities and pathogenic microbes via several mechanisms: agglutination and exclusion from the epithelial surface, neutralization, or via host immunity and complement activation. Given the crucial role of SIg as a microbial scavenger, some pathogens also evolved ways to modulate and utilize pIgR and SIg to facilitate infection. This review will cover the regulation of the pIgR/SIg cycle, mechanisms of SIg-mediated mucosal protection as well as pathogen utilization of SIg.

Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death

PubMed Central

Zhu, Xiaohong; Caplan, Jeffrey; Mamillapalli, Padmavathi; Czymmek, Kirk; Dinesh-Kumar, Savithramma P

2010-01-01

Programmed cell death (PCD) initiated at the pathogen-infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca2+-ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N- and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptor-mediated PCD. Our results indicate that ER-Ca2+-ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response. PMID:20075858

Effect of golden needle mushroom (Flammulina velutipes) stem waste on laying performance, calcium utilization, immune response and serum immunity at early phase of production.

PubMed

Mahfuz, Shad; Song, Hui; Liu, Zhongjun; Liu, Xinyu; Diao, Zipeng; Ren, Guihong; Guo, Zhixin; Cui, Yan

2018-05-01

This experiment was conducted to evaluate the effects of golden needle mushroom ( Flammulina velutipes ) stem waste (FVW), on organic eggs production, calcium utilization, antibody response, serum immunoglobulin, and serum cytokine concentration at early phase of production in laying hens. A total 210, 19 weeks old aged ISA Brown layers were randomly assigned into 5 equal treatment groups, with 7 replications of 6 hens each. Dietary treatment included a standard basal diet as control; antibiotic (0.05% flavomycin); 2% FVW; 4% FVW; and 6% FVW. The experimental duration was 10 weeks. There was no significant differences (p>0.05) on hen day egg production, egg weight, egg mass, feed intake, and feed conversion ratio (FCR) among experimental groups. Unmarketable eggs were significantly lower (p<0.05) both in 4% FVW and 6% FVW fed groups than control group. The calcium retention and calcium in egg shell deposition were significantly higher (p<0.05) in FVW inclusion groups than control and antibiotic groups. Antibody titers against Newcastle diseases were significantly higher (p<0.05) in 6% FVW fed group (except combined with 4% FVW at day 147) and infectious bronchitis were significantly higher (p< 0.05) in FVW fed groups (except 2% FVW and 4% FVW at day 161) than control and antibiotic groups. Serum immunoglobulin sIgA was significantly higher (p<0.05) in all levels of FVW and IgG was significantly higher (p<0.05) in 4% FVW than control and antibiotic groups. Serum cytokine concentration interleukin-2 (IL-2) was significantly higher (p<0.05) in 6% FVW; IL-6 and tumor necrotic factor-α were significantly higher (p<0.05) both in 4% FVW and 6% FVW than control and antibiotic groups; IL-4 was significantly higher (p<0.05) in antibiotic, 2% FVW and 4% FVW fed groups than control. F. velutipes mushroom waste can be used as a novel substitute for antibiotic for organic egg production and sound health status in laying hens.

Effects of dietary CLA supplementation, parity and different concentrate levels before calving on immunoglobulin G1, G2 and M concentrations in dairy cows.

PubMed

Eger, Melanie; Horn, Jana; Hussen, Jamal; Schuberth, Hans-Joachim; Scharf, Maria; Meyer, Ulrich; Dänicke, Sven; Bostedt, Hartwig; Breves, Gerhard

2017-10-01

Peripartal dairy cows exhibit a higher susceptibility for infectious diseases, which might be linked to the negative energy balance occurring at the onset of lactation. A dietary supplementation of conjugated linoleic acids (CLA) may reduce milk fat yield and subsequently lower the energy deficit. The utilization of immunoglobulins (Ig) for colostrogenesis might impair humoral immunity in peripartal dairy cows; therefore this study investigated the effects of a CLA supplement, parity and different dietary energy levels on plasma and colostrum IgG1, IgG2 and IgM levels in dairy cows and their calves. Blood samples were collected from 64 cows from 21days before until 56days after parturition and colostrum samples for the first 3days of lactation. Plasma immunoglobulin concentrations of 19 calves were determined before colostrum uptake. Neither plasma IgG1, nor IgG2 levels were affected by CLA or dietary energy level. However, immunoglobulin levels were affected by parity. Heifers possessed the lowest IgG1 concentrations. IgG2 concentrations were highest in cows with 2 lactations prior to parturition and in heifers after parturition. Plasma IgM levels were characterized by a sharp decrease 3days prior to parturition and were scarcely affected by the feeding regimen or parity. Generally, immunoglobulin levels appear to be mostly independent from the peripartal energy balance of the cows and are not influenced by dietary CLA. However, pronounced differences among parities for IgG1 and IgG2 were revealed which should be further evaluated. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mutual independence of alkaline- and calcium-mediated signalling in Aspergillus fumigatus refutes the existence of a conserved druggable signalling nexus.

PubMed

Loss, Omar; Bertuzzi, Margherita; Yan, Yu; Fedorova, Natalie; McCann, Bethany L; Armstrong-James, Darius; Espeso, Eduardo A; Read, Nick D; Nierman, William C; Bignell, Elaine M

2017-12-01

Functional coupling of calcium- and alkaline responsive signalling occurs in multiple fungi to afford efficient cation homeostasis. Host microenvironments exert alkaline stress and potentially toxic concentrations of Ca 2+ , such that highly conserved regulators of both calcium- (Crz) and pH- (PacC/Rim101) responsive signalling are crucial for fungal pathogenicity. Drugs targeting calcineurin are potent antifungal agents but also perturb human immunity thereby negating their use as anti-infectives, abrogation of alkaline signalling has, therefore, been postulated as an adjunctive antifungal strategy. We examined the interdependency of pH- and calcium-mediated signalling in Aspergillus fumigatus and found that calcium chelation severely impedes hyphal growth indicating a critical requirement for this ion independently of ambient pH. Transcriptomic responses to alkaline pH or calcium excess exhibited minimal similarity. Mutants lacking calcineurin, or its client CrzA, displayed normal alkaline tolerance and nuclear translocation of CrzA was unaffected by ambient pH. Expression of a highly conserved, alkaline-regulated, sodium ATPase was tolerant of genetic or chemical perturbations of calcium-mediated signalling, but abolished in null mutants of the pH-responsive transcription factor PacC, and PacC proteolytic processing occurred normally during calcium excess. Taken together our data demonstrate that in A. fumigatus the regulatory hierarchy governing alkaline tolerance circumvents calcineurin signalling. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity*

PubMed Central

Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T.

2011-01-01

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-d-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP. PMID:21296883

Effect of broiler litter ash and flue gas desulfurization gypsum on yield, calcium, phosphorus, copper, iron, manganese and zinc uptake by peanut

USDA-ARS?s Scientific Manuscript database

Peanut (Arachis hyogaea) is an important oil seed crop that is grown as a principle source of edible oil and vegetable protein. Over 1.6 million acres of peanuts were planted in the United States during 2012. Peanuts require large amounts of calcium (Ca) and phosphorus (P). In 2010, over 10 milli...

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

NASA Technical Reports Server (NTRS)

Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

2003-01-01

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

The multivariate egg: quantifying within- and among-clutch correlations between maternally derived yolk immunoglobulins and yolk androgens using multivariate mixed models.

PubMed

Postma, Erik; Siitari, Heli; Schwabl, Hubert; Richner, Heinz; Tschirren, Barbara

2014-03-01

Egg components are important mediators of prenatal maternal effects in birds and other oviparous species. Because different egg components can have opposite effects on offspring phenotype, selection is expected to favour their mutual adjustment, resulting in a significant covariation between egg components within and/or among clutches. Here we tested for such correlations between maternally derived yolk immunoglobulins and yolk androgens in great tit (Parus major) eggs using a multivariate mixed-model approach. We found no association between yolk immunoglobulins and yolk androgens within clutches, indicating that within clutches the two egg components are deposited independently. Across clutches, however, there was a significant negative relationship between yolk immunoglobulins and yolk androgens, suggesting that selection has co-adjusted their deposition. Furthermore, an experimental manipulation of ectoparasite load affected patterns of covariance among egg components. Yolk immunoglobulins are known to play an important role in nestling immune defence shortly after hatching, whereas yolk androgens, although having growth-enhancing effects under many environmental conditions, can be immunosuppressive. We therefore speculate that variation in the risk of parasitism may play an important role in shaping optimal egg composition and may lead to the observed pattern of yolk immunoglobulin and yolk androgen deposition across clutches. More generally, our case study exemplifies how multivariate mixed-model methodology presents a flexible tool to not only quantify, but also test patterns of (co)variation across different organisational levels and environments, allowing for powerful hypothesis testing in ecophysiology.

Agonist-stimulated cobalt uptake provides selective visualization of neurons expressing AMPA- or kainate-type glutamate receptors in the retina.

PubMed

Pourcho, Roberta G; Qin, Pu; Goebel, Dennis J; Fyk-Kolodziej, Bozena

2002-12-16

Fast-acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) -selective and kainate-selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium-permeable channels. Treatment with both agonists resulted in staining of A- and B-type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA-stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate-stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types. Copyright 2002 Wiley-Liss, Inc.

The Mitochondrial Calcium Uniporter: Mice can live and die without it

PubMed Central

Harrington, Josephine L; Murphy, Elizabeth

2014-01-01

Calcium is of critical importance to mitochondrial and cell function, and calcium signaling is highly localized in the cell. When stimulated, mitochondria are capable of rapidly taking up calcium, affecting both matrix energetics within mitochondria and shaping the amplitude and frequency of cytosolic calcium “waves”. During pathological conditions a large increase in mitochondrial calcium levels is thought to activate the mitochondrial permeability transition pore, resulting in cell death. The protein responsible for mitochondrial calcium uptake, the mitochondrial calcium uniporter (MCU), was identified in 2011 and its molecular elucidation has stimulated and invigorated research in this area. MCU knockout mice have been created, a variety of other regulators have been identified, and a disease phenotype in humans has been attributed to the loss of a uniporter regulator. In the three years since its molecular elucidation, further research into the MCU has revealed a complex uniporter, and raised many questions about its physiologic and pathologic cell roles. PMID:25451167

Effect of heat shock on ultrastructure and calcium distribution in Lavandula pinnata L. glandular trichomes.

PubMed

Huang, S S; Kirchoff, B K; Liao, J P

2013-02-01

The effects of heat shock (HS) on the ultrastructure and calcium distribution of Lavandula pinnata secretory trichomes are examined using transmission electron microscopy and potassium antimonate precipitation. After 48-h HS at 40°C, plastids become distorted and lack stroma and osmiophilic deposits, the cristae of the mitochondria become indistinct, the endoplasmic reticulum acquires a chain-like appearance with ribosomes prominently attached to the lamellae, and the plasma and organelle membranes become distorted. Heat shock is associated with a decrease in calcium precipitates in the trichomes, while the number of precipitates increases in the mesophyll cells. Prolonged exposure to elevated calcium levels may be toxic to the mesophyll cells, while the lack of calcium in the glands cell may deprive them of the normal protective advantages of elevated calcium levels. The inequality in calcium distribution may result not only from uptake from the transpiration stream, but also from redistribution of calcium from the trichomes to the mesophyll cells.

Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

PubMed Central

Cai, Xixi; Lin, Jiaping; Wang, Shaoyun

2016-01-01

Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives. PMID:28036002

Mechanisms of cell uptake, inflammatory potential and protein corona effects with gold nanoparticles.

PubMed

Li, Yang; Monteiro-Riviere, Nancy A

2016-12-01

To assess inflammation, cellular uptake and endocytic mechanisms of gold nanoparticles (AuNP) in human epidermal keratinocytes with and without a protein corona. Human epidermal keratinocytes were exposed to 40 and 80 nm AuNP with lipoic acid, polyethylene glycol (PEG) and branched polyethyleneimine (BPEI) coatings with and without a protein corona up to 48 h. Inhibitors were selected to characterize endocytosis. BPEI-AuNP showed the greatest uptake, while PEG-AuNP had the least. Protein coronas decreased uptake and affected their mechanism. AuNP uptake was energy-dependent, except for 40 nm lipoic-AuNP. Most AuNP were internalized by clathrin and lipid raft-mediated endocytosis, except for 40 nm PEG was by raft/noncaveolae mediated endocytosis. Coronas inhibited caveolae-mediated-endocytosis with lipoic acid and BPEI-AuNP and altered 40 nm PEG-AuNP from raft/noncaveolae to clathrin. Inflammatory responses decreased with a plasma corona. Results suggest protein coronas significantly affect cellular uptake and inflammatory responses of AuNP.

Reciprocal Regulation of Reactive Oxygen Species and Phospho-CREB Regulates Voltage Gated Calcium Channel Expression during Mycobacterium tuberculosis Infection

PubMed Central

Selvakumar, Arti; Antony, Cecil; Singhal, Jhalak; Tiwari, Brijendra K.; Singh, Yogendra; Natarajan, Krishnamurthy

2014-01-01

Our previous work has demonstrated the roles played by L-type Voltage Gated Calcium Channels (VGCC) in regulating Mycobacterium tuberculosis (M. tb) survival and pathogenesis. Here we decipher mechanisms and pathways engaged by the pathogen to regulate VGCC expression in macrophages. We show that M. tb and its antigen Rv3416 use phospho-CREB (pCREB), Reactive Oxygen Species (ROS), Protein Kinase C (PKC) and Mitogen Activated Protein Kinase (MAPK) to modulate VGCC expression in macrophages. siRNA mediated knockdown of MyD88, IRAK1, IRAK2 or TRAF6 significantly inhibited antigen mediated VGCC expression. Inhibiting Protein Kinase C (PKC) or MEK-ERK1/2 further increased VGCC expression. Interestingly, inhibiting intracellular calcium release upregulated antigen mediated VGCC expression, while inhibiting extracellular calcium influx had no significant effect. siRNA mediated knockdown of transcription factors c-Jun, SOX5 and CREB significantly inhibited Rv3416 mediated VGCC expression. A dynamic reciprocal cross-regulation between ROS and pCREB was observed that in turn governed VGCC expression with ROS playing a limiting role in the process. Further dissection of the mechanisms such as the interplay between ROS and pCREB would improve our understanding of the regulation of VGCC expression during M. tb infection. PMID:24797940

AN IP3 RECEPTOR-SENSITIVE CALCIUM STORE MEDIATES DISTURBANCES IN INTRACELLULAR CALCIUM UPON EXPOSURE TO AROCLOR 1254 AND ORTHO-SUBSTITUTED PCBS.

EPA Science Inventory

An IP3 Receptor-Sensitive Ca2+ Store Mediates Disturbances In Intracellular Ca2+ Upon Exposure To Aroclor 1254 And Ortho-Substituted PCBs JR Inglefield, WR Mundy, and TJ Shafer. Neurotoxicol. Div., NHEERL, US EPA, RTP, NC. Sponsor: L Birnbaum

Polychlorinated biphenyls (...

Development of a Radiolabeled Amlodipine Analog for L-type Calcium Channel Imaging.

PubMed

Firouzyar, Tahereh; Jalilian, Amir Reza; Aboudzadeh, Mohammad Reza; Sadeghpour, Hossein; Pooladi, Mehrban; Shafiee-Ardestani, Mahdi; Khalaj, Ali

2017-01-01

The non-invasive imaging and quantification of L-type calcium channels (also known as dihydropyridine channels) in living tissues is of great interest in diagnosis of congestive heart failure, myocardial hypertrophy, irritable bowel syndrome etc. Technetium-99m labeled amlodipine conjugate ([99mTc]-DTPA-AMLO) was prepared starting freshly eluted (<1 h) 99mTechnetium pertechnetate (86.5 MBq) and conjugated DTPAAMLO at pH 5 in 30 min at room temperature in high radiochemical purity (>99%, RTLC; specific activity: 55-60 GBq/mmol). The calcium channel blockade activity (CCBA) and apoptosis/necrosis assay of DTPA-amlodipine conjugate evaluations were performed for the conjugate. Log P, stability, bio-distribution and imaging studies were performed for the tracer followed by biodistribution studies as well as imaging. The conjugate demonstrated low toxicity on MCF-7 cells and CCBA (at µm level) compared to the amlodipine. The tracer was stable up to 4 h in final production and presence of human serum and log P (-0.49) was consistent with a water soluble complex. The tracer was excreted through kidneys and liver as expected for dihydropyridines; excluding excretory organs, calcium channel rich smooth muscle cells; including colon, intestine and lungs which demonstrated significant uptake. SPECT images supported the bio-distribution data up to 4 h. significant uptake of [99mTc]-DTPA-AMLO was obtained in calcium channel rich organs. The complex can be a candidate for further SPECT imaging for L-type calcium channels. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

PubMed

Lakhan, Ram; Said, Hamid M

2017-04-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway

PubMed Central

Lakhan, Ram

2017-01-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr78Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway. PMID:28052864

Carbamylated low-density lipoprotein attenuates glucose uptake via a nitric oxide-mediated pathway in rat L6 skeletal muscle cells.

PubMed

Choi, Hye-Jung; Lee, Kyoung Jae; Hwang, Eun Ah; Mun, Kyo-Cheol; Ha, Eunyoung

2015-07-01

Carbamylation is a cyanate-mediated posttranslational modification. We previously reported that carbamylated low-density lipoprotein (cLDL) increases reactive oxygen species and apoptosis via a lectin-like oxidized LDL receptor mediated pathway in human umbilical vein endothelial cells. A recent study reported an association between cLDL and type 2 diabetes mellitus (T2DM). In the current study, the effects of cLDL on glucose transport were explored in skeletal muscle cells. The effect of cLDL on glucose uptake, glucose transporter 4 (GLUT4) translocation, and signaling pathway were examined in cultured rat L6 muscle cells using 2-deoxyglucose uptake, immunofluorescence staining and western blot analysis. The quantity of nitric oxide (NO) was evaluated by the Griess reaction. The effect of native LDL (nLDL) from patients with chronic renal failure (CRF-nLDL) on glucose uptake was also determined. It was observed that cLDL significantly attenuated glucose uptake and GLUT4 translocation to the membrane, which was mediated via the increase in inducible nitric oxide synthase (iNOS)-induced NO production. Tyrosine nitration of the insulin receptor substrate-1 (IRS‑1) was increased. It was demonstrated that CRF-nLDL markedly reduced glucose uptake compared with nLDL from healthy subjects. Collectively, these findings indicate that cLDL, alone, attenuates glucose uptake via NO-mediated tyrosine nitration of IRS‑1 in L6 rat muscle cells and suggests the possibility that cLDL is involved in the pathogenesis of T2DM.

Microscale characterization of dissolved organic matter production and uptake in marine microbial mat communities

NASA Technical Reports Server (NTRS)

Paerl, H. W.; Bebout, B. M.; Joye, S. B.; Des Marais, D. J.

1993-01-01

Intertidal marine microbial mats exhibited biologically mediated uptake of low molecular weight dissolved organic matter (DOM), including D-glucose, acetate, and an L-amino acid mixture at trace concentrations. Uptake of all compounds occurred in darkness, but was frequently enhanced under natural illumination. The photosystem 2 inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) generally failed to inhibit light-stimulated DOM uptake. Occasionally, light plus DCMU-amended treatments led to uptake rates higher than light-incubated samples, possibly due to phototrophic bacteria present in subsurface anoxic layers. Uptake was similar with either 3H- or 14C-labeled substrates, indicating that recycling of labeled CO2 via photosynthetic fixation was not interfering with measurements of light-stimulated DOM uptake. Microautoradiographs showed a variety of pigmented and nonpigmented bacteria and, to a lesser extent, cyanobacteria and eucaryotic microalgae involved in light-mediated DOM uptake. Light-stimulated DOM uptake was often observed in bacteria associated with sheaths and mucilage surrounding filamentous cyanobacteria, revealing a close association of organisms taking up DOM with photoautotrophic members of the mat community. The capacity for dark- and light-mediated heterotrophy, coupled to efficient retention of fixed carbon in the mat community, may help optimize net production and accretion of mats, even in oligotrophic waters.

Management of food allergy: a survey of Australian paediatricians.

PubMed

Morawetz, Deborah Y; Hiscock, Harriet; Allen, Katrina J; Davies, Sarah; Danchin, Margie H

2014-06-01

The prevalence of Immunoglobulin E (IgE)-mediated food allergy in the developed world is increasing, overwhelming tertiary allergy services. Alternative models of care are required. General paediatricians could provide this care but may require further training to do so. We aimed to determine Australian general paediatricians': (i) knowledge and management of IgE-mediated food allergy; (ii) access to and use of diagnostic services; and (iii) interest in further training. Members of the Australian Paediatric Research Network completed an online survey in 2011/12. A case study elicited paediatrician's knowledge of diagnostic history taking, testing and key management principles. Study-designed questions assessed paediatricians' current practice, access to allergy services and interest in further training. One hundred sixty-eight (43%) of 390 paediatricians responded; 93 paediatricians reported managing food allergy. Diagnostic and management practices varied widely. Paediatricians had high levels of agreement (>90%) for only three of 13 questions pertaining to diagnosis and management. Only 56 (61%) correctly identified that a diagnosis of IgE-mediated food allergy requires a history consistent with a clinical reaction and a positive specific serum IgE antibody or skin prick test result. Reported waiting times for tertiary allergy services ranged from 5.4 (private) to 10.6 months (public). Most (91%) paediatricians expressed interest in further training. General paediatricians would benefit from further training if they are to practice allergy care as their diagnosis and management is often inconsistent with international guidelines. Training could be delivered online to maximise reach and uptake. If effective, such a model could relieve some of the burden experienced by Australian tertiary allergy services. © 2014 The Authors. Journal of Paediatrics and Child Health © 2014 Paediatrics and Child Health Division (Royal Australasian College of Physicians).

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Gut immunity in a protochordate involves a secreted immunoglobulin-type mediator binding host chitin and bacteria.

PubMed

Dishaw, Larry J; Leigh, Brittany; Cannon, John P; Liberti, Assunta; Mueller, M Gail; Skapura, Diana P; Karrer, Charlotte R; Pinto, Maria R; De Santis, Rosaria; Litman, Gary W

2016-02-15

Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.

Intravenous immunoglobulin and Alzheimer's disease immunotherapy.

PubMed

Solomon, Beka

2007-02-01

Amyloid-beta peptide (Abeta) contributes to the acute progression of Alzheimer's disease (AD) and has become the main target for therapeutics. Active immunization with Abeta in individuals with AD has been efficacious; however, some patients developed side effects, possibly related to an autoimmune response. Evidence that intravenous immunoglobulin (IVIg), an FDA-approved purified immunoglobulin fraction from normal human donor blood, shows promise of passive immunotherapy for AD is reviewed. Investigations into the molecular effects of IVIg on Abeta clearance, using the BV-2 cellular microglia line, demonstrate that IVIg dissolves Abeta fibrils in vitro, increases cellular tolerance to Abeta, enhances microglial migration toward Abeta deposits, and mediates phagocytosis of Abeta. Preliminary clinical results indicate that IVIg, which contains natural antibodies against the Abeta, warrants further study into its potential to deliver a controlled immune attack on the peptide, avoiding the immune toxicities that have had a negative impact on the first clinical trials of vaccine against Abeta.

Intravenous Immunoglobulin in the Management of Lupus Nephritis

PubMed Central

Wenderfer, Scott E.; Thacker, Trisha

2012-01-01

The occurrence of nephritis in patients with systemic lupus erythematosus is associated with increased morbidity and mortality. The pathogenesis of lupus nephritis is complex, involving innate and adaptive cellular and humoral immune responses. Autoantibodies in particular have been shown to be critical in the initiation and progression of renal injury, via interactions with both Fc-receptors and complement. One approach in the management of patients with lupus nephritis has been the use of intravenous immunoglobulin. This therapy has shown benefit in the setting of many forms of autoantibody-mediated injury; however, the mechanisms of efficacy are not fully understood. In this paper, the data supporting the use of immunoglobulin therapy in lupus nephritis will be evaluated. In addition, the potential mechanisms of action will be discussed with respect to the known involvement of complement and Fc-receptors in the kidney parenchyma. Results are provocative and warrant additional clinical trials. PMID:23056926

Characterization of keratinocyte differentiation induced by ascorbic acid: protein kinase C involvement and vitamin C homeostasis.

PubMed

Savini, Isabella; Catani, Maria Valeria; Rossi, Antonello; Duranti, Guglielmo; Melino, Gerry; Avigliano, Luciana

2002-02-01

Epidermal keratinocytes undergo differentiation in response to several stimuli to form the cornified envelope, a structure that contributes to the barrier function of skin. Although differentiation has been extensively analyzed, the precise role of vitamin C during this process is still not defined. Ascorbic acid, besides acting as a radical scavenger, has been shown to promote mesenchymal differentiation. In this study, we found that keratinocytes grown in ascorbate-supplemented medium developed a differentiated phenotype, as demonstrated by enhanced expression of marker genes and increase in cornified envelope content. The pro-differentiating effects of ascorbate were mediated by the protein-kinase-C-dependent induction of activating protein 1 DNA binding activity; indeed, down-modulation of protein kinase C activity abolished differentiation triggered by ascorbic acid. Although vitamin C appeared to regulate the same signaling pathway modulated by calcium, a classical in vitro inducer of epidermal differentiation, nonetheless terminally differentiated keratinocytes exhibited different ascorbate homeostasis and cellular antioxidant status. Indeed, we found that, unlike calcium, differentiation promoted by ascorbate was accompanied by (i) an enhanced ascorbate transport, due to overexpression of specific transporters, (ii) a great efficiency of dehydroascorbate uptake, and (iii) an increase in glutathione content with respect to proliferating cells. Ascorbic acid may be useful to promote epidermal differentiation, avoiding depletion of hydrophilic antioxidant stores.

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes.

PubMed

Maxwell, Joshua T; Blatter, Lothar A

2012-12-01

The widely accepted paradigm for cytosolic Ca(2+) wave propagation postulates a 'fire-diffuse-fire' mechanism where local Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca(2+) release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca(2+) wave. A recent challenge to this paradigm proposed the requirement for an intra-SR 'sensitization' Ca(2+) wave that precedes the cytosolic Ca(2+) wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca(2+)](i); rhod-2) and intra-SR ([Ca(2+)](SR); fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca(2+)](i) at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca(2+)](SR) was observed. This transient elevation of [Ca(2+)](SR) could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μM isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca(2+)](SR), whereas inhibition of SERCA (3 μM cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca(2+) uptake by SERCA into the SR facilitates the propagation of cytosolic Ca(2+) waves via luminal sensitization of the RyR, and supports a novel paradigm of a 'fire-diffuse-uptake-fire' mechanism for Ca(2+) wave propagation in cardiac myocytes.

Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stall, Richard; Ramos, Joseph; Kent Fulcher, F.

Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated thatmore » MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.« less

Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes

PubMed Central

Maxwell, Joshua T; Blatter, Lothar A

2012-01-01

The widely accepted paradigm for cytosolic Ca2+ wave propagation postulates a ‘fire-diffuse-fire’ mechanism where local Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca2+ release channels diffuses towards and activates neighbouring release sites, resulting in a propagating Ca2+ wave. A recent challenge to this paradigm proposed the requirement for an intra-SR ‘sensitization’ Ca2+ wave that precedes the cytosolic Ca2+ wave and primes RyRs from the luminal side to CICR. Here, we tested this hypothesis experimentally with direct simultaneous measurements of cytosolic ([Ca2+]i; rhod-2) and intra-SR ([Ca2+]SR; fluo-5N) calcium signals during wave propagation in rabbit ventricular myocytes, using high resolution fluorescence confocal imaging. The increase in [Ca2+]i at the wave front preceded depletion of the SR at each point along the calcium wave front, while during this latency period a transient increase of [Ca2+]SR was observed. This transient elevation of [Ca2+]SR could be identified at individual release junctions and depended on the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Increased SERCA activity (β-adrenergic stimulation with 1 μm isoproterenol (isoprenaline)) decreased the latency period and increased the amplitude of the transient elevation of [Ca2+]SR, whereas inhibition of SERCA (3 μm cyclopiazonic acid) had the opposite effect. In conclusion, the data provide experimental evidence that local Ca2+ uptake by SERCA into the SR facilitates the propagation of cytosolic Ca2+ waves via luminal sensitization of the RyR, and supports a novel paradigm of a ‘fire-diffuse-uptake-fire’ mechanism for Ca2+ wave propagation in cardiac myocytes. PMID:22988145

Linking Fruit Ca Uptake Capacity to Fruit Growth and Pedicel Anatomy, a Cross-Species Study

PubMed Central

Song, Wenpei; Yi, Junwen; Kurniadinata, Odit F.; Wang, Huicong; Huang, Xuming

2018-01-01

Calcium (Ca) in flesh fruits is important for quality formation and maintenance. Most studies on fruit Ca focus on one species. This study attempted to understand some universal relations to fruit Ca uptake across species. Calcium contents in fruit tissues were analyzed in different fruits, including three cultivars of litchi, two cultivars each of grape and citrus, and one cultivar each of loquat, apple, pear, Indian jujube, and longan. In situ Ca distribution was revealed with electron probe and xylem functionality visualized by dye tracing. Fruit Ca uptake rate and activity were calculated and correlated with fruit growth and pedicel anatomy. The results showed that fruit Ca uptake rate was the highest in pomes (loquat, apple, and pear), followed by Indian jujube drupe, arillate fruits (litchis and longan) and citrus, while grape berries were the lowest. Fruit Ca uptake rate showed a strong positive correlation to growth rate. However, Ca uptake activity, reflecting Ca uptake rate relative to growth, was the highest in arillate fruits and loquat and lowest in grape berries, and had a poor correlation with fruit growth rate. In all fruits, Ca concentration in the pedicel was higher than in the fruit, and they displayed a good positive correlation. In the pedicel, Ca was most abundant in the phloem. Dye tracing showed that xylem function loss occurred with maturation in all species/varieties. Apple had the poorest xylem functionality with the least development of secondary xylem, but its Ca uptake rate was among the highest. Vessel density, size and area in the pedicel showed no correlation with fruit Ca uptake rate. It is concluded that: (1) fruit growth may be a key determinant of Ca uptake; (2) the universal pattern of Ca being higher in the pedicel than in the fruit indicates existence of a pedicel-fruit “bottleneck” effect in Ca transport across species; (3) xylem functionality loss with fruit maturation is also a universal event; (4) in the pedicel, Ca is more distributed in the phloem; (5) vessel morphology in the pedicel is not rate-limiting for fruit Ca uptake; (6) phloem pathway might contribute to fruit Ca uptake. PMID:29868049

Parallel pathways of potassium transport in the alga Hydrodictyon reticulatum. Effects of calcium.

PubMed

Nespůrková, L; Rybová, R; Janácek, K

1987-06-01

Inflow of potassium ions into the alga Hydrodictyon reticulatum is reduced in the dark, the reduction being accompanied by a change in the selectivity pattern with respect to alkali metal ions, observed in competition experiments and evaluated by the gnostic analysis as described by Kovanic. This suggests that in the light a special mechanism of potassium uptake with a characteristic selectivity is switched on. This mechanism can be also suppressed by too high (2 mmol/l) or too low (EGTA) concentration of calcium ions in the medium. Since the same applies to the light-induced alkalinization of the algal surroundings it seems that the light-induced potassium uptake is related to the light-induced alkalinization, e.g., via exchange of external potassium cations for intracellular protons.

Role of calcium in the constriction of isolated cerebral arteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wendling, W.W.

1987-01-01

Calcium entry blockers (CEB) have been used in the experimental treatment or prevention of many cerebrovascular disorders including stroke, post-ischemic hypoperfusion after cardiac arrest, cerebral vasospasm after subarachnoid hemorrhage, and migraine headache. However, the mechanism of action of these drugs on the cerebral circulation is poorly understood. This study examined the effects of calcium antagonists, Ca/sup 2 +/-deficient solutions, and vasocostrictors on cerebrovascular tone and /sup 45/Ca fluxes, to determine the role of calcium in cerebral arterial constriction. A Scatchard plot of /sup 45/Ca binding to BMCA showed that Ca/sup 2 +/ was bound at either low or high affinitymore » binding sties. The four vasoconstrictors (potassium, serotonin, PGF/sub 2 ..cap alpha../, or SQ-26,655) each increased low affinity /sup 45/Ca uptake into BMCA. The results demonstrate that: (1) Potassium and serotonin constrict BMCA mainly by promoting Ca/sup 2 +/ influx through CEB-sensitive channels; (2) PGF/sub 2 ..cap alpha../ and SQ-26,655 constrict BMCA in part by promoting Ca/sup 2 +/ influx through CEB-sensitive channels, and in part by releasing Ca/sup 2 +/ from depletable internal stores; (3) The major action of CEB on BMCA is to block vasoconstrictor-induced Ca/sup 2 +/ uptake through both potential-operated (K/sup +/-stimulated) and receptor-operated channels.« less

The role microbial sulfate reduction in the direct mediation of sedimentary authigenic carbonate precipitation

NASA Astrophysics Data System (ADS)

Turchyn, A. V.; Walker, K.; Sun, X.

2016-12-01

The majority of modern deep marine sediments are bathed in water that is undersaturated with respect to calcium carbonate. However, within marine sediments changing chemical conditions, driven largely by the microbial oxidation of organic carbon in the absence of oxygen, lead to supersaturated conditions and drive calcium carbonate precipitation. This sedimentary calcium carbonate is often called `authigenic carbonate', and is found in the form of cements and disseminated crystals within the marine sedimentary pile. As this precipitation of this calcium carbonate is microbially mediated, identifying authigenic carbonate within the geological record and understanding what information its geochemical and/or isotopic signature may hold is key for understanding its importance and what information it may contain past life. However, the modern controls on authigenic carbonate precipitation remain enigmatic because the myriad of microbially mediated reactions occurring within sediments both directly and indirectly impact the proton balance. In this submission we present data from 25 ocean sediment cores spanning the globe where we explore the deviation from the stoichiometrically predicted relationships among alkalinity, calcium and sulfate concentrations. In theory for every mol of organic carbon reduced by sulfate, two mol of alkalinity is produced, and to precipitate subsurface calcium carbonate one mol of calcium is used to consume two mol of alkalinity. We use this data with a model to explore changes in carbonate saturation state with depth below the seafloor. Alkalinity changes in the subsurface are poorly correlated with changes in calcium concentrations, however calcium concentrations are directly and tightly coupled to changes in sulfate concentrations in all studied sites. This suggests a direct role for sulfate reducing bacteria in the precipitation of subsurface carbonate cements.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.

PubMed

Maxwell, Joshua T; Tsai, Chin-Hsien; Mohiuddin, Tahmina A; Kwong, Jennifer Q

2018-04-27

Ca 2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca 2+ from mitochondria is important for determining the role of mitochondrial Ca 2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca 2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca 2+ uptake using the Ca 2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca 2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca 2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca 2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca 2+ in permeabilized cells using the Ca 2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca 2+ . Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca 2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca 2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Calcium transport in the early conceptus and associated maternal tissues in the rabbit

PubMed Central

McIntosh, J. E. A.; Lutwak-Mann, C.

1974-01-01

1. The kinetics of calcium transport were studied in unmated (oestrous) and pregnant rabbits in the first half of gestation, with the aim of establishing evidence of hormonal (ovarian) influence on the pattern of transport. 2. The following tissues were examined at short- (45min and 2h) and long-duration (4, 16 and 48h) intervals after parenteral administration of 45Ca or 47Ca: maternal blood plasma, endometrium, uterine fluid, placental tissues, two developmentally disparate stages of rabbit conceptus, namely the unattached blastocyst and the early post-implantation foetus, and bone (femur). 3. Marked variability in calcium content characterized rabbit tissues and body fluids. 4. Compartmental analysis was applied to measurements of specific radioactivity. Oestrous endometrium had the largest rapidly exchanging calcium fraction (turnover time of 12min) and the highest value for calcium flux (500μg of Ca exchanged/h per g fresh wt. of tissue). A marked downward gradient in values of flux existed between the progestational endometrium, uterine fluid and blastocyst; there was a similar gradient between placental tissues and foetus. 5. An hormonal influence on calcium transport was evident in (i) the decrease in specific radioactivity of rabbit blood plasma with advancing pregnancy, (ii) the extraordinarily rapid calcium transport between blood plasma and endometrium, especially in the oestrous stage, and (iii) the effectiveness of ovarian hormone substitution in ovariectomized rabbits. 6. The very low specific radioactivity recorded for bone indicated that only a minute fraction of its calcium was exchanging with that of blood plasma under the experimental conditions examined. 7. The rate of uptake of 45Ca by rabbit blastocysts growing in vitro was one-tenth of that of 22Na, or that recorded for calcium in vivo. 8. Inhibition of carbonic anhydrase activity with acetazolamide in vivo, in maternal erythrocytes, endometrium and placental tissues, produced no appreciable changes in calcium uptake in these tissues or other systems examined as a routine on either day 6 or days 12–14 of gestation. PMID:4840840

TRPV3 channels mediate strontium-induced mouse egg activation

PubMed Central

Carvacho, Ingrid; Lee, Hoi Chang; Fissore, Rafael A.; Clapham, David E.

2014-01-01

SUMMARY In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a Transient Receptor Potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, as TrpV3−/− eggs failed to permeate Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is the major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

PubMed Central

Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland

2016-01-01

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress. DOI: http://dx.doi.org/10.7554/eLife.19850.001 PMID:27919320

NGP1-01, a multi-targeted polycyclic cage amine, attenuates brain endothelial cell death in iron overload conditions.

PubMed

Lockman, J A; Geldenhuys, W J; Jones-Higgins, M R; Patrick, J D; Allen, D D; Van der Schyf, C J

2012-12-13

Development and progression of neurodegenerative disorders have, amongst other potential causes, been attributed to a disruption of iron regulatory mechanisms and iron accumulation. Excess extracellular iron may enter cells via nontraditional routes such as voltage-gated calcium channels and N-methyl-d-aspartate (NMDA) receptors leading to intracellular oxidative damage and ultimately mitochondrial failure. Nimodipine, an L-type calcium channel blocker has been shown to reduce iron-induced toxicity in neuronal and brain endothelial cells. Our current study investigates NGP1-01, a multimodal drug acting as an antagonist at both the NMDA receptor and the L-type calcium channel. Our previous studies support NGP1-01 as a promising neuroprotective agent in diseases involving calcium-related excitotoxicity. We demonstrate here that NGP1-01 (1 and 10μM) pretreatment abrogates the effects of iron overload in brain endothelial cells protecting cellular viability. Both concentrations of NGP1-01 were found to attenuate iron-induced reduction in cellular viability to a similar extent, and were statistically significant. To further verify the mechanism, the L-type calcium channel agonist FPL 64176 was administered to promote iron uptake. Addition of NGP1-01 dose-dependently reduced FPL 64176 stimulated uptake of iron. These data support further evaluation of NGP1-01 as a neuroprotective agent, not only in diseases associated with excitotoxicity, but also in those of iron overload. Copyright © 2012 Elsevier B.V. All rights reserved.

Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood–brain barrier

PubMed Central

St-Amour, Isabelle; Paré, Isabelle; Alata, Wael; Coulombe, Katherine; Ringuette-Goulet, Cassandra; Drouin-Ouellet, Janelle; Vandal, Milène; Soulet, Denis; Bazin, Renée; Calon, Frédéric

2013-01-01

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets, the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood–brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay, a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053  per hour) in the cortex, consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally, brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary, our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets. PMID:24045402

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Resende, R.R.; Alves, A.S.; Britto, L.R.G

2008-04-15

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereasmore » intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.« less

Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation

PubMed Central

Amin, Ruhul; Sharma, Sapna; Ratakonda, Sireesha

2013-01-01

Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [14C]oxalate uptake in the presence of an outward Cl− gradient as an assay of Cl−/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y2 receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y2 receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y2 purinergic receptor, PLC, and PKC-δ. PMID:23596171

Zinc-mediated attenuation of hippocampal mossy fiber long-term potentiation induced by forskolin.

PubMed

Ando, Masaki; Oku, Naoto; Takeda, Atsushi

2010-11-01

The rise in presynaptic calcium induced by high-frequency stimulation activates the calcium-calmodulin-sensitive adenylyl cyclase (AC) 1 followed by the induction of long-term potentiation (LTP) at the hippocampal mossy fiber-CA3 synapse. Zinc is released with glutamate from mossy fiber terminals. However, the role of the zinc in mossy fiber LTP is controversial. In the present study, the mechanism of zinc-mediated attenuation of mossy fiber LTP was examined in that induced by forskolin, an AC activator. Mossy fiber LTP induced by tetanic stimulation (100 Hz for 1 s) was attenuated in the presence of 5 microM ZnCl(2), whereas that induced by forskolin under test stimulation (0.1 Hz) was not attenuated. Forskolin-induced mossy fiber LTP was attenuated by perfusion with 100 microM ZnCl(2) prior to the induction. However, the zinc (100 microM) pre-perfusion did not attenuate mossy fiber LTP induced by Sp-cAMPS, an activator of protein kinase A, under test stimulation. Zinc is necessary to be taken up into mossy fiber boutons for effectively inhibiting AC activity. In hippocampal slices labeled with ZnAF-2 DA, a membrane-permeable zinc indicator, intracellular ZnAF-2 signal was increased during tetanic stimulation in the presence of 5 microM ZnCl(2), but not under test stimulation. Intracellular ZnAF-2 signal was increased under test stimulation in the presence of 100 microM ZnCl(2). These results suggest that zinc taken up into mossy fibers attenuates forskolin-induced mossy fiber LTP via inhibition of AC activity. The significance of endogenous zinc uptake by mossy fibers is discussed focused on tetanus-induced mossy fiber LTP. Copyright 2010 Elsevier Ltd. All rights reserved.

The total synthesis of calcium atorvastatin.

PubMed

Dias, Luiz C; Vieira, Adriano S; Barreiro, Eliezer J

2016-02-21

A practical and convergent asymmetric route to calcium atorvastatin (1) is reported. The synthesis of calcium atorvastatin (1) was performed using the remote 1,5-anti asymmetric induction in the boron-mediated aldol reaction of β-alkoxy methylketone (4) with pyrrolic aldehyde (3) as a key step. Calcium atorvastatin was obtained from aldehyde (3) after 6 steps, with a 41% overall yield.

Synthesis and characterization of iodinated tetrahydroquinolines targeting the G protein-coupled estrogen receptor GPR30.

PubMed

Ramesh, Chinnasamy; Nayak, Tapan K; Burai, Ritwik; Dennis, Megan K; Hathaway, Helen J; Sklar, Larry A; Prossnitz, Eric R; Arterburn, Jeffrey B

2010-02-11

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ER alpha/beta and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC(50) values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with (125)I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics.

Altered carnitine transport in pressure-overload hypertrophied rat hearts

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Rourke, B.; Foster, K.; Reibel, D.K.

1986-03-01

The authors have previously observed reduced carnitine levels in hypertrophied hearts of rats subjected to aortic constriction. In an attempt to determine the mechanism for reduced myocardial carnitine content, carnitine transport was examined in isolated perfused hearts. Hearts were excised from sham-operated and aortic-constricted rats 3 weeks following surgery and perfused at 60 mm Hg aortic pressure with buffer containing various concentrations of L-/sup 14/C-carnitine. Carnitine uptake by control and hypertrophied hearts was linear throughout 30 minutes of perfusion with 40 ..mu..M carnitine. Total carnitine uptake was significantly reduced by 25% in hypertrophied hearts at each time point examined. Themore » reduction in uptake by hypertrophied hearts was also evident when hearts were perfused with 100 or 200 ..mu..M carnitine. When 0.05 mM mersalyl acid was included in the buffer to inhibit the carrier-mediated component of transport, no difference in carnitine uptake was observed indicating that the transport of carnitine by diffusion was unaltered in the hypertrophied myocardium. Carrier-mediated carnitine uptake (total uptake - uptake by diffusion) was significantly reduced by approximately 40% in hypertrophied hearts at all concentrations examined. Thus, the reduction in carnitine content in the pressure-overload hypertrophied rat heart appears to be due to a reduction in carrier-mediated carnitine uptake by the heart.« less

Immunoglobulin superfamily members encoded by viruses and their multiple roles in immune evasion.

PubMed

Farré, Domènec; Martínez-Vicente, Pablo; Engel, Pablo; Angulo, Ana

2017-05-01

Pathogens have developed a plethora of strategies to undermine host immune defenses in order to guarantee their survival. For large DNA viruses, these immune evasion mechanisms frequently rely on the expression of genes acquired from host genomes. Horizontally transferred genes include members of the immunoglobulin superfamily, whose products constitute the most diverse group of proteins of vertebrate genomes. Their promiscuous immunoglobulin domains, which comprise the building blocks of these molecules, are involved in a large variety of functions mediated by ligand-binding interactions. The flexible structural nature of the immunoglobulin domains makes them appealing targets for viral capture due to their capacity to generate high functional diversity. Here, we present an up-to-date review of immunoglobulin superfamily gene homologs encoded by herpesviruses, poxviruses, and adenoviruses, that include CD200, CD47, Fc receptors, interleukin-1 receptor 2, interleukin-18 binding protein, CD80, carcinoembryonic antigen-related cell adhesion molecules, and signaling lymphocyte activation molecules. We discuss their distinct structural attributes, binding properties, and functions, shaped by evolutionary pressures to disarm specific immune pathways. We include several novel genes identified from extensive genome database surveys. An understanding of the properties and modes of action of these viral proteins may guide the development of novel immune-modulatory therapeutic tools. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calcium bioavailability of vegetarian diets in rats: potential application in a bioregenerative life-support system.

PubMed

Nickel, K P; Nielsen, S S; Smart, D J; Mitchell, C A; Belury, M A

1997-01-01

Calcium bioavailability of vegetarian diets containing various proportions of candidate crops for a controlled ecological life-support system (CELSS) was determined by femur 45Ca uptake. Three vegetarian diets and a control diet were labeled extrinsically with 45Ca and fed to 5-wk old male rats. A fifth group of rats fed an unlabeled control diet received an intraperitoneal (IP) injection of 45Ca. There was no significant difference in mean calcium absorption of vegetarian diets (90.80 +/- 5.23%) and control diet (87.85 +/- 5.25%) when calculated as the percent of an IP dose. The amounts of phytate, oxalate, and dietary fiber in the diets did not affect calcium absorption.

Calcium bioavailability of vegetarian diets in rats: potential application in a bioregenerative life-support system

NASA Technical Reports Server (NTRS)

Nickel, K. P.; Nielsen, S. S.; Smart, D. J.; Mitchell, C. A.; Belury, M. A.

1997-01-01

Calcium bioavailability of vegetarian diets containing various proportions of candidate crops for a controlled ecological life-support system (CELSS) was determined by femur 45Ca uptake. Three vegetarian diets and a control diet were labeled extrinsically with 45Ca and fed to 5-wk old male rats. A fifth group of rats fed an unlabeled control diet received an intraperitoneal (IP) injection of 45Ca. There was no significant difference in mean calcium absorption of vegetarian diets (90.80 +/- 5.23%) and control diet (87.85 +/- 5.25%) when calculated as the percent of an IP dose. The amounts of phytate, oxalate, and dietary fiber in the diets did not affect calcium absorption.

Protective effect of an egg yolk-derived immunoglobulin (IgY) against Prevotella intermedia-mediated gingivitis.

PubMed

Hou, Y-Y; Zhen, Y-H; Wang, D; Zhu, J; Sun, D-X; Liu, X-T; Wang, H-X; Liu, Y; Long, Y-Y; Shu, X-H

2014-04-01

To investigate the effects of an egg yolk-derived immunoglobulin (IgY) specific to Prevotella intermedia in vitro and in vivo. An IgY specific to P. intermedia was produced by immunizing hens with formaldehyde-inactivated P. intermedia and showed high titres when subjected to an ELISA. The obtained IgY inhibited the growth of P. intermedia in a dose-dependent manner at concentrations from 1 to 20 mg ml(-1) in Center for Disease Control and Prevention liquid medium. Forty rats were challenged with P. intermedia on gingivae and then randomly divided into four groups, which were syringed respectively with phosphate-buffered saline, 1 mg ml(-1) of tinidazole, 20 mg ml(-1) of nonspecific IgY and 20 mg ml(-1) of the IgY specific to P. intermedia at a dosage of 300 μl per day. Gingival index (GI), plaque index (PI), bleeding on probing (BOP), counts of white blood cell (WBC) and histopathological slide of the gums were measured after treatment for 15 days. The gingivitis rats treated with the IgY specific to P. intermedia showed significantly decreased GI, PI, BOP and WBC (P < 0·05). Gum histopathology of the treated rats demonstrated a superior protective effect of the specific IgY on P. intermedia-mediated gingivitis. A new immunoglobulin specific to P. intermedia was developed from egg yolk. This specific IgY can dose-dependently inhibit the growth of P. intermedia and protect rats from gingivitis induced by P. intermedia. The new IgY has potential for the treatment of P. intermedia-mediated gingivitis. © 2013 The Society for Applied Microbiology.

A new synthesis for antibody-mediated immunity

PubMed Central

Casadevall, Arturo; Pirofski, Liise-anne

2013-01-01

The view that immunoglobulins function largely by potentiating neutralization, cytotoxicity or phagocytosis is being replaced by a new synthesis whereby antibodies participate in all aspects of the immune response, from protecting the host at the earliest time of encounter with a microbe to later challenges. Perhaps the most transformative concept is that immunoglobulins manifest emergent properties, from their structure and function as individual molecules to their interactions with microbial targets and the host immune system. Given that emergent properties are neither reducible to first principles nor predictable, there is a need for new conceptual approaches for understanding antibody function and mechanisms of antibody immunity. PMID:22179281

The effects of crustacean cardioactive peptide on locust oviducts are calcium-dependent.

PubMed

Donini, Andrew; Lange, Angela B

2002-04-01

The role of calcium as a second messenger in the crustacean cardioactive peptide (CCAP)-induced contractions of the locust oviducts was investigated. Incubation of the oviducts in a calcium-free saline containing, a preferential calcium cation chelator, or an extracellular calcium channel blocker, abolished CCAP-induced contractions, indicating that the effects of CCAP on the oviducts are calcium-dependent. In contrast, sodium free saline did not affect CCAP-induced contractions. Co-application of CCAP to the oviducts with preferential L-type voltage-dependent calcium channel blockers reduced CCAP-induced contractions by 32-54%. Two preferential T-type voltage-dependent calcium channel blockers both inhibited CCAP-induced oviduct contractions although affecting different components of the contractions. Amiloride decreased the tonic component of CCAP-induced contractions by 40-55% and flunarizine dihydrochloride decreased the frequency of CCAP-induced phasic contractions by as much as 65%, without affecting tonus. Flunarizine dihydrochloride did not alter the proctolin-induced contractions of the oviducts. Results suggest that the actions of CCAP are partially mediated by voltage-dependent calcium channels similar to vertebrate L-type and T-type channels. High-potassium saline does not abolish CCAP-induced contractions indicating the presence of receptor-operated calcium channels that mediate the actions of CCAP on the oviducts. The involvement of calcium from intracellular stores in CCAP-induced contractions of the oviducts is likely since, an intracellular calcium antagonist decreased CCAP-induced contractions by 30-35%.

Intravenous immunoglobulins – understanding properties and mechanisms

PubMed Central

Durandy, A; Kaveri, S V; Kuijpers, T W; Basta, M; Miescher, S; Ravetch, J V; Rieben, R

2009-01-01

High-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcγR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled ‘IVIg – Understanding properties and mechanisms’ at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26–28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcγRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy. PMID:19883419

Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers

PubMed Central

Rodriguez, E. R.; Skojec, Diane V.; Tan, Carmela D.; Zachary, Andrea A.; Kasper, Edward K.; Conte, John V.; Baldwin, William M.

2005-01-01

Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56–163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant. PMID:16212640

Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene cluster.

PubMed Central

Brownlee, A G; Arst, H N

1983-01-01

In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 microM was estimated. In uptake assays, the crnA1 mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnA1 reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction. PMID:6350263

Carbocalcitonin treatment in Sudeck's atrophy.

PubMed

Nuti, R; Vattimo, A; Martini, G; Turchetti, V; Righi, G A

1987-02-01

The efficacy of new calcitonin, the amino analog of eel calcitonin (carboCT) on Sudeck's atrophy of the foot was investigated in 14 patients. CarboCT was administered at the dose of 40 Medical Research Council (MRC) units per day, and the duration of treatment was two to ten months. No adverse effects were noted. Bone pain and local edema decreased associated with improvement of motility. CarboCT induced a slight decrease in plasma calcium, plasma phosphate, and 24-hour urinary calcium excretion. An increase in cAMP/Cr ratio, an index of parathyroid function, was also observed (probably a manifestation of the hypocalcemic effect of calcitonin and secondary parathyroid stimulation). The whole body retention of 99mTc-MDP represents a valuable index of bone turnover, it decreased progressively and significantly on treatment. A dynamic study of local bone uptake of 99mTC-MDP was performed in eight patients. After carboCT therapy, statistically significant decreases in local blood flow, early uptake, and delayed uptake were appreciated in the involved foot. These findings lead to the conclusion that carboCT is effective in the treatment of Sudeck's atrophy.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.

PubMed

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-05-01

Mitochondrial calcium ([Ca 2+ ] m ) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca 2+ ] m uptake upon SK channel activation as detected by time lapse mitochondrial Ca 2+ measurements with the Ca 2+ -binding mitochondria-targeted aequorin and FRET-based [Ca 2+ ] m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca 2+ ] m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

PubMed Central

Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

2017-01-01

Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death. PMID:28282037

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

PubMed Central

Mandal, Abhishek; Das, Sushmita; Roy, Saptarshi; Ghosh, Ayan Kumar; Sardar, Abul Hasan; Verma, Sudha; Saini, Savita; Singh, Ruby; Abhishek, Kumar; Kumar, Ajay; Mandal, Chitra; Das, Pradeep

2016-01-01

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis. PMID:26808657

Mechanism of cytotoxic action of perfluorinated acids. III. Disturbance in Ca{sup 2+} homeostasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kleszczynski, Konrad; Skladanowski, Andrzej C., E-mail: acskla@gumed.edu.pl

The global distribution of perfluorinated acids (PFAs) in industry and in household is well known. Their increasing environmental occurrence and biomagnification in the living organisms have drawn growing interests in efforts to describe precisely the mechanisms of action in vitro and in vivo. Our previous investigations widely described lipophilicity-dependent cytotoxicity of PFAs as well as the effect of perfluorination of carbon chain on depolarization of plasma membrane potential, acidification or mitochondrial dysfunctions. In this study we presented in dose- and time-dependent manner the impact of PFAs on calcium homeostasis in HCT116 cells. Comparative analysis of cytosolic [Ca{sup 2+}]{sub c} andmore » mitochondrial calcium [Ca{sup 2+}]{sub m} carried out by flow cytometry revealed distinct uptake of calcium into mitochondria in correlation to increasing lipophilicity of PFAs. Massive accumulation of [Ca{sup 2+}]{sub m} was not accompanied by equivalent loss of [Ca{sup 2+}]{sub c}. Indeed, moderate changes of [Ca{sup 2+}]{sub c} were observed after incubation with 400 {mu}M PFDoDA reaching 29.83% and 49.17% decrease at 4th and 72nd hour, respectively. At the same time, mitochondrial calcium uptake increased from 2- to more than 4-fold comparing with non-treated cells. Incubation with non-fluorinated decanoic acid (DA) did not cause any changes in calcium homeostasis. Presented data show that PFAs-induced perturbations in calcium distribution seem to be a missing link related to mitochondria dysfunction playing a crucial role in determination of apoptotic cell death. Complete scheme for the mechanism of cytotoxic action of PFAs has been included.« less

Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Noriho; Hayashi, Shizu; Gosselink, John

2007-12-01

Exposure to ambient air pollution particles with a diameter of < 10 {mu}m (PM{sub 10}) has been associated with increased cardiopulmonary morbidity and mortality. We have shown that human bronchial epithelial cells (HBECs) exposed to PM{sub 10} produce pro-inflammatory mediators that contribute to a local and systemic inflammatory response. Changes in intracellular calcium concentrations ([Ca{sup 2+}]{sub i}) have been demonstrated to regulate several functions of the airway epithelium including the production of pro-inflammatory mediators. The aim of the present study was to determine the nature and mechanism of calcium responses induced by PM{sub 10} in HBECs and its relationship tomore » cytokine synthesis. Methods: Primary HBECs were exposed to urban air pollution particles (EHC-93) and [Ca{sup 2+}]{sub i} responses were measured using the fluoroprobe (Fura-2). Cytokine levels were measured at mRNA and protein levels using real-time PCR and ELISA. Results: PM{sub 10} increased [Ca{sup 2+}]{sub i} in a dose-dependent manner. This calcium response was reduced by blocking the influx of calcium into cells (i.e. calcium-free medium, NiCl{sub 2}, LaCl{sub 3}). PM{sub 10} also decreased the activity of calcium pumps. PM{sub 10} increased the production of IL-1{beta}, IL-8, GM-CSF and LIF. Preincubation with intracellular calcium chelator (BAPTA-AM) attenuated IL-1{beta} and IL-8 production, but not GM-CSF and LIF production. Conclusion: We conclude that exposure to PM{sub 10} induces an increase in cytosolic calcium and cytokine production in bronchial epithelial cells. Our results also suggest that PM{sub 10} induces the production of pro-inflammatory mediators via either intracellular calcium-dependent (IL-1{beta}, IL-8) or -independent (GM-CSF, LIF) pathways.« less

Vitamin D-mediated calcium absorption in patients with clinically stable Crohn's disease: a pilot study

USDA-ARS?s Scientific Manuscript database

Vitamin D is the critical hormone for intestinal absorption of calcium. Optimal calcium absorption is important for proper mineralization of bone in the prevention of osteoporosis and osteoporotic fractures, among other important functions. Diseases associated with gut inflammation, such as Crohn's ...

Divalent cation mobility throughout exponential growth and sporulation of Bacillus megaterium.

PubMed

Krueger, W B; Kolodziej, B J

1978-01-01

Each of the five elements considered was taken up by Bacillus megaterium during exponential growth. Initial Mg and Mn uptake was rapid and ended by mid-log. For Ca, Fe, and Zn, uptake continued throughout exponential growth. Elements were released from the cells immediately following initial uptake. For Mn, egression continued to t2, with release of 36% of total accumulated. Secondary uptake followed immediately and continued through stage V. Magnesium egression continued to t1 with release of 33% accumulated. Secondary uptake began by t5 (stage IV) and continued slowly through sporulation. Calcium egression ceased by t4 with release of 25% total accumulated. Secondary uptake began by t6 (stage V) and continued until depleted. Zinc egression stopped by t5 with release of 34% accumulated with some secondary uptake by stage V. Iron egression terminated at t4 with release of 59% of total accumulated. This was followed by secondary uptake after t12 (stage VI).

Targeting Chronic and Neuropathic Pain: The N-type Calcium Channel Comes of Age

PubMed Central

Snutch, Terrance P.

2005-01-01

Summary: The rapid entry of calcium into cells through activation of voltage-gated calcium channels directly affects membrane potential and contributes to electrical excitability, repetitive firing patterns, excitation-contraction coupling, and gene expression. At presynaptic nerve terminals, calcium entry is the initial trigger mediating the release of neurotransmitters via the calcium-dependent fusion of synaptic vesicles and involves interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex of synaptic release proteins. Physiological factors or drugs that affect either presynaptic calcium channel activity or the efficacy of calcium-dependent vesicle fusion have dramatic consequences on synaptic transmission, including that mediating pain signaling. The N-type calcium channel exhibits a number of characteristics that make it an attractive target for therapeutic intervention concerning chronic and neuropathic pain conditions. Within the past year, both U.S. and European regulatory agencies have approved the use of the cationic peptide Prialt for the treatment of intractable pain. Prialt is the first N-type calcium channel blocker approved for clinical use and represents the first new proven mechanism of action for chronic pain intervention in many years. The present review discusses the rationale behind targeting the N-type calcium channel, some of the limitations confronting the widespread clinical application of Prialt, and outlines possible strategies to improve upon Prialt's relatively narrow therapeutic window. PMID:16489373

Mast cells and basophils are essential for allergies: mechanisms of allergic inflammation and a proposed procedure for diagnosis

PubMed Central

He, Shao-heng; Zhang, Hui-yun; Zeng, Xiao-ning; Chen, Dong; Yang, Ping-chang

2013-01-01

The current definition of allergy is a group of IgE-mediated diseases. However, a large portion of patients with clinical manifestations of allergies do not exhibit elevated serum levels of IgE (sIgEs). In this article, three key factors, ie soluble allergens, sIgEs and mast cells or basophils, representing the causative factors, messengers and primary effector cells in allergic inflammation, respectively, were discussed. Based on current knowledge on allergic diseases, we propose that allergic diseases are a group of diseases mediated through activated mast cells and/or basophils in sensitive individuals, and allergic diseases include four subgroups: (1) IgE dependent; (2) other immunoglobulin dependent; (3) non-immunoglobulin mediated; (4) mixture of the first three subgroups. According to our proposed definition, pseudo-allergic-reactions, in which mast cell or basophil activation is not mediated via IgE, or to a lesser extent via IgG or IgM, should be non-IgE-mediated allergic diseases. Specific allergen challenge tests (SACTs) are gold standard tests for diagnosing allergies in vivo, but risky. The identification of surface membrane activation markers of mast cells and basophils (CD203c, CCR3, CD63, etc) has led to development of the basophil activation test (BAT), an in vitro specific allergen challenge test (SACT). Based on currently available laboratory allergy tests, we here propose a laboratory examination procedure for allergy. PMID:23974516

Nitric oxide-mediated inhibition of taurocholate uptake involves S-nitrosylation of NTCP.

PubMed

Schonhoff, Christopher M; Ramasamy, Umadevi; Anwer, M Sawkat

2011-02-01

The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.

Chronic exposure to trichloroethene causes early onset of SLE-like disease in female MRL +/+ mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai Ping; Koenig, Rolf; Boor, Paul J.

2008-04-01

Trichloroethene (TCE) exacerbates the development of autoimmune responses in autoimmune-prone MRL +/+ mice. Although TCE-mediated autoimmune responses are associated with an increase in serum immunoglobulins and autoantibodies, the underlying mechanism of autoimmunity is not known. To determine the progression of TCE-mediated immunotoxicity, female MRL +/+ mice were chronically exposed to TCE through the drinking water (0.5 mg/ml of TCE) for various periods of time. Serum concentrations of antinuclear antibodies increased after 36 and 48 weeks of TCE exposure. Histopathological analyses showed lymphocyte infiltration in the livers of MRL +/+ mice exposed to TCE for 36 or 48 weeks. Lymphocyte infiltrationmore » was also apparent in the pancreas, lungs, and kidneys of mice exposed to TCE for 48 weeks. Immunoglobulin deposits in kidney glomeruli were found after 48 weeks of exposure to TCE. Our results suggest that chronic exposure to TCE promotes inflammation in the liver, pancreas, lungs, and kidneys, which may lead to SLE-like disease in MRL +/+ mice.« less

Direct interactions of OCA-B and TFII-I regulate immunoglobulin heavy-chain gene transcription by facilitating enhancer-promoter communication.

PubMed

Ren, Xiaodi; Siegel, Rachael; Kim, Unkyu; Roeder, Robert G

2011-05-06

B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in Igh promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated Igh promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3' enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3' enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism. Copyright © 2011 Elsevier Inc. All rights reserved.

Direct interactions of OCA-B and TFII-I regulate immunoglobulin heavy-chain gene transcription by facilitating enhancer-promoter communication

PubMed Central

Ren, Xiaodi; Siegel, Rachael; Kim, Unkyu; Roeder, Robert G.

2011-01-01

Summary B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in IgH promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated IgH promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3′enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3′enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism. PMID:21549311

TRPV4 calcium-permeable channel is a novel regulator of oxidized LDL-induced macrophage foam cell formation.

PubMed

Goswami, Rishov; Merth, Michael; Sharma, Shweta; Alharbi, Mazen O; Aranda-Espinoza, Helim; Zhu, Xiaoping; Rahaman, Shaik O

2017-09-01

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory arterial disease, is the most dominant underlying pathology. Macrophages are thought to orchestrate atherosclerosis by generating lipid-laden foam cells and by secreting inflammatory mediators. Emerging data support a role for a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function, vascular elasticity, and atherogenesis. However, the identity of the plasma membrane mechanosensor and the mechanisms by which pro-atherogenic signals are transduced/maintained are unknown. We have obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family and a known mechanosensor, is the likely mediator of oxidized low-density lipoprotein (oxLDL)-dependent macrophage foam cell formation, a critical process in atherogenesis. Specifically, we found that: i) genetic ablation of TRPV4 or pharmacologic inhibition of TRPV4 activity by a specific antagonist blocked oxLDL-induced macrophage foam cell formation, and ii) TRPV4 deficiency prevented pathophysiological range matrix stiffness or scratch-induced exacerbation of oxLDL-induced foam cell formation. Mechanistically, we found that: i) plasma membrane localization of TRPV4 was sensitized to the increasing level of matrix stiffness, ii) lack of foam cell formation in TRPV4 null cells was not due to lack of expression of CD36, a major receptor for oxLDL, and iii) TRPV4 channel activity regulated oxLDL uptake but not its binding on macrophages. Altogether, these findings identify a novel role for TRPV4 in regulating macrophage foam cell formation by modulating uptake of oxLDL. These findings suggest that therapeutic targeting of TRPV4 may provide a selective approach to the treatment of atherosclerosis. Copyright © 2017. Published by Elsevier Inc.

Antidiabetic Property of Symplocos cochinchinensis Is Mediated by Inhibition of Alpha Glucosidase and Enhanced Insulin Sensitivity

PubMed Central

Antu, Kalathookunnel Antony; Riya, Mariam Philip; Mishra, Arvind; Anilkumar, Karunakaran S.; Chandrakanth, Chandrasekharan K.; Tamrakar, Akhilesh K.; Srivastava, Arvind K.; Raghu, K. Gopalan

2014-01-01

The study is designed to find out the biochemical basis of antidiabetic property of Symplocos cochinchinensis (SC), the main ingredient of ‘Nisakathakadi’ an Ayurvedic decoction for diabetes. Since diabetes is a multifactorial disease, ethanolic extract of the bark (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90% ethanol) were evaluated by in vitro methods against multiple targets relevant to diabetes such as the alpha glucosidase inhibition, glucose uptake, adipogenic potential, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPP-IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition (IC50 value-82.07±2.10 µg/mL), insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F (3.5 fold increase) and reduced triglyceride accumulation (22% decrease) in 3T3L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells (59.57% decrease) with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence and quantity of bioactives (beta-sitosterol, phloretin 2′glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. We conclude that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with moderate antiglycation and antioxidant activity. PMID:25184241

Abscisic acid triggers whole-plant and fruit-specific mechanisms to increase fruit calcium uptake and prevent blossom end rot development in tomato fruit.

PubMed

de Freitas, Sergio Tonetto; Shackel, Kenneth A; Mitcham, Elizabeth J

2011-05-01

Calcium (Ca) uptake into fruit and leaves is dependent on xylemic water movement, and hence presumably driven by transpiration and growth. High leaf transpiration is thought to restrict Ca movement to low-transpiring tomato fruit, which may increase fruit susceptibility to the Ca-deficiency disorder, blossom end rot (BER). The objective of this study was to analyse the effect of reduced leaf transpiration in abscisic acid (ABA)-treated plants on fruit and leaf Ca uptake and BER development. Tomato cultivars Ace 55 (Vf) and AB2 were grown in a greenhouse environment under Ca-deficit conditions and plants were treated weekly after pollination with water (control) or 500 mg l(-1) ABA. BER incidence was completely prevented in the ABA-treated plants and reached values of 30-45% in the water-treated controls. ABA-treated plants had higher stem water potential, lower leaf stomatal conductance, and lower whole-plant water loss than water-treated plants. ABA treatment increased total tissue and apoplastic water-soluble Ca concentrations in the fruit, and decreased Ca concentrations in leaves. In ABA-treated plants, fruit had a higher number of Safranin-O-stained xylem vessels at early stages of growth and development. ABA treatment reduced the phloem/xylem ratio of fruit sap uptake. The results indicate that ABA prevents BER development by increasing fruit Ca uptake, possibly by a combination of whole-plant and fruit-specific mechanisms.

Intestinal absorption and renal reabsorption of calcium throughout postnatal development

PubMed Central

Beggs, Megan R

2017-01-01

Calcium is vital for many physiological functions including bone mineralization. Postnatal deposition of calcium into bone is greatest in infancy and continues through childhood and adolescence until peek mineral density is reached in early adulthood. Thereafter, bone mineral density remains static until it eventually declines in later life. A positive calcium balance, i.e. more calcium absorbed than excreted, is crucial to bone deposition during growth and thus to peek bone mineral density. Dietary calcium is absorbed from the intestine into the blood. It is then filtered by the renal glomerulus and either reabsorbed by the tubule or excreted in the urine. Calcium can be (re)absorbed across intestinal and renal epithelia via both transcellular and paracellular pathways. Current evidence suggests that significant intestinal and renal calcium transport changes occur throughout development. However, the molecular details of these alterations are incompletely delineated. Here we first briefly review the current model of calcium transport in the intestine and renal tubule in the adult. Then, we describe what is known with regard to calcium handling through postnatal development, and how alterations may aid in mediating a positive calcium balance. The role of transcellular and paracellular calcium transport pathways and the contribution of specific intestinal and tubular segments vary with age. However, the current literature highlights knowledge gaps in how specifically intestinal and renal calcium (re)absorption occurs early in postnatal development. Future research should clarify the specific changes in calcium transport throughout early postnatal development including mediators of these alterations enabling appropriate bone mineralization. Impact statement This mini review outlines the current state of knowledge pertaining to the molecules and mechanisms maintaining a positive calcium balance throughout postnatal development. This process is essential to achieving optimal bone mineral density in early adulthood, thereby lowering the lifetime risk of osteoporosis. PMID:28346014

Salmonella typhimurium IroN and FepA Proteins Mediate Uptake of Enterobactin but Differ in Their Specificity for Other Siderophores

PubMed Central

Rabsch, Wolfgang; Voigt, Wolfgang; Reissbrodt, Rolf; Tsolis, Renée M.; Bäumler, Andreas J.

1999-01-01

Salmonella typhimurium possesses two outer membrane receptor proteins, IroN and FepA, which have been implicated in the uptake of enterobactin. To determine whether both receptors have identical substrate specificities, fepA and iroN mutants and a double mutant were characterized. While both receptors transported enterobactin, the uptake of corynebactin and myxochelin C was selectively mediated by IroN and FepA, respectively. PMID:10348879

Effects of serum immunoglobulins from patients with complex regional pain syndrome (CRPS) on depolarisation-induced calcium transients in isolated dorsal root ganglion (DRG) neurons.

PubMed

Reilly, Joanne M; Dharmalingam, Backialakshmi; Marsh, Stephen J; Thompson, Victoria; Goebel, Andreas; Brown, David A

2016-03-01

Complex regional pain syndrome (CRPS) is thought to have an auto-immune component. One such target recently proposed from the effects of auto-immune IgGs on Ca(2+) transients in cardiac myocytes and cell lines is the α1-adrenoceptor. We have tested whether such IgGs exerted comparable effects on nociceptive sensory neurons isolated from rat dorsal root ganglia. Depolarisation-induced [Ca(2+)]i transients were generated by applying 30 mM KCl for 2 min and monitored by Fura-2 fluorescence imaging. No IgGs tested (including 3 from CRPS patients) had any significant effect on these [Ca(2+)]i transients. However, IgG from one CRPS patient consistently and significantly reduced the K(+)-induced response of cells that had been pre-incubated for 24h with a mixture of inflammatory mediators (1 μM histamine, 5-hydroxytryptamine, bradykinin and PGE2). Since this pre-incubation also appeared to induce a comparable inhibitory response to the α1-agonist phenylephrine, this is compatible with the α1-adrenoceptor as a target for CRPS auto-immunity. A mechanism whereby this might enhance pain is suggested. Copyright © 2015. Published by Elsevier Inc.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

PubMed

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-04-01

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

PubMed Central

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-01-01

Abstract The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges. PMID:21300746

Astroglial Glutamate Signaling and Uptake in the Hippocampus

PubMed Central

Rose, Christine R.; Felix, Lisa; Zeug, Andre; Dietrich, Dirk; Reiner, Andreas; Henneberger, Christian

2018-01-01

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses. PMID:29386994

Characterization of the porcine neonatal Fc receptor—potential use for trans-epithelial protein delivery

PubMed Central

Stirling, Catrina M A; Charleston, Bryan; Takamatsu, Haru; Claypool, Steven; Lencer, Wayne; Blumberg, Richard S; Wileman, Thomas E

2005-01-01

The neonatal Fc receptor transports maternal immunoglobulin across the gut wall and has the potential to deliver genetically engineered proteins bearing immunoglobulin Fc domains across the gut to the mucosal immune system. Here we have characterized the porcine neonatal Fc receptor and tested its utility as a model system to study this kind of protein delivery. The complete DNA sequence obtained from an EST revealed 70–80% homology to mouse and human receptors, respectively, and tyrptophan and di-leucine endocytosis motifs were identified in the cytoplasmic tail. Reverse transcription–polymerase chain reaction analysis showed expression of the receptor mRNA in gut, liver, kidney and spleen tissue, aortic endothelial cells and monocytes. Pig kidney cell lines showed saturable pH-dependent binding and uptake of porcine immunoglobulin G (IgG) and also bovine, mouse and human IgG. Polyclonal antibodies raised against the receptor immunoprecipitated a protein of 40 000 MW when the cDNA was expressed in cells and the receptor required assembly with porcine β2-microglobulin for transport from the endoplasmic reticulum to recycling and early endosomes. Immunohistochemical analysis showed the receptor expressed in epithelial cells of the gut of young and adult animals. The ability of the receptor to deliver immunoglobulin across the gut was demonstrated by feeding piglets bovine colostrum as a source of bovine IgG. Bovine IgG was delivered into the pig circulation. Pigs express the neonatal Fc receptor and the receptor has the potential to deliver protein antigens to the pig immune system. PMID:15804291

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport

PubMed Central

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) – 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG5000-DSPE]/maleimide [M]-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%–45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport. PMID:24940060

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport.

PubMed

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG₅₀₀₀-DSPE]/maleimide [M]-PEG₅₀₀₀-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG₅₀₀₀-DSPE/PEG₅₀₀₀-Glu2C₁₈ at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport.

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

PubMed

Asmat, Tauseef M; Tenenbaum, Tobias; Jonsson, Ann-Beth; Schwerk, Christian; Schroten, Horst

2014-01-01

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Modulation of cardiomyocyte activity using pulsed laser irradiated gold nanoparticles

PubMed Central

Gentemann, Lara; Kalies, Stefan; Coffee, Michelle; Meyer, Heiko; Ripken, Tammo; Heisterkamp, Alexander; Zweigerdt, Robert; Heinemann, Dag

2016-01-01

Can photothermal gold nanoparticle mediated laser manipulation be applied to induce cardiac contraction? Based on our previous work, we present a novel concept of cell stimulation. A 532 nm picosecond laser was employed to heat gold nanoparticles on cardiomyocytes. This leads to calcium oscillations in the HL-1 cardiomyocyte cell line. As calcium is connected to the contractility, we aimed to alter the contraction rate of native and stem cell derived cardiomyocytes. A contraction rate increase was particularly observed in calcium containing buffer with neonatal rat cardiomyocytes. Consequently, the study provides conceptual ideas for a light based, nanoparticle mediated stimulation system. PMID:28101410

Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts

NASA Technical Reports Server (NTRS)

Reich, K. M.; McAllister, T. N.; Gudi, S.; Frangos, J. A.

1997-01-01

Interstitial fluid flow may play a role in load-induced bone remodeling. Previously, we have shown that fluid flow stimulates osteoblast production of cAMP inositol trisphosphate (IP3), and PGE2. Flow-induced increases in cAMP and IP3 were shown to be a result of PG production. Thus, PGE2 production appears to be an important component in fluid flow induced signal transduction. In the present study, we investigated the mechanism of flow-induced PGE2 synthesis. Flow-induced a 20-fold increase in PGE2 production in osteoblasts. Increases were also observed with ALF4-(10mM) (98-fold), an activator of guanidine nucleotide-binding proteins (G proteins), and calcium ionophore A23187 (2 microM) (100-fold) in stationary cells. We then investigated whether flow stimulation is mediated by G proteins and increases in intracellular calcium. Flow-induced PGE2 production was inhibited by the G protein inhibitors GDP beta S (100 microM) and pertussis toxin (1 microgram/ml) by 83% and 72%, respectively. Chelation of extracellular calcium by EGTA (2 mM) and intracellular calcium by quin-2/AM (30 microM) blocked flow stimulation by 87% and 67%, respectively. These results suggest that G proteins and calcium play an important role in mediating mechanochemical signal transduction in osteoblasts.

Radiological study of the effect of low calcium diet on the mineral metabolism of bone tissue. With reference to mineralization in callus (in Japanese)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, K.

1972-01-01

Deficiency of available food material due either to poor diet or to malabsorption may adversely affect the skeleton. To study the affection, DDN mice were fed low calcium diet to induce low calcium state corresponding to malabsorption of calcium from the intestine. The femur was fractured manually. Then, calcium deposition in the callus was observed by microradiography and tracer technics with /sup 47/Ca. Increase of the body weight in mice fed low calcium diet was much slower than in the control. The affection of the low calcium diet on bone tissue appeared as a decrease of precipitation of calcium salt.more » This tendency was also observed in callus, Tracer study with /sup 47/Ca was performed in mice fed the low calcium diet for 24 days. Incorporation activity of calcium was generally high in each organ except the kidney. Callus in the site of the fracture in mice fed a low calcium diet was formed to the same degree as the control, although the amount of precipitated calcium in it was significantly poorer. In summary, insufficient mineralization in relation to osteogenesis occurred when the supply of the requisite electrolytes was insufficient or inappropriate. On the other hand, the uptake rate of calcinm in the callus was elevated even in the calcium deficient state. (auth)« less

Calcium Biofortification: Three Pronged Molecular Approaches for Dissecting Complex Trait of Calcium Nutrition in Finger Millet (Eleusine coracana) for Devising Strategies of Enrichment of Food Crops

PubMed Central

Sharma, Divya; Jamra, Gautam; Singh, Uma M.; Sood, Salej; Kumar, Anil

2017-01-01

Calcium is an essential macronutrient for plants and animals and plays an indispensable role in structure and signaling. Low dietary intake of calcium in humans has been epidemiologically linked to various diseases which can have serious health consequences over time. Major staple food-grains are poor source of calcium, however, finger millet [Eleusine coracana (L.) Gaertn.], an orphan crop has an immense potential as a nutritional security crop due to its exceptionally high calcium content. Understanding the existing genetic variation as well as molecular mechanisms underlying the uptake, transport, accumulation of calcium ions (Ca2+) in grains is of utmost importance for development of calcium bio-fortified crops. In this review, we have discussed molecular mechanisms involved in calcium accumulation and transport thoroughly, emphasized the role of molecular breeding, functional genomics and transgenic approaches to understand the intricate mechanism of calcium nutrition in finger millet. The objective is to provide a comprehensive up to date account of molecular mechanisms regulating calcium nutrition and highlight the significance of bio-fortification through identification of potential candidate genes and regulatory elements from finger millet to alleviate calcium malnutrition. Hence, finger millet could be used as a model system for explaining the mechanism of elevated calcium (Ca2+) accumulation in its grains and could pave way for development of nutraceuticals or designer crops. PMID:28144246

Calcium Biofortification: Three Pronged Molecular Approaches for Dissecting Complex Trait of Calcium Nutrition in Finger Millet (Eleusine coracana) for Devising Strategies of Enrichment of Food Crops.

PubMed

Sharma, Divya; Jamra, Gautam; Singh, Uma M; Sood, Salej; Kumar, Anil

2016-01-01

Calcium is an essential macronutrient for plants and animals and plays an indispensable role in structure and signaling. Low dietary intake of calcium in humans has been epidemiologically linked to various diseases which can have serious health consequences over time. Major staple food-grains are poor source of calcium, however, finger millet [ Eleusine coracana (L.) Gaertn.], an orphan crop has an immense potential as a nutritional security crop due to its exceptionally high calcium content. Understanding the existing genetic variation as well as molecular mechanisms underlying the uptake, transport, accumulation of calcium ions (Ca 2+ ) in grains is of utmost importance for development of calcium bio-fortified crops. In this review, we have discussed molecular mechanisms involved in calcium accumulation and transport thoroughly, emphasized the role of molecular breeding, functional genomics and transgenic approaches to understand the intricate mechanism of calcium nutrition in finger millet. The objective is to provide a comprehensive up to date account of molecular mechanisms regulating calcium nutrition and highlight the significance of bio-fortification through identification of potential candidate genes and regulatory elements from finger millet to alleviate calcium malnutrition. Hence, finger millet could be used as a model system for explaining the mechanism of elevated calcium (Ca 2+ ) accumulation in its grains and could pave way for development of nutraceuticals or designer crops.

The Regulatory Functions of Calcium and the Potential Role of Calcium in Mediating Gravitational Responses in Cells and Tissues

NASA Technical Reports Server (NTRS)

Roux, S. J. (Editor)

1983-01-01

The hypothesis that calcium plays an important part in regulating cellular response to gravity and to other environmental stimuli is explored. Topics covered include the role of calmodulin and other proteins, gravitropic responses, bone demineralization during space flight, and intracellular communication.

Interaction of zinc with dental mineral.

PubMed

Ingram, G S; Horay, C P; Stead, W J

1992-01-01

As some currently available toothpastes contain zinc compounds, the reaction of zinc with dental mineral and its effect on crystal growth rates were studied using three synthetic calcium-deficient hydroxyapatites (HAP) as being representative of dental mineral. Zinc was readily acquired by all HAP samples in the absence of added calcium, the amount adsorbed being proportional to the HAP surface area; about 9 mumol Zn/m2 was adsorbed at high zinc concentrations. As zinc was acquired, calcium was released, consistent with 1:1 Ca:Zn exchange. Soluble calcium reduced zinc uptake and similarly, calcium post-treatment released zinc. Pretreatment of HAP with 0.5 mM zinc reduced its subsequent ability to undergo seeded crystal growth, as did extracts of a toothpaste containing 0.5% zinc citrate, even in the presence of saliva. The reverse reaction, i.e. displacement of adsorbed zinc by salivary levels of calcium, however, indicates the mechanism by which zinc can reduce calculus formation in vivo by inhibiting plaque mineralisation without adversely affecting the anti-caries effects of fluoride.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding[OPEN

PubMed Central

Vincent, Thomas R.; Avramova, Marieta; Canham, James; Higgins, Peter; Bilkey, Natasha; Mugford, Sam T.; Pitino, Marco; Toyota, Masatsugu

2017-01-01

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction. PMID:28559475

Monosaccharide uptake by erythrocytes of the embryonic and adult chicken.

PubMed

Ingermann, R L; Stock, M K; Metcalfe, J; Bissonnette, J M

1985-01-01

Rates of monosaccharide uptake by adult and 10-18 day old embryonic chicken erythrocytes were quantitated. The rate of carrier-mediated, stereospecific transport decreased 28% from day 10 to day 14 of incubation and was unchanged thereafter. At no time, however, did the rate of carrier-mediated transport by embryonic erythrocytes differ significantly from that of the adult cells. The rate of transfer by simple diffusion was 3-5 fold faster in embryonic than in adult erythrocytes. Uptake by simple diffusion decreased slightly as the embryo developed. Chronic hyperoxic incubation (70% O2) had little influence on total monosaccharide uptake by embryonic erythrocytes.

Fetuin mediates hepatic uptake of negatively charged nanoparticles via scavenger receptor.

PubMed

Nagayama, Susumu; Ogawara, Ken-ichi; Minato, Keiko; Fukuoka, Yoshiko; Takakura, Yoshinobu; Hashida, Mitsuru; Higaki, Kazutaka; Kimura, Toshikiro

2007-02-01

We tried to evaluate the possible involvement of fetuin in the scavenger receptors (SRs)-mediated hepatic uptake of polystyrene nanospheres with the size of 50 nm (NS-50), which has surface negative charge (zeta potential=-21.8+/-2.3 mV). The liver perfusion studies in rats revealed that the hepatic uptake of NS-50 pre-coated with fetuin (NS-50-fetuin) was significantly inhibited by poly inosinic acid (poly I), a typical inhibitor of SRs, whereas that of plain NS-50 or NS-50 pre-coated with BSA (NS-50-BSA) was not. The uptake of NS-50-fetuin by cultured Kupffer cells was also significantly inhibited by poly I, and anti-class A scavenger receptors (SR-A) antibody, suggesting that fetuin on NS-50 mediated the recognition and internalization of NS-50 by Kupffer cells and at least SR-A would be responsible for the uptake. Taken that Western blot analysis confirmed that fetuin certainly adsorbed on the surface of NS-50 after the incubation of NS-50 with serum, the results obtained in the present study indicate that fetuin would be one of the serum proteins that were substantially involved in the hepatic uptake of NS-50 via SRs.

Endogenous protein and enzyme fragments induce immunoglobulin E-independent activation of mast cells via a G protein-coupled receptor, MRGPRX2.

PubMed

Tatemoto, K; Nozaki, Y; Tsuda, R; Kaneko, S; Tomura, K; Furuno, M; Ogasawara, H; Edamura, K; Takagi, H; Iwamura, H; Noguchi, M; Naito, T

2018-05-01

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E-dependent and E-independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas-related G protein-coupled receptors (MRGPRs). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin-10 fragment) that act as bioactive peptides and induce immunoglobulin E-independent mast cell activation via MRGPRX2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX2 responds various as-yet-unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells. © 2018 The Foundation for the Scandinavian Journal of Immunology.

Regulation of transmural transport of amino acid/metal conjugates by dietary calcium in crustacean digestive tract.

PubMed

Abdel-Malak, Rania; Ahearn, Gregory A

2014-03-01

Effects of luminal Ca(2+) and Mn(2+) on transmural mucosal to serosal (MS) transport of (3) H-L-leucine were characterized in the isolated and perfused intestine of the American lobster, Homarus americanus. (3) H-L-leucine MS transport in the presence of 20 µM Mn(2+) was a sigmoidal function of luminal amino acid concentration, following the Hill equation for multisite cooperative, carrier-mediated, transport. Luminal Ca(2+) was a non-competitive inhibitor of Mn(2+) -stimulated (3) H-L-leucine MS flux. Amino acid transport was hyperbolically stimulated by luminal Ca(2+) or Mn(2+). During 20 µM Mn(2+) -stimulation of (3) H-L-leucine MS flux, addition of 25 mM Ca(2+) strongly reduced amino acid transport Jmax , without affecting amino acid binding properties. Hyperbolic luminal Mn(2+) stimulation of 20 µM (3) H-L-leucine MS flux was also strongly inhibited by 25 mM luminal Ca(2+) , significantly reducing 20 µM (3) H-L-leucine Jmax . Increasing the luminal concentration of verapamil, a calcium channel blocker, significantly increased MS transport of 20 µM (3) H-L-leucine in the presence of 100 nM Mn(2+) by reducing diffusional Ca(2+) uptake into intestinal epithelial cells through verapamil-sensitive channels. A model is proposed supporting the concept of molecular mimicry, whereby (3) H-L-leucine enters lobster intestinal epithelial cells by one or more amino acid-specific transporters and by a dipeptide-like transporter that is capable of binding and transporting peptide molecular mimics (bis-complexes) between Ca(2+) or Mn(2+) and (3) H-L-leucine using the membrane potential as a major driving force for the transport event. According to the model, Ca(2+) entry through apical Ca(2+) channels regulates the magnitude of the membrane potential and therefore the size of the driving force for bis-complex uptake. © 2013 Wiley Periodicals, Inc.

A longitudinal study of human milk composition in the second year postpartum: implications for human milk banking.

PubMed

Perrin, Maryanne T; Fogleman, April D; Newburg, David S; Allen, Jonathan C

2017-01-01

While the composition of human milk has been studied extensively in the first year of lactation, there is a paucity of data regarding human milk composition beyond one year postpartum. Policies vary at milk banks around the world regarding how long lactating women are eligible to donate their milk. The primary purpose of this study is to describe longitudinal changes in human milk composition in the second year postpartum to support the development of evidence based guidelines regarding how long lactating women can donate human milk to a milk bank. Nineteen lactating women in North Carolina provided monthly milk samples from 11 months to 17 months postpartum (N = 131), and two non-profit milk banks provided (N = 33) pooled, unpasteurized milk samples from 51 approved donors less than one year postpartum. There was a significant increase (P < 0.05) in the concentration of total protein, lactoferrin, lysozyme, Immunoglobulin A, oligosaccharides and sodium in longitudinal samples of mother's milk between 11 and 17 months postpartum, while zinc and calcium concentrations declined, and no changes were observed in lactose, fat, iron and potassium. Human milk in the second year postpartum contained significantly higher concentrations of total protein, lactoferrin, lysozyme and Immunoglobulin A, than milk bank samples, and significantly lower concentrations of zinc, calcium, iron and oligosaccharides. Accepting milk bank donations beyond one year postpartum is a potential strategy for increasing the supply of donor milk, but may require mineral fortification. © 2016 John Wiley & Sons Ltd.

Stone former urine proteome demonstrates a cationic shift in protein distribution compared to normal.

PubMed

Kolbach-Mandel, Ann M; Mandel, Neil S; Hoffmann, Brian R; Kleinman, Jack G; Wesson, Jeffrey A

2017-08-01

Many urine proteins are found in calcium oxalate stones, yet decades of research have failed to define the role of urine proteins in stone formation. This urine proteomic study compares the relative amounts of abundant urine proteins between idiopathic calcium oxalate stone forming and non-stone forming (normal) cohorts to identify differences that might correlate with disease. Random mid-morning urine samples were collected following informed consent from 25 stone formers and 14 normal individuals. Proteins were isolated from urine using ultrafiltration. Urine proteomes for each sample were characterized using label-free spectral counting mass spectrometry, so that urine protein relative abundances could be compared between the two populations. A total of 407 unique proteins were identified with the 38 predominant proteins accounting for >82% of all sample spectral counts. The most highly abundant proteins were equivalent in stone formers and normals, though significant differences were observed in a few moderate abundance proteins (immunoglobulins, transferrin, and epidermal growth factor), accounting for 13 and 10% of the spectral counts, respectively. These proteins contributed to a cationic shift in protein distribution in stone formers compared to normals (22% vs. 18%, p = 0.04). Our data showing only small differences in moderate abundance proteins suggest that no single protein controls stone formation. Observed increases in immunoglobulins and transferrin suggest increased inflammatory activity in stone formers, but cannot distinguish cause from effect in stone formation. The observed cationic shift in protein distribution would diminish protein charge stabilization, which could lead to protein aggregation and increased risk for crystal aggregation.

Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

PubMed

Yuan, Wei; Sanda, Miloslav; Wu, Jing; Koomen, John; Goldman, Radoslav

2015-02-26

Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC-MS-MRM assay to quantify glycoforms of IgG subclasses 1-4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indicate that HCC patients have increased circulating IgG1, IgG3, IgA1, and IgM compared to healthy controls; comparison of HCC and CIR patients shows that HCC patients have significantly higher concentration of IgG1 and IgM but lower concentration of IgG2. An increase in galactose-deficient core fucosylated glycoforms was consistently observed in CIR and HCC patients. The FA2G0 and FA2BG0 glycoforms increase approximately 2-fold in all IgG subclasses accompanied by a decrease in the FA2G2 glycoform. Fucosylation changes are less pronounced but we have detected increased degree of fucosylation in the IgG1 and IgG3 glycoforms. In conclusion, we have optimized a sensitive and selective LC-MS-MRM method for the quantification of immunoglobulin subclasses and their site specific glycoforms, demonstrating that both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC. We have demonstrated that both quantities and glycoforms of immunoglobulin subclasses change significantly in liver disease progression to HCC through quantitative study of immunoglobulin subclasses and their site specific glycoforms using a sensitive and selective LC-MS-MRM method. Redistribution of the glycoforms of specific immunoglobulin subclasses could have important implications for receptor mediated responses affecting the progression of liver disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Adolescent Attitudes toward Influenza Vaccination and Vaccine Uptake in a School-Based Influenza Vaccination Intervention: A Mediation Analysis

ERIC Educational Resources Information Center

Painter, Julia E.; Sales, Jessica M.; Pazol, Karen; Wingood, Gina M.; Windle, Michael; Orenstein, Walter A.; DiClemente, Ralph J.

2011-01-01

Background: School-based vaccination programs may provide an effective strategy to immunize adolescents against influenza. This study examined whether adolescent attitudes toward influenza vaccination mediated the relationship between receipt of a school-based influenza vaccination intervention and vaccine uptake. Methods: Participants were…

Intracellular and transdermal protein delivery mediated by non-covalent interactions with a synthetic guanidine-rich molecular carrier.

PubMed

Im, Jungkyun; Das, Sanket; Jeong, Dongjun; Kim, Chang-Jin; Lim, Hyun-Suk; Kim, Ki Hean; Chung, Sung-Kee

2017-08-07

The impermeability of the cell plasma membrane is one of the major barriers for protein transduction into mammalian cells, and it also limits the use of proteins as therapeutic agents. Protein transduction has usually been achieved based on certain invasive processes or cell penetrating peptides (CPP). Herein we report our study in which a synthetic guanidine-rich molecular carrier is used as a delivery vector for intracellular and transdermal delivery of proteins. First a sorbitol-based molecular carrier having 8 guanidine units (Sor-G8) was synthesized, and then was simply mixed with a cargo protein of varying sizes to form the non-covalent complex of carrier-cargo proteins. These ionic complexes were shown to have efficient cellular uptake properties. The optimum conditions including the molar ratio between cargo protein and carrier, and the treatment time have been defined. Several protein cargoes were successfully examined with differing sizes and molecular weights: green fluorescent protein (MW 27kDa), albumin (66kDa), concanavalin A (102kDa), and immunoglobulin G (150kDa). These non-covalent complexes were also found to have excellent transdermal penetration ability into the mouse skin. The skin penetration depth was studied histologically by light microscopy as well as two-photon microscopy thus generating a depth profile. These complexes were largely found in the epidermis and dermis layers, i.e. down to ca. 100μm depth of the mouse skin. Our synthetic Sor-G8 carrier was found to be substantially more efficient that Arg8 in both the intracellular transduction and the transdermal delivery of proteins. The mechanism of the cellular uptake of the complex was briefly studied, and the results suggested macropinocytosis. Copyright © 2017 Elsevier B.V. All rights reserved.

CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

PubMed

Paes, Marcia C; Silveira, Alan B; Ventura-Martins, Guilherme; Luciano, Monalisa; Coelho, Marsen G P; Todeschini, Adriane R; Bianconi, M Lucia; Atella, Georgia C; Silva-Neto, Mário A C

2015-10-01

Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation. © 2015 Wiley Periodicals, Inc.

Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo

PubMed Central

Quinn, Stephen J.; Thomsen, Alex R. B.; Pang, Jian L.; Kantham, Lakshmi; Bräuner-Osborne, Hans; Pollak, Martin; Goltzman, David

2013-01-01

Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calcium's role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR; PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] despite no change in FGF23, suggesting direct regulation of 1,25(OH)2D3 synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dl. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium × phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full-length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium × phosphorus product remains within a physiological range. PMID:23233539

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

PubMed Central

Mandal, Amritlal; Delamere, Nicholas A.

2010-01-01

Purpose. Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90RSK and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation. Methods. Cytoplasmic calcium concentration ([Ca2+]i) was measured in cultured rat optic nerve astrocytes loaded with fura-2. The cells were placed in a closed imaging chamber and subjected to an HP increase of 15 mm Hg. Protein phosphorylation was detected by Western blot analysis. Results. The increase of HP caused an immediate slow increase in [Ca2+]i. The response persisted in calcium-free solution and when nickel chloride (4 mM) was added to suppress channel-mediated calcium entry. Previous depletion of the ER calcium stores by cyclopiazonic acid abolished the HP-induced calcium level increase. The HP-induced increase persisted in cells exposed to xestospongin C, an inhibitor of IP3R-mediated calcium release. In contrast, ryanodine receptor (RyR) antagonist ruthenium red (10 μM) or dantrolene (25 μM) inhibited the HP-induced calcium increase. The HP-induced calcium increase was abolished when ryanodine-sensitive calcium stores were pre-depleted with caffeine (3 mM). HP caused ERK1/2 phosphorylation. The magnitude of the ERK1/2 phosphorylation response was reduced by ruthenium red and dantrolene. Conclusions. Increasing HP causes calcium release from a ryanodine-sensitive cytoplasmic store and subsequent ERK1/2 activation. Calcium store release appears to be a required early step in the initial astrocyte response to an HP increase. PMID:20071675

The cation transporters rOCT1 and rOCT2 interact with bicarbonate but play only a minor role for amantadine uptake into rat renal proximal tubules.

PubMed

Goralski, Kerry B; Lou, Ganlu; Prowse, Matthew T; Gorboulev, Valentin; Volk, Christopher; Koepsell, Hermann; Sitar, Daniel S

2002-12-01

In renal proximal tubules, the organic cation transporters rOCT1 and rOCT2 are supposed to mediate the first step in organic cation secretion. We investigated whether previously described differences in amantadine and tetraethylammonium (TEA) uptake into isolated renal proximal tubules could be explained by differences in their transport by rOCT1 and rOCT2. By expressing rOCT1 and rOCT2 in Xenopus oocytes and HEK 293 cells, we demonstrated that both transporters translocated amantadine. In Xenopus oocytes, the inhibitory potency of several rOCT1/2 inhibitors was similar for amantadine compared to TEA uptake and supports amantadine transport by rOCT1 and rOCT2. In proximal tubules, procainamide, quinine, cyanine(863), choline, and guanidine in concentrations that inhibit rOCT1/2-mediated TEA or amantadine uptake in Xenopus oocytes exhibited no effect on amantadine uptake. At variance, these inhibitors blocked TEA uptake into proximal tubules. Amantadine and TEA transport were sensitive to modulation by 25 mM bicarbonate. The effect of bicarbonate on organic cation transport was dependent on substrate (amantadine or TEA), cell system (oocytes, HEK 293 cells, or proximal tubules), and transporter (rOCT1 or rOCT2). In proximal tubules, only amantadine uptake was stimulated by bicarbonate. The data suggested that rat renal proximal tubules contain an organic cation transporter in addition to rOCT1 and rOCT2 that mediates amantadine uptake and requires bicarbonate for optimal function. TEA uptake by the basolateral membrane may be mediated mainly by rOCT1 and rOCT2, but these transporters may be in a different functional or regulatory state when expressed in cells or oocytes compared with expression in vivo.

Lipopolysaccharide-Specific but Not Anti-Flagellar Immunoglobulin A Monoclonal Antibodies Prevent Salmonella enterica Serotype Enteritidis Invasion and Replication within HEp-2 Cell Monolayers

PubMed Central

Iankov, Ianko D.; Petrov, Dragomir P.; Mladenov, Ivan V.; Haralambieva, Iana H.; Mitov, Ivan G.

2002-01-01

The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope of Salmonella lipopolysaccharide antigen alone prevented S. enterica serotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms. PMID:11854252

Mutations, kataegis, and translocations in B lymphocytes: towards a mechanistic understanding of AID promiscuous activity

PubMed Central

Casellas, Rafael; Basu, Uttiya; Yewdell, William T.; Chaudhuri, Jayanta; Robbiani, Davide F.; Di Noia, Javier M.

2016-01-01

As B cells engage in the immune response they express the deaminase AID to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens, However, AID must be tightly controlled in B cells to minimize off-targeting mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the crucial question of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity. PMID:26898111

Calcium-Mediated Apoptosis and Apoptotic Sensitization in Prostate Cancer

DTIC Science & Technology

2004-06-01

calcium- sensitive protease calpain, stimulating two distinct pathways that regulate phosphotyrosine-initiated cell signaling ( PTP1B ) or directly...trigger apoptosis (caspase 7). The role of caspase 7 and PTP1B in PC cell death and survival signaling was investigated using dominant negatives, siRNA...of a calpain-proteolyzed variant of PTP1B (tPTP1B) had minimal impact on growth-factor or cytokine-mediated tyrosine phosphorylation or cell

Calcium carbonate mineralization mediated by in vitro cultured mantle cells from Pinctada fucata.

PubMed

Kong, Wei; Li, Shiguo; Xiang, Liang; Xie, Liping; Zhang, Rongqing

2015-08-07

Formation of the molluscan shell is believed to be an extracellular event mediated by matrix proteins. We report calcium carbonate mineralization mediated by Pinctada fucata mantle cells. Crystals only appeared when mantle cells were present in the crystallization solution. These crystals were piled up in highly ordered units and showed the typical characteristics of biomineralization products. A thin organic framework was observed after dissolving the crystals in EDTA. Some crystals had etched surfaces with a much smoother appearance than other parts. Mantle cells were observed to be attached to some of these smooth surfaces. These results suggest that mantle cells may be directly involved in the nucleation and remodeling process of calcium carbonate mineralization. Our result demonstrate the practicability of studying the mantle cell mechanism of biomineralization and contribute to the overall understanding of the shell formation process. Copyright © 2015 Elsevier Inc. All rights reserved.

Uptake of Silicon by Sugarcane from Applied Sources May Not Reflect Plant-Available Soil Silicon and Total Silicon Content of Sources.

PubMed

Keeping, Malcolm G

2017-01-01

Soils of the tropics and sub-tropics are typically acid and depleted of soluble sources of silicon (Si) due to weathering and leaching associated with high rainfall and temperatures. Together with intensive cropping, this leads to marginal or deficient plant Si levels in Si-accumulating crops such as rice and sugarcane. Although such deficiencies can be corrected with exogenous application of Si sources, there is controversy over the effectiveness of sources in relation to their total Si content, and their capacity to raise soil and plant Si concentrations. This study tested the hypothesis that the total Si content and provision of plant-available Si from six sources directly affects subsequent plant Si uptake as reflected in leaf Si concentration. Two trials with potted cane plants were established with the following Si sources as treatments: calcium silicate slag, fused magnesium (thermo) phosphate, volcanic rock dust, magnesium silicate, and granular potassium silicate. Silicon sources were applied at rates intended to achieve equivalent elemental soil Si concentrations; controls were untreated or lime-treated. Analyses were conducted to determine soil and leaf elemental concentrations. Among the sources, calcium silicate produced the highest leaf Si concentrations, yet lower plant-available soil Si concentrations than the thermophosphate. The latter, with slightly higher total Si than the slag, produced substantially greater increases in soil Si than all other products, yet did not significantly raise leaf Si above the controls. All other sources did not significantly increase soil or leaf Si concentrations, despite their high Si content. Hence, the total Si content of sources does not necessarily concur with a product's provision of soluble soil Si and subsequent plant uptake. Furthermore, even where soil pH was raised, plant uptake from thermophosphate was well below expectation, possibly due to its limited liming capacity. The ability of the calcium silicate to provide Si while simultaneously and significantly increasing soil pH, and thereby reducing reaction of Si with exchangeable Al 3+ , is proposed as a potential explanation for the greater Si uptake into the shoot from this source.

Uptake of Silicon by Sugarcane from Applied Sources May Not Reflect Plant-Available Soil Silicon and Total Silicon Content of Sources

PubMed Central

Keeping, Malcolm G.

2017-01-01

Soils of the tropics and sub-tropics are typically acid and depleted of soluble sources of silicon (Si) due to weathering and leaching associated with high rainfall and temperatures. Together with intensive cropping, this leads to marginal or deficient plant Si levels in Si-accumulating crops such as rice and sugarcane. Although such deficiencies can be corrected with exogenous application of Si sources, there is controversy over the effectiveness of sources in relation to their total Si content, and their capacity to raise soil and plant Si concentrations. This study tested the hypothesis that the total Si content and provision of plant-available Si from six sources directly affects subsequent plant Si uptake as reflected in leaf Si concentration. Two trials with potted cane plants were established with the following Si sources as treatments: calcium silicate slag, fused magnesium (thermo) phosphate, volcanic rock dust, magnesium silicate, and granular potassium silicate. Silicon sources were applied at rates intended to achieve equivalent elemental soil Si concentrations; controls were untreated or lime-treated. Analyses were conducted to determine soil and leaf elemental concentrations. Among the sources, calcium silicate produced the highest leaf Si concentrations, yet lower plant-available soil Si concentrations than the thermophosphate. The latter, with slightly higher total Si than the slag, produced substantially greater increases in soil Si than all other products, yet did not significantly raise leaf Si above the controls. All other sources did not significantly increase soil or leaf Si concentrations, despite their high Si content. Hence, the total Si content of sources does not necessarily concur with a product's provision of soluble soil Si and subsequent plant uptake. Furthermore, even where soil pH was raised, plant uptake from thermophosphate was well below expectation, possibly due to its limited liming capacity. The ability of the calcium silicate to provide Si while simultaneously and significantly increasing soil pH, and thereby reducing reaction of Si with exchangeable Al3+, is proposed as a potential explanation for the greater Si uptake into the shoot from this source. PMID:28555144

Treatment of cashew extracts with Aspergillopepsin reduces IgE binding to cashew allergens

USDA-ARS?s Scientific Manuscript database

Cashew nuts can cause serious and sometimes life threatening reactions in people that suffer from food allergies. These reactions are mediated by immunoglobulin E binding (IgE) to allergenic cashew proteins. Enzymes from Aspergillus fungal species are used in many industrial and pharmaceutical appli...

AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway.

PubMed

Li, Xiao C; Hopfer, Ulrich; Zhuo, Jia L

2009-11-01

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.

Protease-mediated Inflammation: An In Vitro Human Keratinocyte-based Screening Tool for Anti-inflammatory Drug Nanocarrier Systems

NASA Astrophysics Data System (ADS)

Frombach, Janna; Lohan, Silke B.; Lemm, Davina; Gruner, Paul; Hasler, Julia; Ahlberg, Sebastian; Blume-Peytavi, Ulrike; Unbehauen, Michael; Haag, Rainer; Meinke, Martina C.; Vogt, Annika

2018-05-01

Refined encapsulation approaches in dermatotherapy gain increased interest. There is need of reproducible in vitro systems representing disease features to screen drug delivery systems for preclinical assessment. Inflammatory human skin diseases are commonly accompanied by abnormal epidermal differentiation and barrier impairment. Serine proteases (SPs) and their inhibitors play a critical role in such dysfunctional differentiation. SPs also initiate cellular pathways via activation of protease-activated receptors, which contribute to inflammation. Thus, function and activity of SPs should be considered for the design of new therapies of such disorders. Herein, we established a novel simplified cell culture model, based on SP-mediated inflammation suitable to assess nanocarriers loaded with anti-inflammatory drugs. SP-mediated inflammation and the regulatory effect of free or encapsulated dexamethasone were determined by measuring interleukin-6 and interleukin-8 in culture medium of HaCaT (human adult low calcium temperature)-keratinocytes. Additionally, radical formation was analyzed by electron paramagnetic resonance spectroscopy. Cellular uptake of core-multishell nanocarriers was investigated by fluorescence microscopy. Cytotoxicity of all additives was determined by a viability assay. SP-Stimulation of keratinocytes resulted in increased radical production and release of inflammatory cytokines without affecting cell viability. Induced inflammation was successfully downregulated by addition of free or encapsulated dexamethasone. SP-addition can be used as inflammatory stimulus in cell culture to mimic effects of aberrant enzymatic activities found in skin of atopic dermatitis patients. The set-up is appropriate as a preliminary test to examine the effectiveness of new molecules or delivery-systems to counteract serine protease-mediated inflammatory processes prior to skin studies.

Pre-incubation with cyclosporine A potentiates its inhibitory effects on pitavastatin uptake mediated by recombinantly expressed cynomolgus monkey hepatic organic anion transporting polypeptide.

PubMed

Takahashi, Tsuyoshi; Ohtsuka, Tatsuyuki; Uno, Yasuhiro; Utoh, Masahiro; Yamazaki, Hiroshi; Kume, Toshiyuki

2016-11-01

Cyclosporine A, an inhibitor of hepatic organic anion transporting polypeptides (OATPs), reportedly increased plasma concentrations of probe substrates, although its maximum unbound blood concentrations were lower than the experimental half-maximal inhibitory (IC 50 ) concentrations. Pre-incubation with cyclosporine A in vitro before simultaneous incubation with probes has been reported to potentiate its inhibitory effects on recombinant human OATP-mediated probe uptake. In the present study, the effects of cyclosporine A and rifampicin on recombinant cynomolgus monkey OATP-mediated pitavastatin uptake were investigated in pre- and simultaneous incubation systems. Pre-incubation with cyclosporine A, but not with rifampicin, decreased the apparent IC 50 values on recombinant cynomolgus monkey OATP1B1- and OATP1B3-mediated pitavastatin uptake. Application of the co-incubated IC 50 values toward R values (1 + [unbound inhibitor] inlet to the liver, theoretically maximum /inhibition constant) in static models, 1.1 in monkeys and 1.3 in humans, for recombinant cynomolgus monkey and human OATP1B1-mediated pitavastatin uptake might result in the poor prediction of drug interaction magnitudes. In contrast, the lowered IC 50 values after pre-incubation with cyclosporine A provided better prediction with R values of 3.9 for monkeys and 2.7 for humans when the estimated maximum cyclosporine A concentrations at the inlet to the liver were used. These results suggest that the enhanced inhibitory potential of perpetrator medicines by pre-incubation on cynomolgus monkey OATP-mediated pitavastatin uptake in vitro could be of value for the precise estimation of drug interaction magnitudes in silico, in accordance with the findings from pre-administration of inhibitors on pitavastatin pharmacokinetics validated in monkeys. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation.

PubMed

An, Dong; Hao, Feng; Hu, Chen; Kong, Wei; Xu, Xuemin; Cui, Mei-Zhen

2017-01-01

Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.

PubMed

Fulcher, F Kent; Smith, Bethany T; Russ, Misty; Patel, Yashomati M

2008-10-15

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty

2008-10-15

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform viamore » MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.« less

Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecay, T.W.; Valentich, J.D.

1991-03-01

Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of {sup 3}H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases inmore » inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.« less

A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+/cation transport in yeast

PubMed Central

Yadav, Akhilesh K.; Shankar, Alka; Jha, Saroj K.; Kanwar, Poonam; Pandey, Amita; Pandey, Girdhar K.

2015-01-01

In plant cell, cations gradient in cellular compartments is maintained by synergistic action of various exchangers, pumps and channels. The Arabidopsis exchanger family members (AtCCX3 and AtCCX5) were previously studied and belong to CaCA (calcium cation exchangers) superfamily while none of the rice CCXs has been functionally characterized for their cation transport activities till date. Rice genome encode four CCXs and only OsCCX2 transcript showed differential expression under abiotic stresses and Ca2+ starvation conditions. The OsCCX2 localized to tonoplast and suppresses the Ca2+ sensitivity of K667 (low affinity Ca2+ uptake deficient) yeast mutant under excess CaCl2 conditions. In contrast to AtCCXs, OsCCX2 expressing K667 yeast cells show tolerance towards excess Na+, Li+, Fe2+, Zn2+ and Co2+ and suggest its ability to transport both mono as well as divalent cations in yeast. Additionally, in contrast to previously characterized AtCCXs, OsCCX2 is unable to complement yeast trk1trk2 double mutant suggesting inability to transport K+ in yeast system. These finding suggest that OsCCX2 having distinct metal transport properties than previously characterized plant CCXs. OsCCX2 can be used as potential candidate for enhancing the abiotic stress tolerance in plants as well as for phytoremediation of heavy metal polluted soil. PMID:26607171

Ascorbic Acid Efflux and Re-uptake in Endothelial Cells: Maintenance of Intracellular Ascorbate

PubMed Central

May, James M.; Qu, Zhi-chao

2013-01-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70–80% of ascorbate to the medium over several hours at 37 °C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel. PMID:19148707

Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate.

PubMed

May, James M; Qu, Zhi-chao

2009-05-01

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

Effect of treatment with depot somatostatin analogue octreotide on primary hyperparathyroidism (PHP) in multiple endocrine neoplasia type 1 (MEN1) patients.

PubMed

Faggiano, Antongiulio; Tavares, Lidice Brandao; Tauchmanova, Libuse; Milone, Francesco; Mansueto, Gelsomina; Ramundo, Valeria; De Caro, Maria Laura Del Basso; Lombardi, Gaetano; De Rosa, Gaetano; Colao, Annamaria

2008-11-01

In patients with multiple endocrine neoplasia type 1 (MEN1), expression of somatostatin receptor (SST) in parathyroid adenomas and effectiveness of therapy with somatostatin analogues on primary hyperparathyroidism (PHP) have been scarcely investigated. To evaluate the effects of depot long acting octreotide (OCT-LAR) in patients with MEN1-related PHP. Eight patients with a genetically confirmed MEN1, presenting both PHP and duodeno-pancreatic neuroendocrine tumours (NET), were enrolled. The initial treatment was OCT-LAR 30 mg every 4 weeks. This therapy was established to stabilize the duodeno-pancreatic NET before to perform parathyroidectomy for PHP. Before OCT-LAR therapy, a SST scintigraphy was performed in all patients. SST subtype 2A immunohistochemistry was performed on parathyroid tumour samples from three patients undergone parathyroidectomy after OCT-LAR therapy. Serum concentrations of PTH, calcium and phosphorus as well as the 24-h urine calcium : creatinine ratio and the renal threshold phosphate concentration were evaluated before and after OCT-LAR. After OCT-LAR therapy, hypercalcaemia and hypercalciuria normalized in 75% and 62.5% of patients, respectively, and serum phosphorus and renal threshold phosphate significantly increased. Serum PTH concentrations significantly decreased in all patients and normalized in two of them. SST subtype 2A immunostaining was found in all parathyroid adenomas investigated, while SST scintigraphy showed a positive parathyroid tumour uptake in three of eight patients (37.5%). Six months of OCT-LAR therapy controlled hypercalcaemia and hypercalciuria in two-thirds of patients with MEN1-related PHP. Direct OCT-LAR effects mediated by binding to SST expression on parathyroid tumour cells are likely the main mechanism to explain the activity of this compound on calcium and phosphorus abnormalities in MEN1 PHP.

Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes.

PubMed

Callamaras, N; Sun, X P; Ivorra, I; Parker, I

1998-09-01

1. The mechanisms underlying hemispheric asymmetry of the inositol 1, 4,5-trisphosphate (InsP3)-calcium signalling pathway in Xenopus oocytes were examined by fluorescence imaging of calcium signals and recording calcium-activated Cl- currents (ICl,Ca) evoked by intracellular calcium injections and photorelease of InsP3. 2. The maximal ICl,Ca evoked by strong photorelease of InsP3 was 8 times greater in the animal than the vegetal hemisphere, but the average threshold amounts of InsP3 required to evoke detectable currents were similar in each hemisphere. 3. Currents evoked by injections of calcium were about 2.5 times greater near the animal pole than near the vegetal pole, whereas fluorescence signals evoked by injections were similar in each hemisphere. 4. Calcium waves were evoked by photolysis flashes of similar strengths in both hemispheres of albino oocytes, but peak calcium levels evoked by supramaximal stimuli were 70 % greater in the animal hemisphere. 5. Elementary calcium release events (puffs) in the animal hemisphere had amplitudes about double that in the vegetal hemisphere, and more often involved coupled release from adjacent sites. Calcium release sites were more closely packed in the animal hemisphere, with a mean spacing of about 1.5 micro m compared with 2.25 micro m in the vegetal hemisphere. 6. The larger amplitude of currents mediated by InsP3 in the animal hemisphere, therefore, involves an increased flux of calcium at individual release units, a more dense packing of release units and a higher density of Cl- channels.

Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes

PubMed Central

Callamaras, Nick; Sun, Xiao-Ping; Ivorra, Isabel; Parker, Ian

1998-01-01

The mechanisms underlying hemispheric asymmetry of the inositol 1,4,5-trisphosphate (InsP3)-calcium signalling pathway in Xenopus oocytes were examined by fluorescence imaging of calcium signals and recording calcium-activated Cl− currents (ICl,Ca) evoked by intracellular calcium injections and photorelease of InsP3. The maximal ICl,Ca evoked by strong photorelease of InsP3 was 8 times greater in the animal than the vegetal hemisphere, but the average threshold amounts of InsP3 required to evoke detectable currents were similar in each hemisphere. Currents evoked by injections of calcium were about 2.5 times greater near the animal pole than near the vegetal pole, whereas fluorescence signals evoked by injections were similar in each hemisphere. Calcium waves were evoked by photolysis flashes of similar strengths in both hemispheres of albino oocytes, but peak calcium levels evoked by supramaximal stimuli were 70% greater in the animal hemisphere. Elementary calcium release events (puffs) in the animal hemisphere had amplitudes about double that in the vegetal hemisphere, and more often involved coupled release from adjacent sites. Calcium release sites were more closely packed in the animal hemisphere, with a mean spacing of about 1.5 μm compared with 2.25 μm in the vegetal hemisphere. The larger amplitude of currents mediated by InsP3 in the animal hemisphere, therefore, involves an increased flux of calcium at individual release units, a more dense packing of release units and a higher density of Cl− channels. PMID:9706018

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

PubMed

Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

2013-08-01

Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cell-specific vacuolar calcium storage mediated by "CAX1" regulates apoplastic calcium concentration, gas exchange, and plant productivity in "Arabidopsis"

USDA-ARS?s Scientific Manuscript database

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from "Arabidopsis thaliana" leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen...

Including carrier-mediated transport in oral uptake prediction of nutrients and pharmaceuticals in humans.

PubMed

O'Connor, Isabel A; Veltman, Karin; Huijbregts, Mark A J; Ragas, Ad M J; Russel, Frans G M; Hendriks, A Jan

2014-11-01

Most toxicokinetic models consider passive diffusion as the only mechanism when modeling the oral uptake of chemicals. However, the overall uptake of nutrients and xenobiotics, such as pharmaceuticals and environmental pollutants, can be increased by influx transport proteins. We incorporated carrier-mediated transport into a one-compartment toxicokinetic model originally developed for passive diffusion only. The predictions were compared with measured oral uptake efficiencies of nutrients and pharmaceuticals, i.e. the fraction of the chemical reaching systemic circulation. Including carrier-mediated uptake improved model predictions for hydrophilic nutrients (RMSE=10% vs. 56%, Coefficient of Efficiency CoE=0.5 vs. -9.6) and for pharmaceuticals (RMSE=21% vs. 28% and CoE=-0.4 vs. -1.1). However, the negative CoE for pharmaceuticals indicates that further improvements are needed. Most important in this respect is a more accurate estimation of vMAX and KM as well as the determination of the amount of expressed and functional transport proteins both in vivo and in vitro. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of basal ganglia calcification on its glucose metabolism and dopaminergic function in idiopathic hypoparathyroidism.

PubMed

Modi, Sagar; Arora, Geetanjali; Bal, Chandra Shekhar; Sreenivas, Vishnubhatla; Kailash, Suparna; Sagar, Rajesh; Goswami, Ravinder

2015-10-01

The functional significance of basal ganglia calcification (BGC) in idiopathic hypoparathyroidism (IH) is not clear. To assess the effect of BGC on glucose metabolism and dopaminergic function in IH. (18) F-FDG and (99m) Tc-TRODAT-1 nuclear imaging were performed in 35 IH patients with (n = 26) and without (n = 9) BGC. Controls were subjects without hypoparathyroidism or BGC (nine for (18) F-FDG and 12 for (99m) Tc-TRODAT-1). Relationship of the glucose metabolism and dopaminergic function was assessed with the neuropsychological and biochemical abnormalities. (18) F-FDG uptake in IH patients with calcification at caudate and striatum was less than that of IH patients without calcification (1·06 ± 0·13 vs 1·24 ± 0·09, P = <0·0001 and 1·06 ± 0·09 vs 1·14 ± 0·08, P = 0·03, respectively). (18) F-FDG uptake did not correlate with neuropsychological dysfunctions. (18) F-FDG uptake in IH without BGC was significantly lower than that of controls. The mean (99m) Tc-TRODAT-1 uptake at basal ganglia was comparable between IH with and without BGC and between IH without BGC and controls. Serum calcium-phosphorus ratio maintained by the patients correlated with (18) F-FDG uptake at striatum (r = 0·57, P = 0·001). For every 0·1 unit reduction in calcium-phosphorus ratio, (18) F-FDG uptake decreased by 2·5 ± 0·68% (P = 0·001). BGC was associated with modest reduction (15%) in (18) F-FDG uptake at basal ganglia in IH but did not affect dopaminergic function. (18) F-FDG uptake did not correlate with neuropsychological dysfunctions. Interestingly, chronic hypocalcaemia-hyperphosphataemia also contributed to reduction in (18) F-FDG uptake which was independent of BGC. © 2014 John Wiley & Sons Ltd.

Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

PubMed

Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

2015-07-01

In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

DTIC Science & Technology

2000-03-31

touted as inhibitors of the PMCAs, but their effects have yet to be thoroughly examined on all P-type ATPases [23, 125]. SERCAs: a family ofP·type ATPases... effective in reducing the sensitivity to TG by 1aOO-fold [151]. The potent, seledive blockade of Ca2+ accumulation in the ER by TG has made it possible...accumulation while the calcium ionophore A23187 is 100%) effective [140]. Thus, 10-150/0 of net Ca2+ uptake into brain microsomal compartments is

Formation of Fe(III) oxyhydroxide colloids in freshwater and brackish seawater, with incorporation of phosphate and calcium

NASA Astrophysics Data System (ADS)

Gunnars, Anneli; Blomqvist, Sven; Johansson, Peter; Andersson, Christian

2002-03-01

The formation of Fe(III) oxyhydroxide colloids by oxidation of Fe(II) and their subsequent aggregation to larger particles were studied in laboratory experiments with natural water from a freshwater lake and a brackish coastal sea. Phosphate was incorporated in the solid phase during the course of hydrolysis of iron. The resulting precipitated amorphous Fe(III) oxyhydroxide phases were of varying composition, depending primarily on the initial dissolved Fe/P molar ratio, but with little influence by salinity or concentration of calcium ions. The lower limiting Fe/P ratio found for the solid phase suggests the formation of a basic Fe(III) phosphate compound with a stoichiometric Fe/P ratio of close to two. This implies that an Fe/P stoichiometry of ≈2 ultimately limits the capacity of precipitating Fe(III) to fix dissolved phosphate at oxic/anoxic boundaries in natural waters. In contrast to phosphorus, the uptake of calcium seemed to be controlled by sorption processes at the surface of the iron-rich particles formed. This uptake was more efficient in freshwater than in brackish water, suggesting that salinity restrains the uptake of calcium by newly formed Fe(III) oxyhydroxides in natural waters. Moreover, salinity enhanced the aggregation rate of the colloids formed. The suspensions were stabilised by the presence of organic matter, although this effect was less pronounced in seawater than in freshwater. Thus, in seawater of 6 to 33 ‰S, the removal of particles was fast (removal half time < 200 h), whereas the colloidal suspensions formed in freshwater were stable (removal half time > 900 h). Overall, oxidation of Fe(II) and removal of Fe(III) oxyhydroxide particles were much faster in seawater than in freshwater. This more rapid turnover results in lower iron availability in coastal seawater than in freshwater, making iron more likely to become a limiting element for chemical scavenging and biologic production.

Decreased contraction induced by endothelium-derived contracting factor in prolonged treatment of rat renal artery with endoplasmic reticulum stress inducer.

PubMed

Ando, Makoto; Matsumoto, Takayuki; Taguchi, Kumiko; Kobayashi, Tsuneo

2018-05-04

Recent evidence suggests that endoplasmic reticulum (ER) stress is involved in the regulation of various physiological functions, including those of the vascular system. However, the relationship between ER stress and vascular function is poorly understood. The endothelial cells control the vascular tone by releasing endothelium-derived relaxing factors and contracting factors (EDCFs). We hypothesized that tunicamycin, an inducer of ER stress, modifies endothelium-dependent contraction and prostaglandins (PGs), a major class of EDCFs, induced contractions in the rat renal artery in rats. An organ-culture technique was used to purely investigate the effects of ER stress on the vascular tissue. We observed that tunicamycin treatment (20 μg/mL for 23 ± 1 h) did not affect acetylcholine (ACh)-induced relaxation and decreased EDCF-mediated contractions under nitric oxide synthase (NOS) inhibition induced by ACh, ATP, or A23187 (a calcium ionophore) in the renal arteries. Under NOS inhibition, U46619 (a thromboxane A 2 mimetic)- and beraprost (a prostacyclin analog)-induced contractions were also decreased in the renal arteries of the tunicamycin-treated group (vs. vehicle), while PGE 2 - and PGF 2α -induced contractions were similar between the groups. Tunicamycin treatment slightly enhanced the contractions induced by phenylephrine, an α 1 adrenoceptor ligand. Isotonic high-K + -induced contractions were similar between the vehicle- and tunicamycin-treated groups. Another ER stress inducer, thapsigargin (4 μmol/L for 23 ± 1 h), also caused substantial reduction of ACh-induced EDCF-mediated contraction (vs. vehicle-treated group). In the cultured renal arteries, tunicamycin and thapsigargin increased the expression of binding immunoglobulin protein (BiP), an ER stress marker. In conclusion, ER stress induction directly affects renal arterial function, especially in reducing EDCF-mediated contractions.

Vasotropic light-chain amyloidosis and ischaemic cholangiopathy.

PubMed

Johnston, Emma L; Wilkinson, Mark; Knisely, A S

2015-06-25

A 75-year-old woman was incidentally found to have deranged liver function tests (LFTs). She was well, apart from 2 years of dyspnoea. Investigations had revealed atrial fibrillation and a right pleural effusion, without identified aetiology. On examination, the only finding was a palpable liver edge. Initial blood and ultrasound screening suggested no cause. The patient underwent liver biopsy. Microscopy showed κ-immunoglobulin light chains deposited exclusively in portal tracts, within blood vessel and bile duct walls. This pattern, although unusual, raised the possibility of κ-light chain disease. Serum electrophoresis was normal, as were serum immunoglobulin values. Serum concentrations of κ-light chains were elevated and microscopy of aspirated bone marrow found light-chain deposits with 10% plasmacytosis. Serum amyloid P (SAP) scintigraphy demonstrated splenic uptake. Myeloma, κ-light chain, with light-chain amyloidosis was diagnosed. The patient has responded well to cyclophosphamide, bortazomib and dexamethasone chemotherapy, and her LFTs are now nearly normal. 2015 BMJ Publishing Group Ltd.

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

PubMed Central

Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

2000-01-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages. PMID:10864653

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

PubMed

Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

2000-07-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

Rituximab for Treatment of Membranoproliferative Glomerulonephritis and C3 Glomerulopathies

PubMed Central

2017-01-01

Membranoproliferative glomerulonephritis (MPGN) is a histological pattern of injury resulting from predominantly subendothelial and mesangial deposition of immunoglobulins or complement factors with subsequent inflammation and proliferation particularly of the glomerular basement membrane. Recent classification of MPGN is based on pathogenesis dividing MPGN into immunoglobulin-associated MPGN and complement-mediated C3 glomerulonephritis (C3GN) and dense deposit disease (DDD). Current guidelines suggest treatment with steroids, cytotoxic agents with or without plasmapheresis only for subjects with progressive disease, that is, nephrotic range proteinuria and decline of renal function. Rituximab, a chimeric B-cell depleting anti-CD20 antibody, has emerged in the last decade as a treatment option for patients with primary glomerular diseases such as minimal change disease, focal-segmental glomerulosclerosis, or idiopathic membranous nephropathy. However, data on the use of rituximab in MPGN, C3GN, and DDD are limited to case reports and retrospective case series. Patients with immunoglobulin-associated and idiopathic MPGN who were treated with rituximab showed partial and complete responses in the majorities of cases. However, rituximab was not effective in few cases of C3GN and DDD. Despite promising results in immunoglobulin-associated and idiopathic MPGN, current evidence on this treatment remains weak, and controlled and prospective data are urgently needed. PMID:28573137

Regulation of cardiomyocyte autophagy by calcium

PubMed Central

Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F. G.; Hill, Joseph A.

2016-01-01

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. PMID:26884385

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells

PubMed Central

Azizi, Paymon M.; Zyla, Roman E.; Guan, Sha; Wang, Changsen; Liu, Jun; Bolz, Steffen-Sebastian; Heit, Bryan; Klip, Amira; Lee, Warren L.

2015-01-01

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel–derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent. PMID:25540431

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

PubMed

Miyanokoshi, Miki; Yokosawa, Takumi; Wakasugi, Keisuke

2018-06-01

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Adult Sexual Experiences as a Mediator Between Child Abuse and Current Secretory Immunoglobulin A Levels.

PubMed

Waldron, Jonathan C; Scarpa, Angela; Kim-Spoon, Jungmeen; Coe, Christopher L

2016-03-01

The current study investigated whether a history of child abuse is a predictor of adult immune status, with unwanted adult sexual experiences as a proximal mediator. Participants included 89 young adult women (M(age) = 19.24) who were classified as having experienced no child abuse, child physical abuse, or child sexual abuse, based upon self-reported victimization history before 14 years of age. Participants also reported on unwanted sexual experiences in young adulthood and provided four saliva samples, which were collected over two consecutive days to determine secretory immunoglobulin A (sIgA). Age and negative life events were considered as covariates. The results indicated that adult sexual victimization partially mediated the relationship between child abuse (physical and sexual) and sIgA. Specifically, child abuse experiences predicted more adult sexual victimization experiences, which in turn predicted lower sIgA levels. These findings support long-term health effects of victimization, and suggest that the influence of child abuse on sIgA may be perpetuated through adult victimization. Prevention efforts should aim to empower child maltreatment survivors with skills to prevent adult re-victimization. By thwarting future unwanted sexual experiences in adulthood, individuals will be better protected from the health impairments associated with early abuse experiences. © The Author(s) 2014.

Differences in dissolved cadmium and zinc uptake among stream insects: Mechanistic explanations

USGS Publications Warehouse

Buchwalter, D.B.; Luoma, S.N.

2005-01-01

This study examined the extent to which dissolved Cd and Zn uptake rates vary in several aquatic insect taxa commonly used as indicators of ecological health. We further attempted to explain the mechanisms underlying observed differences. By comparing dissolved Cd and Zn uptake rates in several aquatic insect species, we demonstrated that species vary widely in these processes. Dissolved uptake rates were not related to gross morphological features such as body size or gill size-features that influence water permeability and therefore have ionoregulatory importance. However, finer morphological features, specifically, the relative numbers of ionoregulatory cells (chloride cells), appeared to be related to dissolved metal uptake rates. This observation was supported by Michaelis-Menten type kinetics experiments, which showed that dissolved Cd uptake rates were driven by the numbers of Cd transporters and not by the affinities of those transporters to Cd. Calcium concentrations in exposure media similarly affected Cd and Zn uptake rates in the caddisfly Hydropsyche californica. Dissolved Cd and Zn uptake rates strongly co-varied among species, suggesting that these metals are transported by similar mechanisms.

Antibody uptake into neurons occurs primarily via clathrin-dependent Fcγ receptor endocytosis and is a prerequisite for acute tau protein clearance.

PubMed

Congdon, Erin E; Gu, Jiaping; Sait, Hameetha B R; Sigurdsson, Einar M

2013-12-06

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r(2) = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤ 78%, p < 0.0001) and slices (≤ 35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.

Mini-dystrophin Expression Down-regulates Overactivation of G Protein–mediated IP3 Signaling Pathway in Dystrophin-deficient Muscle Cells

PubMed Central

Balghi, Haouaria; Sebille, Stéphane; Constantin, Bruno; Patri, Sylvie; Thoreau, Vincent; Mondin, Ludivine; Mok, Elise; Kitzis, Alain; Raymond, Guy; Cognard, Christian

2006-01-01

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(−) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(−) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(−) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(−) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling. PMID:16446505

Increased technetium-99 m hydroxy diphosphonate soft tissue uptake on bone scintigraphy in chronic kidney disease patients with secondary hyperparathyroidism: correlation with hyperphosphataemia.

PubMed

Enevoldsen, Lotte Hahn; Heaf, James; Højgaard, Liselotte; Zerahn, Bo; Hasbak, Philip

2017-03-01

In bone scan patients with dialysis-treated chronic kidney disease (CKD) and hyperparathyroidism, soft tissue accumulation of technetium-99 m hydroxy/methylene diphosphonate (Tc-99 m-HDP/MDP) has been reported primarily in case reports and usually explained by hypercalcaemia and/or hyperphosphataemia. As human vascular smooth muscle cells produce hydroxyapatite during cell culture with increased phosphate levels and as Tc-99 m-HDP/MDP primarily binds to hydroxyapatite, we hypothesized that soft tissue accumulation would be found in patients with hyperphosphataemia. We identified 63 CKD patients diagnosed with secondary hyperparathyroidism admitted for Tc-99 m-HDP bone scan. Baseline characteristics and mean concentrations of biochemical markers (including P-calcium and P-phosphate) taken 0-3 months prior to the bone scans were collected. Soft tissue uptake was detected on bone scans in 37 of 63 (59%) patients. Primary locations were in the heart (27/37 = 73%), muscles (12/37 = 32%), lung (9/37 = 24%) and gastrointestinal tract (6/37 = 16%), and 13 of 37 (35%) patients had simultaneous uptake in more than one location. Regarding biochemical markers, patients with soft tissue uptake only differed from patients without in terms of plasma phosphate levels (1·95 ± 0·15 (n = 37) versus 1·27 ± 0·08 (n = 26), P = 0·0012). All patients with myocardial uptake (n = 27) had a coronary arteriography-verified history of coronary artery disease (CAD), whereas CAD was only present in six of the 36 patients without myocardial uptake. In conclusion, dialysis-treated CKD patients with secondary hyperparathyroidism have a high incidence of soft tissue uptake, and this finding is strongly correlated with elevated phosphate, but not calcium values. © 2015 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

The effect of albumin on podocytes: The role of the fatty acid moiety and the potential role of CD36 scavenger receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pawluczyk, I.Z.A., E-mail: izap1@le.ac.uk; John Walls Renal Unit, Leicester General Hospital Leicester; Pervez, A.

Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified. Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA{sub +FA}) or depleted of them (HSA{sub −FA}). Receptor-mediated endocytosis of FITC-HSA{sub +FA} over 60 min was 5 times greater than that of FITC-HSA{sub −FA}. 24 h exposure of podocytes to albuminmore » up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA{sub −FA.} Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake. These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin. - Highlights: • The fatty acid moiety is essential for receptor mediated endocytosis of albumin. • Fatty acid depleted albumin is more pathogenic to podocytes. • CD36 is not directly involved in albumin uptake by podocytes.« less

Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwartz, Justin; Holmuhamedov, Ekhson; Zhang, Xun

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50 μM of each tetracycline-derived compound 20 min prior to exposure to 500 μM iodoacetic acid plus 1 mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs–Ringer–HEPES buffer at pH 6.2 for 4 h prior to reoxygenation at pH 7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca{sup 2+} uptake wasmore » measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca{sup 2+} uptake and suppressed the Ca{sup 2+}-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca{sup 2+} uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU. - Highlights: • Minocycline and doxycycline are the only cytoprotective tetracyclines of those tested • Cytoprotective tetracyclines inhibit the MPT and mitochondrial calcium and iron uptake. • Cytoprotective tetracyclines protect by inhibiting the MCU.« less

Nitrate Utilization by the Diatom Skeletonema costatum

PubMed Central

Serra, Juan L.; Llama, Maria J.; Cadenas, Eduardo

1978-01-01

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased. PMID:16660652

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

PubMed

Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2012-04-15

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together, these results suggest that L-FABP, particularly in the absence of SCP-2, plays a significant role in HDL-mediated cholesterol uptake in cultured primary hepatocytes.

Signaling complexes of voltage-gated calcium channels

PubMed Central

Turner, Ray W; Anderson, Dustin

2011-01-01

Voltage-gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage-gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead form complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily. PMID:21832880

Apoptosis-mediated endothelial toxicity but not direct calcification or functional changes in anti-calcification proteins defines pathogenic effects of calcium phosphate bions

NASA Astrophysics Data System (ADS)

Kutikhin, Anton G.; Velikanova, Elena A.; Mukhamadiyarov, Rinat A.; Glushkova, Tatiana V.; Borisov, Vadim V.; Matveeva, Vera G.; Antonova, Larisa V.; Filip'Ev, Dmitriy E.; Golovkin, Alexey S.; Shishkova, Daria K.; Burago, Andrey Yu.; Frolov, Alexey V.; Dolgov, Viktor Yu.; Efimova, Olga S.; Popova, Anna N.; Malysheva, Valentina Yu.; Vladimirov, Alexandr A.; Sozinov, Sergey A.; Ismagilov, Zinfer R.; Russakov, Dmitriy M.; Lomzov, Alexander A.; Pyshnyi, Dmitriy V.; Gutakovsky, Anton K.; Zhivodkov, Yuriy A.; Demidov, Evgeniy A.; Peltek, Sergey E.; Dolganyuk, Viatcheslav F.; Babich, Olga O.; Grigoriev, Evgeniy V.; Brusina, Elena B.; Barbarash, Olga L.; Yuzhalin, Arseniy E.

2016-06-01

Calcium phosphate bions (CPB) are biomimetic mineralo-organic nanoparticles which represent a physiological mechanism regulating the function, transport and disposal of calcium and phosphorus in the human body. We hypothesised that CPB may be pathogenic entities and even a cause of cardiovascular calcification. Here we revealed that CPB isolated from calcified atherosclerotic plaques and artificially synthesised CPB are morphologically and chemically indistinguishable entities. Their formation is accelerated along with the increase in calcium salts-phosphates/serum concentration ratio. Experiments in vitro and in vivo showed that pathogenic effects of CPB are defined by apoptosis-mediated endothelial toxicity but not by direct tissue calcification or functional changes in anti-calcification proteins. Since the factors underlying the formation of CPB and their pathogenic mechanism closely resemble those responsible for atherosclerosis development, further research in this direction may help us to uncover triggers of this disease.

Single channel recording of a mitochondrial calcium uniporter.

PubMed

Wu, Guangyan; Li, Shunjin; Zong, Guangning; Liu, Xiaofen; Fei, Shuang; Shen, Linda; Guan, Xiangchen; Yang, Xue; Shen, Yuequan

2018-01-29

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca 2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca 2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria. Copyright © 2018 Elsevier Inc. All rights reserved.

Neuronal uptake of anti-Hu antibody, but not anti-Ri antibody, leads to cell death in brain slice cultures.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Jaskowski, Troy D; Carlson, Noel G

2014-09-17

Anti-Hu and anti-Ri antibodies are paraneoplastic immunoglobulin (Ig)G autoantibodies which recognize cytoplasmic and nuclear antigens present in all neurons. Although both antibodies produce similar immunohistological labeling, they recognize different neuronal proteins. Both antibodies are associated with syndromes of central nervous system dysfunction. However, the neurological deficits associated with anti-Hu antibody are associated with neuronal death and are usually irreversible, whereas neurological deficits in patients with anti-Ri antibody may diminish following tumor removal or immunosuppression. To study the effect of anti-Hu and anti-Ri antibodies on neurons, we incubated rat hippocampal and cerebellar slice cultures with anti-Hu or anti-Ri sera from multiple patients. Cultures were evaluated in real time for neuronal antibody uptake and during prolonged incubation for neuronal death. To test the specificity of anti-Hu antibody cytotoxic effect, anti-Hu serum IgG was incubated with rat brain slice cultures prior to and after adsorption with its target Hu antigen, HuD. We demonstrated that: 1) both anti-Hu and anti-Ri antibodies were rapidly taken up by neurons throughout both cerebellum and hippocampus; 2) antibody uptake occurred in living neurons and was not an artifact of antibody diffusion into dead cells; 3) intracellular binding of anti-Hu antibody produced neuronal cell death, whereas uptake of anti-Ri antibody did not affect cell viability during the period of study; and 4) adsorption of anti-Hu antisera against HuD greatly reduced intraneuronal IgG accumulation and abolished cytotoxicity, confirming specificity of antibody-mediated neuronal death. Both anti-Hu and anti-Ri antibodies were readily taken up by viable neurons in slice cultures, but the two antibodies differed markedly in terms of their effects on neuronal viability. The ability of anti-Hu antibodies to cause neuronal death could account for the irreversible nature of paraneoplastic neurological deficits in patients with this antibody response. Our results raise questions as to whether anti-Ri antibody might initially induce reversible neuronal dysfunction, rather than causing cell death. The ability of IgG antibodies to access and react with intracellular neuronal proteins could have implications for other autoimmune diseases involving the central nervous system.

Nanoparticle-based strategy for personalized B-cell lymphoma therapy

PubMed Central

Martucci, Nicola M; Migliaccio, Nunzia; Ruggiero, Immacolata; Albano, Francesco; Calì, Gaetano; Romano, Simona; Terracciano, Monica; Rea, Ilaria; Arcari, Paolo; Lamberti, Annalisa

2016-01-01

B-cell lymphoma is associated with incomplete response to treatment, and the development of effective strategies targeting this disease remains challenging. A new personalized B-cell lymphoma therapy, based on a site-specific receptor-mediated drug delivery system, was developed in this study. Specifically, natural silica-based nanoparticles (diatomite) were modified to actively target the antiapoptotic factor B-cell lymphoma/leukemia 2 (Bcl2) with small interfering RNA (siRNA). An idiotype-specific peptide (Id-peptide) specifically recognized by the hypervariable region of surface immunoglobulin B-cell receptor was exploited as a homing device to ensure specific targeting of lymphoma cells. Specific nanoparticle uptake, driven by the Id-peptide, was evaluated by flow cytometry and confocal microscopy and was increased by approximately threefold in target cells compared with nonspecific myeloma cells and when a random control peptide was used instead of Id-peptide. The specific internalization efficiency was increased by fourfold when siRNA was also added to the modified nanoparticles. The modified diatomite particles were not cytotoxic and their effectiveness in downregulation of gene expression was explored using siRNA targeting Bcl2 and evaluated by quantitative real-time polymerase chain reaction and Western blot analyses. The resulting gene silencing observed is of significant biological importance and opens new possibilities for the personalized treatment of lymphomas. PMID:27895482

Steap4 Plays a Critical Role in Osteoclastogenesis in Vitro by Regulating Cellular Iron/Reactive Oxygen Species (ROS) Levels and cAMP Response Element-binding Protein (CREB) Activation*

PubMed Central

Zhou, Jian; Ye, Shiqiao; Fujiwara, Toshifumi; Manolagas, Stavros C.; Zhao, Haibo

2013-01-01

Iron is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis, reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation of osteoclast precursors to mature bone-resorbing osteoclasts. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclasts. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxygen species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function. PMID:23990467

Prevention of allergic rhinitis by ginger and the molecular basis of immunosuppression by 6-gingerol through T cell inactivation.

PubMed

Kawamoto, Yoshiyuki; Ueno, Yuki; Nakahashi, Emiko; Obayashi, Momoko; Sugihara, Kento; Qiao, Shanlou; Iida, Machiko; Kumasaka, Mayuko Y; Yajima, Ichiro; Goto, Yuji; Ohgami, Nobutaka; Kato, Masashi; Takeda, Kozue

2016-01-01

The incidence of allergies has recently been increasing worldwide. Immunoglobulin E (IgE)-mediated hypersensitivity is central to the pathogenesis of asthma, hay fever and other allergic diseases. Ginger (Zingiber officinale Roscoe) and its extracts have been valued for their medical properties including antinausea, antiinflammation, antipyresis and analgesia properties. In this study, we investigated the antiallergic effects of ginger and 6-gingerol, a major compound of ginger, using a mouse allergy model and primary/cell line culture system. In mice with ovalbumin (OVA)-induced allergic rhinitis, oral administration of 2% ginger diet reduced the severity of sneezing and nasal rubbing by nasal sensitization of OVA and suppressed infiltration of mast cells in nasal mucosa and secretion of OVA-specific IgE in serum. 6-Gingerol inhibited the expression of not only Th2 cytokines but also Th1 cytokines in OVA-sensitized spleen cells. Accordingly, 6-gingerol suppressed in vitro differentiation of both Th1 cells and Th2 cells from naïve T cells. In addition, 6-gingerol suppressed both superantigen staphylococcal enterotoxin B (SEB)- and anti-CD3-induced T cell proliferation. 6-Gingerol also abrogated PMA plus ionomycin- and SEB-induced IL-2 production in T cells, suggesting that 6-gingerol affected T cell receptor-mediated signal transduction rather than the antigen-presentation process. Indeed, 6-gingerol inhibited the phosphorylation of MAP kinases, calcium release and nuclear localization of c-fos and NF-κB by PMA and ionomycin stimulation. Thus, our results demonstrate that 6-gingerol suppresses cytokine production for T cell activation and proliferation, thereby not causing B cell and mast cell activation and resulting in prevention or alleviation of allergic rhinitis symptoms. Copyright © 2015 Elsevier Inc. All rights reserved.

Uptake and intra-inclusion accumulation of exogenous immunoglobulin by Chlamydia-infected cells

PubMed Central

Pollack, David V; Croteau, Nancy L; Stuart, Elizabeth S

2008-01-01

Background Obligate intracellular pathogens belonging to the Chlamydiaceae family possess a number of mechanisms by which to manipulate the host cell and surrounding environment. Such capabilities include the inhibition of apoptosis, down-regulation of major histocompatability complex (MHC) and CD1/d gene expression, and the acquisition of host-synthesized nutrients. It is also documented that a limited number of host-derived macromolecules such as β-catenin and sphingomyelin accumulate within the inclusion. Results This report provides evidence that immunoglobulin, inherently present in the extracellular environment in vivo and in vitro, enters infected cells and accumulates within the chlamydial inclusion. Using epi-fluorescent and confocal microscopy, this selective uptake of Ig is shown to occur among human leukocytes in vivo as well as cells cultured in vitro. These findings were confirmed by detection of IgG in the lysate of infected cells by western blot hybridization. Sequestered antibodies appear to be present during the entire course of the chlamydial developmental cycle and are distributed throughout this compartment. IgG pre-labeled with fluorescein, when added to the supernatant of infected cell cultures, was also imported and readily visualized. Accumulation of these molecules within the inclusion and the failure of bovine serum albumin or F(ab')2 fragments to accumulate in a similar manner suggests the process of entry is specific for intact IgG molecules and not a result of pinocytosis, diffusion, or any other mass endocytic event. Conclusion Sequestration of a host cell-derived protein within the chlamydial inclusion, although unexpected, is not an unprecedented occurrence. However, selective accumulation of an exogenous host protein, such as extracellular IgG, has not been previously reported in connection with chlamydial infections. The selectivity of this process may indicate that this uptake plays an important role in pathogen physiology or virulence during infection and the phenomenon itself may give rise to novel diagnostic and therapeutic approaches. PMID:19061499

Prolonged exposure to 1,25(OH)2D3 and high ionized calcium induces FGF-23 production in intestinal epithelium-like Caco-2 monolayer: A local negative feedback for preventing excessive calcium transport.

PubMed

Rodrat, Mayuree; Wongdee, Kannikar; Panupinthu, Nattapon; Thongbunchoo, Jirawan; Teerapornpuntakit, Jarinthorn; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

2018-02-15

Overdose of oral calcium supplement and excessive intestinal calcium absorption can contribute pathophysiological conditions, e.g., nephrolithiasis, vascular calcification, dementia, and cardiovascular accident. Since our previous investigation has indicated that fibroblast growth factor (FGF)-23 could abolish the 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]-enhanced calcium absorption, we further hypothesized that FGF-23 produced locally in the enterocytes might be part of a local negative feedback loop to regulate calcium absorption. Herein, 1,25(OH) 2 D 3 was found to enhance the transcellular calcium transport across the epithelium-like Caco-2 monolayer, and this stimulatory effect was diminished by preceding prolonged exposure to high-dose 1,25(OH) 2 D 3 or high concentration of apical ionized calcium. Pretreatment with a neutralizing antibody for FGF-23 prevented this negative feedback regulation of calcium hyperabsorption induced by 1,25(OH) 2 D 3 . FGF-23 exposure completely abolished the 1,25(OH) 2 D 3 -enhanced calcium transport. Western blot analysis revealed that FGF-23 expression was upregulated in a dose-dependent manner by 1,25(OH) 2 D 3 or apical calcium exposure. Finally, calcium-sensing receptor (CaSR) inhibitors were found to prevent the apical calcium-induced suppression of calcium transport. In conclusion, prolonged exposure to high apical calcium and calcium hyperabsorption were sensed by CaSR, which, in turn, increased FGF-23 expression to suppress calcium transport. This local negative feedback loop can help prevent unnecessary calcium uptake and its detrimental consequences. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system.

PubMed

Loureiro-Dos-Santos, N E; Reis, R A; Kubrusly, R C; de Almeida, O M; Gardino, P F; de Mello, M C; de Mello, F G

2001-05-01

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 microM kainate (T(1/2) : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium-3 sensitive choline uptake and hemicolinium-3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mM N(G)-nitro-L-arginine methyl ester (L-NAME). Veratridine (3 microM) also reduced ChAT by a Ca(2+) dependent, but glutamate independent mechanism and was prevented by 1 microM tetrodotoxin.

Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson's disease.

PubMed

Arduíno, Daniela Moniz; Esteves, A Raquel; Cardoso, Sandra M; Oliveira, Catarina R

2009-09-01

Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

Multiple functions of neuronal plasma membrane neurotransmitter transporters.

PubMed

Raiteri, Luca; Raiteri, Maurizio

2015-11-01

Removal from receptors of neurotransmitters just released into synapses is one of the major steps in neurotransmission. Transporters situated on the plasma membrane of nerve endings and glial cells perform the process of neurotransmitter (re)uptake. Because the density of transporters in the membranes can fluctuate, transporters can determine the transmitter concentrations at receptors, thus modulating indirectly the excitability of neighboring neurons. Evidence is accumulating that neurotransmitter transporters can exhibit multiple functions. Being bidirectional, neurotransmitter transporters can mediate transmitter release by working in reverse, most often under pathological conditions that cause ionic gradient dysregulations. Some transporters reverse to release transmitters, like dopamine or serotonin, when activated by 'indirectly acting' substrates, like the amphetamines. Some transporters exhibit as one major function the ability to capture transmitters into nerve terminals that perform insufficient synthesis. Transporter activation can generate conductances that regulate directly neuronal excitability. Synaptic and non-synaptic transporters play different roles. Cytosolic Na(+) elevations accompanying transport can interact with plasmalemmal or/and mitochondrial Na(+)/Ca(2+) exchangers thus generating calcium signals. Finally, neurotransmitter transporters can behave as receptors mediating releasing stimuli able to cause transmitter efflux through multiple mechanisms. Neurotransmitter transporters are therefore likely to play hitherto unknown roles in multiple therapeutic treatments. Copyright © 2015 Elsevier Ltd. All rights reserved.

Abnormal scintigraphic evolution in AA hepatic amyloidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lomena, F.; Rosello, R.; Pons, F.

1988-03-01

A patient with AA amyloidosis secondary to ankylosing spondylitis showed intense liver uptake of Tc-99m MDP on bone imaging. The biopsy showed hepatic amyloid deposition. A repeat bone scan with Tc-99m MDP 1 year later was negative, although the clinical signs and liver function tests of the patient had not changed. A mechanism might exist, other than the affinity of amyloid to calcium, which would explain the extraosseous uptake of pyrophosphates and diphosphonates in organs and soft tissues affected by systemic amyloidosis.

SaRNA-mediated activation of TRPV5 reduces renal calcium oxalate deposition in rat via decreasing urinary calcium excretion.

PubMed

Zeng, Tao; Duan, Xiaolu; Zhu, Wei; Liu, Yang; Wu, Wenqi; Zeng, Guohua

2018-06-01

Hypercalciuria is a main risk factor for kidney stone  formation. TRPV5 is the gatekeeper protein for mediating calcium transport and reabsorption in the kidney. In the present study, we tested the effect of TRPV5 activation with small activating RNA (saRNA), which could induce gene expression by targeting the promoter of the gene, on ethylene glycol (EG)-induced calcium oxalate (CaOx) crystals formation in rat kidney. Five pairs of RNA activation sequences targeting the promoter of rat TRPV5 were designed and synthesized. The synthesized saRNA with the strongest activating effect was selected, and transcellular calcium transportation was tested by Fura-2 analysis. Subsequently, Sprague-Dawley rats were equally divided into three groups and fed with water, 1% EG for 28 days after injecting the negative control saRNA, 1% EG for 28 days after injecting the selected TRPV5-saRNA, respectively. The CaOx crystal formation and the 24-h urine components were assessed. In vitro study, saRNA ds-320 could significantly activate the expression of TRPV5 and transcellular calcium transportation. In vivo study, after 28 days treatment of EG, rats pre-infected with saRNA ds-320 had lower urinary calcium excretion and renal CaOx crystals formation as compared to that pre-infected with negative control saRNA. Activation of TRVP5 with saRNA ds-320 could inhibit EG-induced calcium oxalate crystals formation via promoting urine calcium reabsorption and decreasing urine calcium excretion in rats.

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

NASA Astrophysics Data System (ADS)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

Immunoglobulin E-Mediated Autoimmunity

PubMed Central

Maurer, Marcus; Altrichter, Sabine; Schmetzer, Oliver; Scheffel, Jörg; Church, Martin K.; Metz, Martin

2018-01-01

The study of autoimmunity mediated by immunoglobulin E (IgE) autoantibodies, which may be termed autoallergy, is in its infancy. It is now recognized that systemic lupus erythematosus, bullous pemphigoid (BP), and chronic urticaria, both spontaneous and inducible, are most likely to be mediated, at least in part, by IgE autoantibodies. The situation in other conditions, such as autoimmune uveitis, rheumatoid arthritis, hyperthyroid Graves’ disease, autoimmune pancreatitis, and even asthma, is far less clear but evidence for autoallergy is accumulating. To be certain of an autoallergic mechanism, it is necessary to identify both IgE autoantibodies and their targets as has been done with the transmembrane protein BP180 and the intracellular protein BP230 in BP and IL-24 in chronic spontaneous urticaria. Also, IgE-targeted therapies, such as anti-IgE, must have been shown to be of benefit to patients as has been done with both of these conditions. This comprehensive review of the literature on IgE-mediated autoallergy focuses on three related questions. What do we know about the prevalence of IgE autoantibodies and their targets in different diseases? What do we know about the relevance of IgE autoantibodies in different diseases? What do we know about the cellular and molecular effects of IgE autoantibodies? In addition to providing answers to these questions, based on a broad review of the literature, we outline the current gaps of knowledge in our understanding of IgE autoantibodies and describe approaches to address them. PMID:29686678

Toxic shock syndrome: major advances in pathogenesis, but not treatment.

PubMed

Low, Donald E

2013-07-01

Toxic shock syndrome (TSS) is primarily the result of a superantigen-mediated cytokine storm and M protein-mediated neutrophil activation, resulting in the release of mediators leading to respiratory failure, vascular leakage, and shock. Mortality for streptococcal TSS still hovers at 50%. There is evidence to support a role for intravenous immunoglobulin (IVIG) in the treatment of streptococcal TSS. An observational study suggests that an initial conservative surgical approach combined with the use of immune modulators, such as IVIG, may reduce the morbidity associated with extensive surgical exploration in hemodynamically unstable patients without increasing mortality. Copyright © 2013 Elsevier Inc. All rights reserved.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

PubMed

Back, Moon Jung; Lee, Hae Kyung; Lee, Joo Hyun; Fu, Zhicheng; Son, Mi Won; Choi, Sang Zin; Go, Hyo Sang; Yoo, Sungjae; Hwang, Sun Wook; Kim, Dae Kyong

2016-11-16

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Binding analysis of carbon nanoparticles to human immunoglobulin G: Elucidation of the cytotoxicity of CNPs and perturbation of immunoglobulin conformations

NASA Astrophysics Data System (ADS)

Zhang, Shengrui; Yang, Haitao; Ji, Xiaohui; Wang, Qin

2016-02-01

The chemical compositions, sizes and fluorescent properties of synthesized carbon nanoparticles (CNPs) were characterized. Escherichia coli (E. coli) cells were used as a model to study the cytotoxicity of CNPs, and the results of the cellular uptake of CNPs yielded excellent results: the CNPs demonstrated good biocompatibility and were non-toxic to the growth of the E. coli cells. Moreover, to assess the potential toxicity of CNPs to human health, the binding behavior of CNPs with human immunoglobulin G (HIgG) was examined by fluorescence quenching spectroscopy, synchronous fluorescence spectroscopy and circular dichroism spectroscopy under physiological conditions. The fluorescence quenching constants and parameters for the interaction at different temperatures had been calculated according to Scatchard. The thermodynamic parameters, such as enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG), were calculated, and the results indicated strong static quenching and showed that van der Waals forces, hydrogen bonds and hydrophobic interactions were the predominant intermolecular forces stabilizing the CNP-HIgG complex. Synchronous fluorescence and circular dichroism spectra provided information regarding the conformational alteration of HIgG in the presence of CNPs. These findings help to characterize the interactions between CNPs and HIgG, which may clarify the potential risks and undesirable health effects of CNPs, as well as the related cellular trafficking and systemic translocation.

Processing of fish lg heavy chain transcripts diverse splicing patterns and unusual nonsense mediated decay

USDA-ARS?s Scientific Manuscript database

Alternate pathways of RNA processing play an important role in the expression of the secreted (S) and membrane (Mb) forms of immunoglobulin (Ig) heavy (H) chain isotypes in all vertebrates. Interestingly, while the differential splicing mechanism and the splice sites that generate the two forms of I...

Ikaros controls isotype selection during immunoglobulin class switch recombination.

PubMed

Sellars, MacLean; Reina-San-Martin, Bernardo; Kastner, Philippe; Chan, Susan

2009-05-11

Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG(2b) and IgG(2a), and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the gamma2b and gamma2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3' enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of gamma2b and gamma2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.

Effect of TEI-9874, an inhibitor of immunoglobulin E production, on allergen-induced asthmatic model in rats.

PubMed

Nonaka, T; Mitsuhashi, H; Takahashi, K; Sugiyama, H; Kishimoto, T

2000-08-25

As TEI-9874, 2-(4-(6-cyclohexyloxy-2-naphtyloxy)phenylacetamide)ben zoic acid reduces allergen-specific immunoglobulin E (IgE) production by human peripheral blood mononuclear cells in vitro, we evaluated its potency on an allergen-induced asthmatic model in Brown-Norway rats. Inhaled ovalbumin induced the immediate-phase asthmatic response, the late-phase asthmatic response, the infiltration of leukocytes into bronchoalveolar lavage fluid, and an increase of serum anti-ovalbumin IgE. These parameters were suppressed by the treatment with TEI-9874 (3, 10, and 30 mg/kg p.o.). The ovalbumin-induced airway hyperresponsiveness was prevented by TEI-9874 (30 mg/kg p.o.). Furthermore, the suppression of the immediate-phase asthmatic response and the late-phase asthmatic response by TEI-9874 was almost completely extinguished by the exogenous administration of rat anti-ovalbumin antiserum. These results indicate that the efficacy of TEI-9874 on the asthmatic response is mainly mediated by the suppression of allergen-specific IgE production and TEI-9874 appears to be a good candidate as therapy for IgE-mediated allergic asthma.

B Cells and Humoral Immunity in Atherosclerosis

PubMed Central

Tsiantoulas, Dimitrios; Diehl, Cody J.; Witztum, Joseph L.; Binder, Christoph J.

2014-01-01

Insights into the important contribution of inflammation and immune functions in the development and progression of atherosclerosis have greatly improved our understanding of this disease. Although the role of T cells has been extensively studied for decades, only recently has the role of B cells gained more attention. Recent studies have identified differential effects of different B-cell subsets and helped to clarify the still poorly understood mechanisms by which these act. B1 cells have been shown to prevent lesion formation, whereas B2 cells have been suggested to promote it. Natural IgM antibodies, mainly derived from B1 cells, have been shown to mediate atheroprotective effects, but the functional role of other immunoglobulin classes, particularly IgG, still remains elusive. In this review, we will focus on recent insights on the role of B cells and various immunoglobulin classes and how these may mediate their effects in atherosclerotic lesion formation. Moreover, we will highlight potential therapeutic approaches focusing on B-cell depletion that could be used to translate experimental evidence to human disease. PMID:24855199

Diabetic Hyperglycemia: Link to Impaired Glucose Transport in Pancreatic β Cells

NASA Astrophysics Data System (ADS)

Unger, Roger H.

1991-03-01

Glucose uptake into pancreatic β cells by means of the glucose transporter GLUT-2, which has a high Michaelis constant, is essential for the normal insulin secretory response to hyperglycemia. In both autoimmune and nonautoimmune diabetes, this glucose transport is reduced as a consequence of down-regulation of the normal β-cell transporter. In autoimmune diabetes, circulating immunoglobulins can further impair this glucose transport by inhibiting functionally intact transporters. Insights into mechanisms of the unresponsiveness of β cells to hyperglycemia may improve the management and prevention of diabetes.

Kinetic properties of the sodium-calcium exchanger in rat brain synaptosomes.

PubMed Central

Fontana, G; Rogowski, R S; Blaustein, M P

1995-01-01

1. The kinetic properties of the internal Na+ (Na+i)- dependent 45Ca2+ influx and external Na+ (Na+o)-dependent 45Ca2+ efflux were determined in isolated rat brain nerve terminals (synaptosomes) under conditions which the concentrations of internal Na+ ([Na+]i), external Na+ ([Na+]o), external Ca2+ (Ca2+]o), and external K+ ([K+]o) were varied. Both fluxes are manifestations of Na(+)-Ca2+ exchange. 2. Ca2+ uptake was augmented by raising [Na+]i and / or lowering [Na+]o. The increase in Ca2+ uptake induced by removing external Na+ was, in most instances, quantitatively equal to the Na+i-dependent Ca2+ uptake. 3. The Na+i-dependent Ca2+ uptake (measured at 1 s) was activated with an apparent half-maximal [Ca2+]o (KCa(o)) of about 0.23 mM. External Na+ inhibited the uptake in a non- competitive manner: increasing [Na+]o from 4.7 to 96 mM reduced the maximal Na+(i)-dependent Ca2+ uptake but did not affect KCa(o). 4. The inhibition of Ca2+ uptake by Na+o was proportional to ([Na+]o)2, and had a Hill coefficient (nH) of approximately 2.0. The mean apparent half-maximal [Na+]o for inhibition (KI(Na)) was about 60mM, and was independent of [Ca2+]o between 0.1 and 1.2mM; this, too, is indicative of non-competitive inhibition. 5. Low concentrations of alkali metal ions (M+) in the medium, including Na+, stimulated the Na+i-dependent uptake. The external Na+ and K+ concentrations required for apparent half-maximal activation (KM(Na) and KM(K), respectively) were 0.12 and 0.10mM. Thus, the relationship between Ca2+ uptake and [Na+]o was biphasic: uptake was stimulated by [Na+]o < or = 10 mM, and inhibited by higher [Na+]o. 6. The calculated maximal Na+i-dependent Ca2+ uptake (Jmax) was about 1530 pmol (mg protein) -1s-1 at 30 degrees C saturating [Ca2+]o and external M+ concentration ([M+]o), and with negligible inhibition by external Na+. 7. Internal Na+ activated the Ca2+ uptake with an apparent half-maximal concentration (KNa(i)) of about 20 mM and a Hill coefficient, nH, of approximately 3.0. 8. The Jmax for the Na+o-dependent efflux of Ca2+ from 45Ca(2+)-loaded synaptosomes treated with carbonyl cyanide p-trifluormethoxy-phenylhydrazone (FCCP) and caffeine (to release stored Ca2+ and raise the internal Ca2+ concentration ([Ca2+]i) was about 1800-2000 pmol (mg protein -1s-1 at 37 degrees C. 9. When the membrane potential (Vm) was reduced (depolarized) by increasing [K+]o, the Na+i-dependent Ca2+ influx increased, and the Na+o-dependent Ca2+ efflux declined. Both fluxes changed about 2-fold per 60 mV change in Vm. This voltage sensitivity corresponds to the movement of one elementary charge through about 60% of the membrane electric field. The symmetry suggests that the voltage-sensitive step is reversible. 10. The Jmax values for both Ca2P influx and efflux correspond to a Na+-Ca2+ exchange-mediated flux of about 425-575 jumol Ca2P (1 cell water)-' s-' or a turnover of about one quarter of the total synaptosome Ca2P in 1 s. We conclude that the Na+-Ca2P exchanger may contribute to Ca2P entry during nerve terminal depolarization; it is likely to be a major mechanism mediating Ca2P extrusion during subsequent repolarization and recovery. PMID:7666363

Calcium modulation of the effects of serotonin, carbachol, and histamine on rabbit ileal ion transport.

PubMed Central

Chough, S. P.; Goldenring, J. R.; Hurst, R. D.; Ballantyne, G. H.; Modlin, I. M.

1993-01-01

In mammalian intestine, a number of secretagogues have been shown to work through either cyclic nucleotide or calcium mediated pathways to elicit ion secretion. Because excessive intestinal electrolyte and fluid secretion is central to the pathogenesis of a variety of diarrheal disorders, understanding of these processes is essential to the development of future clinical treatments. In the current study, the effects of serotonin (5HT), histamine, and carbachol on intestinal ion transport were examined in in vitro preparations of rabbit ileum. All three agonists induced a rapid and transient increase short-circuit current (delta Isc) across ileal mucosa. Inhibition of the delta Isc response of all three agents in chloride-free solution or in the presence of bumetanide confirmed that chloride is the main electrolyte involved in electrogenic ion secretion. Pretreatment of tissue with tetrodotoxin or atropine did not effect secretagogue-mediated electrolyte secretion. While tachyphylaxis of delta Isc response was shown to develop after repeated exposure of a secretagogue to tissue, delta Isc responses after sequential addition of different agonists indicate that cross-tachyphylaxis between agents did not occur. Serotonin, histamine, and carbachol have previously been reported to mediate electrolyte secretion through calcium-dependent pathways. To access the role of extracellular calcium in regulating ion secretion, the effect of verapamil on each agent was tested; verapamil decreased 5HT-induced delta Isc by 65.2% and histamine response by 33.5%, but had no effect on carbachol-elicited chloride secretion. An additive secretory effect was found upon simultaneous exposure of 5HT and carbachol to the system; no other combination of agents produced a significant additive effect. Findings from this study support previous work which has suggested that multiple calcium pathways may be involved in mediating chloride secretion in mammalian intestine. PMID:7716972

Calcium bioavailability and kinetics of calcium ascorbate and calcium acetate in rats.

PubMed

Cai, Jianwei; Zhang, Qinmin; Wastney, Meryl E; Weaver, Connie M

2004-01-01

The objective was to investigate the bioavailability and mechanism of calcium absorption of calcium ascorbate (ASC) and calcium acetate (AC). A series of studies was performed in adult Sprague-Dawley male rats. In the first study, each group of rats (n = 10/group) was assigned to one of the five test meals labeled with (45)Ca: (i) 25 mg calcium as heated ASC or (ii) unheated ASC, (iii) 25 mg calcium as unheated AC, (iv) 3.6 mg Ca as unheated ASC, or (v) unheated AC. Femur uptake indicated better calcium bioavailability from ASC than AC at both calcium loads. A 5-min heat treatment partly reduced bioavailability of ASC. Kinetic studies were performed to further investigate the mechanism of superior calcium bioavailability from ASC. Two groups of rats (n = 10/group) received oral doses of 25 mg Ca as ASC or AC. Each dose contained 20 micro Ci (45)Ca. Two additional groups of rats (n = 10/group) received an intravenous injection (iv) of 10 micro Ci (45)Ca after receiving an unlabeled oral dose of 25 mg calcium as ASC or AC. Sequential blood samples were collected over 48 hrs. Urine and fecal samples were collected every 12 hrs for 48 hrs and were analyzed for total calcium and (45)Ca content. Total calcium and (45)Ca from serum, urine, and feces were fitted by a compartment kinetics model with saturable and nonsaturable absorption pathways by WinSAAM (Windows-based Simulation Analysis and Modeling). The difference in calcium bioavailability between the two salts was due to differences in saturable rather than passive intestinal absorption and not to endogenous secretion or calcium deposition rate. The higher bioavailability of calcium ascorbate was due to a longer transit time in the small intestine compared with ASC.

Histone hyperacetylation and exon skipping: a calcium-mediated dynamic regulation in cardiomyocytes

PubMed Central

Sharma, Alok; Nguyen, Hieu; Cai, Lu; Lou, Hua

2015-01-01

In contrast to cell type-specific pre-mRNA alternative splicing, mechanisms controlling activity-dependent alternative splicing is under-studied and not well understood. In a recent study, we conducted a comprehensive analysis of calcium-mediated mechanism that regulates alternative exon skipping in mouse cardiomyocytes. Our results reveal a strong link between histone hyperacetylation and skipping of cassette exons, and provide support to the kinetic coupling model of the epigenetic regulation of alternative splicing at the chromatin level. PMID:26325491

A procyanidin type A trimer from cinnamon extract attenuates glial cell swelling and the reduction in glutamate uptake following ischemia-like injury in vitro.

PubMed

Panickar, K S; Polansky, M M; Graves, D J; Urban, J F; Anderson, R A

2012-01-27

Dietary polyphenols exert neuroprotective effects in ischemic injury. The protective effects of a procyanidin type A trimer (trimer 1) isolated from a water soluble cinnamon extract (CE) were investigated on key features of ischemic injury, including cell swelling, increased free radical production, increased intracellular calcium ([Ca(2+)](i)), mitochondrial dysfunction, and the reduction in glutamate uptake. Astrocyte (glial) swelling is a major component of cytotoxic brain edema in ischemia and, along with vasogenic edema, may contribute to increased intracranial pressure, brain herniation, and additional ischemic injuries. C6 glial cultures were exposed to oxygen-glucose deprivation (OGD) for 5 h, and cell swelling was determined at 90 min after the end of OGD. OGD-induced increases in glial swelling were significantly blocked by trimer 1, but not by the major nonpolyphenol fractions of CE including cinnamaldehyde and coumarin. Increased free radical production, a contributing factor in cell swelling following ischemic injury, was also significantly reduced by trimer 1. Mitochondrial dysfunction, another key feature of ischemic injury, is hypothesized to contribute to glial swelling. Depolarization of the inner mitochondrial membrane potential (ΔΨ(m)) was assessed using a fluorescent dye (tetramethylrhodamine ethyl ester [TMRE]), and was significantly attenuated by trimer 1 as was OGD-induced increased [Ca(2+)](i). Taken together with our previous observation that blockers of [Ca(2+)](i) reduce cell swelling, our results indicate that trimer 1 may attenuate cell swelling by regulating [Ca(2+)](i). Trimer 1 also significantly attenuated the OGD-induced decrease in glutamate uptake. In addition, cyclosporin A, a blocker of the mitochondrial permeability pore (mPT), but not FK506 (that does not block the mPT), reduced the OGD-induced decline in glutamate uptake indicating a role of the mPT in such effects. Thus, the effects of trimer 1 in attenuating the reduction in glutamate uptake are likely mediated through their action on the mitochondria. Published by Elsevier Ltd on behalf of IBRO.

Investigation of repeated vaccination as a possible cause of glomerular disease in mink.

PubMed

Newman, Shelley Joy; Johnson, Roger; Sears, William; Wilcock, Brian

2002-07-01

The search for antigens capable of causing immune-complex-mediated glomerulonephritis continues. Modified live-virus vaccines commercially available for veterinary use are a possible source. In this study, repeated vaccination of mink with live-virus vaccines was investigated as a model for vaccine-induced glomerular injury. Three groups of 10-wk-old mink, 15 per group, were vaccinated once with 4-way vaccine against distemper, Pseudomonas aeruginosa infection, botulism and mink viral enteritis. Subsequently, all mink in each group each were vaccinated either with the 4-way vaccine, a monovalent canine distemper vaccine, or saline. Glomerular function was assessed at 2-wk intervals by determining the urinary protein:creatinine (P:C) ratio. Kidney sections taken at necropsy, 20 wk after the 1st vaccination, were examined by light and immunofluorescent microscopy for deposition of immunoglobulin and complement. There was no statistically significant difference between the treated and control groups based on average urinary P:C ratio medians. Light microscopic changes were detected in glomeruli, but Fisher's exact test showed no significant differences between any of the treatment groups. Deposition of immunoglobulin but not complement was significantly more frequent (P < 0.05) in the glomeruli of animals that received multiple injections of the 4-way vaccine than in the glomeruli of those given only the monovalent canine distemper vaccine or saline. These findings suggest that repeated vaccination may increase the glomerular deposition of immunoglobulin. Further studies are required to determine if the increased deposition of immunoglobulin contributes to the development of glomerular damage and to identify the antigens driving production of the deposited immunoglobulin.

Calcium transport into the cells of the sea urchin larva in relation to spicule formation

PubMed Central

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas; Ben-Ezra, David; Shpigel, Muki; Addadi, Lia; Weiner, Steve

2016-01-01

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium. PMID:27791140

Calcium transport into the cells of the sea urchin larva in relation to spicule formation.

PubMed

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas; Ben-Ezra, David; Shpigel, Muki; Addadi, Lia; Weiner, Steve

2016-10-24

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.

One Dimensional Finite Element Method Approach to Study Effect of Ryanodine Receptor and Serca Pump on Calcium Distribution in Oocytes

NASA Astrophysics Data System (ADS)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2013-11-01

Oocyte is a female gametocyte or germ cell involved in reproduction. Calcium ions (Ca2+) impact nearly all aspects of cellular life as they play an important role in a variety of cellular functions. Calcium ions contributes to egg activation upon fertilization. Since it is the internal stores which provide most of the calcium signal, much attention has been focused on the intracellular channels. There are mainly two types of calcium channels which release calcium from the internal stores to the cytoplasm in many cell types. These channels are IP3-Receptor and Ryanodine Receptor (RyR). Further it is essential to maintain low cytosolic calcium concentration, the cell engages the Serco/Endoplasmic reticulum Ca2+ ATPases (SERCA) present on the ER or SR membrane for the re-uptake of cytosolic calcium at the expense of ATP hydrolysis. In view of above an attempt has been made to study the effect of the Ryanodine receptor (RyR) and the SERCA pump on the calcium distribution in oocytes. The main aim of this paper is to study the calcium concentration in absence and presence of these parameters. The FEM is used to solve the proposed Mathematical model under appreciate initial and boundary conditions. The program has been developed in MATLAB 7.10 for the entire problem to get numerical results.

CALCIUM BINDING TO INTESTINAL MEMBRANES

PubMed Central

Oschman, James L.; Wall, Betty J.

1972-01-01

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium. The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative. In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes. The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication. They are also observed in osmicated tissue if calcium is added to wash and osmium solutions. Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl2 (as low as 5 x 10-5 M) are added. The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium. Other divalent cations (Mg++, Sr++, Ba++, Mn++, Fe++) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not. After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes. The dense regions may thus localize membrane constituents that bind calcium. The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization. PMID:4569411

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria.

PubMed

Kristián, Tibor; Weatherby, Tina M; Bates, Timothy E; Fiskum, Gary

2002-12-01

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.

Antibodies and Their Receptors: Different Potential Roles in Mucosal Defense

PubMed Central

Horton, Rachel E.; Vidarsson, Gestur

2013-01-01

Over recent years it has become increasingly apparent that mucosal antibodies are not only restricted to the IgM and IgA isotypes, but that also other isotypes and particularly IgG can be found in significant quantities at some mucosal surfaces, such as in the genital tract. Their role is more complex than traditionally believed with, among other things, the discovery of novel function of mucosal immunoglobulin receptors. A thorough knowledge in the source and function and mucosal immunoglobulins is particularly important in development of vaccines providing mucosal immunity, and also in the current climate of microbicide development, to combat major world health issues such as HIV. We present here a comprehensive review of human antibody mediated mucosal immunity. PMID:23882268

Regulation of cardiomyocyte autophagy by calcium.

PubMed

Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

2016-04-15

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

Calcium/Calmodulin-Mediated Gravitropic Response in Plants

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.

2002-01-01

Plant organs respond to different physical signals such as gravity, light and touch. Gravity gives plants proper orientation, resulting in the proper form that we take for granted; the roots grow down into soil and shoots grow towards the light. Under microgravity conditions, as in space, plant growth patterns lack a clear sense of direction. Calcium and calmodulin (CaM) play an important role in gravity signal transduction. However, the molecular and biochemical mechanisms involved in gravity signal transduction are not clearly understood. The goal of this project was to gain a fundamental understanding of how calcium/calmodulin-mediated signaling is involved in gravity signal transduction in plants. During the grant period, significant progress was made in elucidating the role of calmodulin and its target proteins in gravitropism.

Improvement of desolvation and resilience of alginate binders for Si-based anodes in a lithium ion battery by calcium-mediated cross-linking.

PubMed

Yoon, Jihee; Oh, Dongyeop X; Jo, Changshin; Lee, Jinwoo; Hwang, Dong Soo

2014-12-14

Si-based anodes in lithium ion batteries (LIBs) have exceptionally high theoretical capacity, but the use of a Si-based anode in LIBs is problematic because the charging-discharging process can fracture the Si particles. Alginate and its derivatives show promise as Si particle binders in the anode. We show that calcium-mediated "egg-box" electrostatic cross-linking of alginate improves toughness, resilience, electrolyte desolvation of the alginate binder as a Si-binder for LIBs. Consequently, the improved mechanical properties of the calcium alginate binder compared to the sodium alginate binder and other commercial binders extend the lifetime and increase the capacity of Si-based anodes in LIBs.

Aluminum Citrate Prevents Renal Injury from Calcium Oxalate Crystal Deposition

PubMed Central

Besenhofer, Lauren M.; Cain, Marie C.; Dunning, Cody

2012-01-01

Calcium oxalate monohydrate crystals are responsible for the kidney injury associated with exposure to ethylene glycol or severe hyperoxaluria. Current treatment strategies target the formation of calcium oxalate but not its interaction with kidney tissue. Because aluminum citrate blocks calcium oxalate binding and toxicity in human kidney cells, it may provide a different therapeutic approach to calcium oxalate-induced injury. Here, we tested the effects of aluminum citrate and sodium citrate in a Wistar rat model of acute high-dose ethylene glycol exposure. Aluminum citrate, but not sodium citrate, attenuated increases in urea nitrogen, creatinine, and the ratio of kidney to body weight in ethylene glycol–treated rats. Compared with ethylene glycol alone, the addition of aluminum citrate significantly increased the urinary excretion of both crystalline calcium and crystalline oxalate and decreased the deposition of crystals in renal tissue. In vitro, aluminum citrate interacted directly with oxalate crystals to inhibit their uptake by proximal tubule cells. These results suggest that treating with aluminum citrate attenuates renal injury in rats with severe ethylene glycol toxicity, apparently by inhibiting calcium oxalate’s interaction with, and retention by, the kidney epithelium. PMID:23138489

[Calcium and vitamin D in bone metabolism: Clinical importance for fracture treatment].

PubMed

Amling, M

2015-12-01

A balanced calcium homeostasis is of critical importance not only for bone remodeling, the physiological process of bone resorption and bone formation that constantly renews bone throughout life but also for normal fracture healing. Given that disturbances of calcium homeostasis are present in 50 % of the German population and that this might result in delayed fracture healing after correct surgical treatment, this paper focusses on calcium and vitamin D in the daily practice in orthopedics and trauma surgery. To ensure the required enteral calcium uptake the following three conditions are required: (1) sufficient calcium intake via the nutrition, (2) a 25-hydroxyvitamin D serum level > 30 µg/l and (3) the presence of sufficient gastric acidification. Given the endemic vitamin D deficiency in Germany as well as the constantly increasing number of people using proton pump inhibitors on a regular basis, it is necessary to closely connect trauma orthopedic surgery and osteological treatment. The first issue to be dealt with is to control and if needed normalize calcium homeostasis in order to allow a normal undisturbed fracture healing process after both conservative as well as operative treatment of fractures.

Monocarboxylate Transporters Mediate Fluorescein Uptake in Corneal Epithelial Cells.

PubMed

Sun, Yi-Chen; Liou, Hau-Min; Yeh, Po-Ting; Chen, Wei-Li; Hu, Fung-Rong

2017-07-01

To determine the presence of monocarboxylate transporter (MCT) in human and rabbit corneal epithelium and its role in transcellular fluorescein transportation in the cornea. The presence of MCTs in human and rabbit corneal epithelium was determined by RT-PCR and immunohistochemistry. Intracellular fluorescein uptake experiment was performed using cultured human corneal epithelial cells (HCECs). The involvement of MCT in fluorescein uptake was determined by addition of MCT inhibitors to HCECs and acute dry eye model on New Zealand albino rabbits by spectrophotometry, corneal impression cytology, and external eye photographs. MCT-1 and MCT-4 were identified in both human and rabbit corneal epithelia. A longer treatment period and a lower pH value in culture medium increased fluorescein uptake in HCECs. Fluorescein uptake in HCECs was decreased following addition of MCT inhibitors in a concentration-dependent manner. Impression cytology under fluorescent microscopy showed intracellular fluorescein staining in the rabbit cornea with acute dry eye treatment that was decreased following topical treatment of MCT inhibitors. Fluorescein ingress in corneal epithelial cells is mediated by the MCT family. Further study of MCT-mediated transport on HCECs may potentially benefit differential diagnosis and contribute better understandings of ocular surface disorders.

Disruption of Calcium Homeostasis During Exercise as a Mediator of Bone Metabolism

DTIC Science & Technology

2015-10-01

Meeting of the American College of Sports Medicine (Appendix A). 15. SUBJECT TERMS calcium homeostasis, exercise, bone resorption, parathyroid hormone ... hormone (PTH). PTH can defend serum Ca by reducing urinary Ca excretion, increasing intestinal Ca absorption, and increasing mobilization of skeletal Ca...certain conditions. It is our contention that disruptions in calcium homeostasis during exercise lead to increases in parathyroid hormone (PTH) and

Fluid-Phase Pinocytosis of Native Low Density Lipoprotein Promotes Murine M-CSF Differentiated Macrophage Foam Cell Formation

PubMed Central

Xu, Qing; Bohnacker, Thomas; Wymann, Matthias P.; Kruth, Howard S.

2013-01-01

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of 125I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on 125I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect 125I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries. PMID:23536783

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

PubMed Central

Wang, Shiyu; Allen, Nickolas; Vickers, Timothy A; Revenko, Alexey S; Sun, Hong; Liang, Xue-hai; Crooke, Stanley T

2018-01-01

Abstract Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs. PMID:29514240

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells.

PubMed

Loh, Lip Nam; McCarthy, Elizabeth M C; Narang, Priyanka; Khan, Naveed A; Ward, Theresa H

2017-11-01

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The tripeptide aldehyde, Boc-DPhe-Phe-Lysinal, is a novel Ca2+ channel inhibitor in pituitary cells.

PubMed

Makara, G B; Rappay, G; Garamvölgyi, V; Nagy, I; Dankó, S; Bajusz, S

1988-06-22

The effect of Boc-DPhe-Phe-Lysinal (Boc-DPPL) on the 45Ca2+ uptake of rat anterior pituitary monolayer cultures was investigated. The compound decreased the basal Ca2+ uptake at 3 x 10(-4) mol/l. The 45Ca2+ uptake stimulated by potassium-induced depolarization was more sensitive to Boc-DPPL inhibition, a slight decrease was seen with 3 x 10(-6) mol/l and there was a half maximal inhibition at 3 x 10(-5) mol/l. Boc-DPPL is known to inhibit pituitary hormone release in similar concentrations, an effect might also be due to its calcium antagonist property.

Calcium uptake in the skin of a freshwater teleost.

PubMed Central

McCormick, S D; Hasegawa, S; Hirano, T

1992-01-01

The skin, particularly the opercular membrane of some teleosts, contains mitochondrion-rich "chloride" cells and has been widely used as a model to study branchial salt-extrusion mechanisms in seawater fish. Skin isolated from the operculum of the freshwater Nile tilapia (Oreochromis niloticus) can transport Ca2+ against an ionic and electrical gradient. Adaptation of Nile tilapia to a low-Ca2+ environment increased the capacity of the opercular membrane to transport Ca2+. The density of mitochondrion-rich cells increased in parallel with Ca2+ transport capacity. The results demonstrate net Ca2+ uptake by vertebrate skin and strongly implicate mitochondrion-rich cells as the site of Ca2+ uptake in fresh water. Images PMID:1565659

Uptake of 2,4-dichlorophenoxyacetic acid by Pseudomonas fluorescens

USGS Publications Warehouse

Wedemeyer, Gary

1966-01-01

Factors influencing the uptake of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D), under conditions in which no net metabolism occurred, were investigated in an effort to determine both the significance of “non-metabolic” uptake as a potential agent in reducing pesticide levels and the mechanisms involved. Uptake of 2,4-D was affected by pH, temperature, and the presence of other organic and inorganic compounds. Uptake was more pronounced at pH values less than 6, which implies that there may be some interaction between charged groups on the cell and the ionized carboxyl group of 2,4-D. Active transport, carrier-mediated diffusion, passive diffusion, and adsorption were considered as possible mechanisms. Though uptake was inhibited by glucose, sodium azide, and fluorodinitrobenzene (but not by uranyl ion), 2,4-D was not accumulated against a concentration gradient, a necessary consequence of an active transport system, nor was isotope counterflow found to occur. Thus, carrier-mediated diffusion was finally precluded, implying that uptake probably occurs by a two-step process: sorption onto the cell wall followed by passive diffusion into the cytoplasm.

The impact on quality of life on families of children on an elimination diet for Non-immunoglobulin E mediated gastrointestinal food allergies.

PubMed

Meyer, Rosan; Godwin, Heather; Dziubak, Robert; Panepinto, Julie A; Foong, Ru-Xin M; Bryon, Mandy; Lozinsky, Adriana Chebar; Reeve, Kate; Shah, Neil

2017-01-01

The impact on health related quality of life (HRQL) has been well studied in children with Immunoglobulin E (IgE)-mediated food allergy. However limited data exists on related quality of life (QOL) of families who have a child suffering from food protein induced non-IgE mediated gastrointestinal allergies. We aimed to establish the QOL of families with children at the beginning of following an elimination diet for non-IgE mediated gastrointestinal food allergies. A prospective, observational study was performed. Parents of children aged 4 weeks-16 years who improved after 4-8 weeks of following an elimination diet for suspected non-IgE mediated allergies were included. The Family Impact Module (FIM) of the Pediatric Quality of Life (PedsQL™) was used and we compared our data to two historical cohorts: one with sickle cell disease and another with intestinal failure. One hundred and twenty three children with a median age of 20 months were included (84 boys). The total FIM Score was 57.43 (SD 22.27) and particularly low for daily activities and worry. Factors that impacted significantly included age ( p  < 0.0001), number of foods excluded ( p  = 0.008), symptom severity ( p  = 0.041) and chronic nasal congestion ( p  = 0.012). Children with non-IgE mediated food allergies had worse scores in all domains ( p  < 0.0001) compared to sickle cell disease and worse physical ( p  = 0.04), emotional ( p  = 0.04) and worry ( p  = 0.01) domains compared to intestinal failure. This study found that parent QOL and family functioning was worse in those families who had a child on an elimination diet for non-IgE mediated allergies compared to those with sickle cell disease and intestinal failure, highlighting the impact this disease has on families.

Role of magnesium on the biomimetic deposition of calcium phosphate

NASA Astrophysics Data System (ADS)

Sarma, Bimal K.; Sarma, Bikash

2016-10-01

Biomimetic depositions of calcium phosphate (CaP) are carried out using simulated body fluid (SBF), calcifying solution and newly developed magnesium containing calcifying solution. Calcium phosphate has a rich phase diagram and is well known for its excellent biocompatibility and bioactivity. The most common phase is hydroxyapatite (HAp), an integral component of human bone and tooth, widely used in orthopedic and dental applications. In addition, calcium phosphate nanoparticles show promise for the targeted drug delivery. The doping of calcium phosphate by magnesium, zinc, strontium etc. can change the protein uptake by CaP nanocrystals. This work describes the role of magnesium on the nucleation and growth of CaP on Ti and its oxide substrates. X-ray diffraction studies confirm formation of HAp nanocrystals which closely resemble the structure of bone apatite when grown using SBF and calcifying solution. It has been observed that magnesium plays crucial role in the nucleation and growth of calcium phosphate. A low magnesium level enhances the crystallinity of HAp while higher magnesium content leads to the formation of amorphous calcium phosphate (ACP) phase. Interestingly, the deposition of ACP phase is rapid when magnesium ion concentration in the solution is 40% of calcium plus magnesium ions concentration. Moreover, high magnesium content alters the morphology of CaP films.

Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production.

PubMed

Uto, Yoshihiro; Kawai, Tomohito; Sasaki, Toshihide; Hamada, Ken; Yamada, Hisatsugu; Kuchiike, Daisuke; Kubo, Kentaro; Inui, Toshio; Mette, Martin; Tokunaga, Ken; Hayakawa, Akio; Go, Akiteru; Oosaki, Tomohiro

2015-08-01

Colostrum contains antibodies, such as immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM), and, therefore, has potent immunomodulating activity. In particular, IgA has an O-linked sugar chain similar to that in the group-specific component (Gc) protein, a precursor of the Gc protein-derived macrophage-activating factor (GcMAF). In the present study, we investigated the macrophage-activating effects of degalactosylated/desialylated bovine colostrum. We detected the positive band in degalactosylated/ desialylated bovine colostrum by western blotting using Helix pomatia agglutinin lectin. We also found that degalactosylated/ desialylated bovine colostrum could significantly enhance the phagocytic activity of mouse peritoneal macrophages in vitro and of intestinal macrophages in vivo. Besides, degalactosylated/desialylated bovine colostrum did not mediate the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Similar to the use of GcMAF, degalactosylated/desialylated bovine colostrum can be used as a potential macrophage activator for various immunotherapies. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Inhibiting the Mitochondrial Calcium Uniporter during Development Impairs Memory in Adult Drosophila.

PubMed

Drago, Ilaria; Davis, Ronald L

2016-09-06

The uptake of cytoplasmic calcium into mitochondria is critical for a variety of physiological processes, including calcium buffering, metabolism, and cell survival. Here, we demonstrate that inhibiting the mitochondrial calcium uniporter in the Drosophila mushroom body neurons (MBn)-a brain region critical for olfactory memory formation-causes memory impairment without altering the capacity to learn. Inhibiting uniporter activity only during pupation impaired adult memory, whereas the same inhibition during adulthood was without effect. The behavioral impairment was associated with structural defects in MBn, including a decrease in synaptic vesicles and an increased length in the axons of the αβ MBn. Our results reveal an in vivo developmental role for the mitochondrial uniporter complex in establishing the necessary structural and functional neuronal substrates for normal memory formation in the adult organism. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Hypogravity's Effect on the Life Cycle of Japanese Quail

NASA Technical Reports Server (NTRS)

Hester, Patricia Y.

1999-01-01

A series of studies were conducted to determine the effect of activities preceding space-flight and during space-flight on quail embryonic development. While the overall development of the quail embryos was evaluated, the report presented herein, focused on calcium utilization or uptake from eggshells by developing embryos during incubation in space and on earth. In the pre-space trials, fertilized quail eggs were subjected to pre-night dynamics including forces of centrifugation, vibration, or a combination of vibration and centrifugation prior to incubation for 6 or 16 days. In another trial, fertile quail eggs were tested for survivability in a refrigerator stowage kit for eggs (RSKE) which was subsequently used to transport the eggs to space. Eggs in the RSKE were subjected to shuttle launch dynamics including G force and random vibration profiles. In the space- flight trials, 48 fertile quail eggs were launched on space shuttle Flight STS-76 and were subsequently incubated in a Slovakian incubator onboard space station, MIR. Two sets of ground controls each with 48 fertile eggs with and without exposure to launch dynamics were initiated 5 days post-launch. There was a laboratory control (incubated in Lyon RX2 incubator at 37.5 C) and a synchronous control (incubated in Lyon RX2 incubator at 39 - 400 C), which simulated the temperature of the space-flight incubator. Following space-flight trials, post-flight trials were conducted where quail eggs were incubated in Lyon RX2 or Slovakian incubators under various temperatures with or without launch dynamics. Eggshells from all study trials were retrieved and analyzed for calcium content to determine if its utilization by developing quail embryos was affected by activities preceding space-flight or during incubation in space under microgravity. Results from the pre-flight and post-flight showed that pre-flight activities and shuttle launch dynamics had no effect on calcium uptake from the eggshell by developing embryos. However, calcium uptake from the eggshell by developing embryos incubated in micro,aravity was impaired by 12.6% when compared to embryos incubated on earth under laboratory control environment. This impairment was unlikely due to factors other than microgravity. In general, calcium utilization by developing embryos increased with age of incubation with the most increase occurring at day 16 of incubation.

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

PubMed

Zhang, Xuemei; Li, Fangping; Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

2015-01-01

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

PubMed Central

Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

2015-01-01

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells. PMID:25902045

Effect of ascorbate and dehydroascorbate on tissue uptake of glucose.

PubMed

Mooradian, A D

1987-09-01

In vitro studies have suggested that ascorbate or dehydroascorbate share with glucose the same tissue-transport carrier. To determine if ascorbic acid or its oxidized form can inhibit tissue uptake of glucose, the brain uptake index (BUI) and muscle uptake index of glucose were determined by single arterial injection tissue-sampling technique. The injectate was either buffered Ringer's solution with varying concentrations of ascorbate, dehydroascorbate (pH 7.4), or 70% serum from individuals on vitamin C supplements. Ascorbic acid over a wide range of concentrations (0-10,000 mg/L) did not reduce the BUI. Ascorbic acid reduced BUI from the control value of 33 +/- 3.2 to 20.1 +/- 2.2% (P less than .01) only at 100,000 mg/L; this effect was probably secondary to osmotic disruption of blood-brain barrier. In contrast, dehydroascorbate inhibited the BUI of glucose from baseline value of 32.8 +/- 1.1 to 10.7 +/- 0.67%, with an estimated Ki of 13.0 mM. Masseter muscle glucose uptake was not significantly altered over a wide range of ascorbate or dehydroascorbate concentrations in the injectate. Dehydroascorbate (7500 mg/L) did not significantly reduce the BUI of [14C]phenylalanine (55.2 +/- 4.4 vs. 62.1 +/- 4.2% in controls). When serum from six individuals on calcium ascorbate (3-5 g/day) was compared with that of nine controls, the BUI was not different (19.3 +/- 1.7 vs. 19.3 +/- 1.1%). Similarly, supplementing the diet of eight healthy volunteers with 1 g calcium ascorbate for 8 days did not alter the BUI of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

PubMed Central

Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2014-01-01

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

Accumulation of phenanthrene by roots of intact wheat (Triticum acstivnm L.) seedlings: passive or active uptake?

PubMed Central

2010-01-01

Background Polycyclic aromatic hydrocarbons (PAHs) are of particular concern due to their hydrophobic, recalcitrant, persistent, potentially carcinogenic, mutagenic and toxic properties, and their ubiquitous occurrence in the environment. Most of the PAHs in the environment are present in surface soil. Plants grown in PAH-contaminated soils or water can become contaminated with PAHs because of their uptake. Therefore, they may threaten human and animal health. However, the mechanism for PAHs uptake by crop roots is little understood. It is important to understand exactly how PAHs are transported into the plant root system and into the human food chain, since it is beneficial in governing crop contamination by PAHs, remedying soils or waters polluted by PAHs with plants, and modeling potential uptake for risk assessment. Results The possibility that plant roots may take up phenanthrene (PHE), a representative of PAHs, via active process was investigated using intact wheat (Triticum acstivnm L.) seedlings in a series of hydroponic experiments. The time course for PHE uptake into wheat roots grown in Hoagland solution containing 5.62 μM PHE for 36 h could be separated into two periods: a fast uptake process during the initial 2 h and a slow uptake component thereafter. Concentration-dependent PHE uptake was characterized by a smooth, saturable curve with an apparent Km of 23.7 μM and a Vmax of 208 nmol g-1 fresh weight h-1, suggesting a carrier-mediated uptake system. Competition between PHE and naphthalene for their uptake by the roots further supported the carrier-mediated uptake system. Low temperature and 2,4-dinitrophenol (DNP) could inhibit PHE uptake equally, indicating that metabolism plays a role in PHE uptake. The inhibitions by low temperature and DNP were strengthened with increasing concentration of PHE in external solution within PHE water solubility (7.3 μM). The contribution of active uptake to total absorption was almost 40% within PHE water solubility. PHE uptake by wheat roots caused an increase in external solution pH, implying that wheat roots take up PHE via a PHE/nH+ symport system. Conclusion It is concluded that an active, carrier-mediated and energy-consuming influx process is involved in the uptake of PHE by plant roots. PMID:20307286

Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

PubMed

Orellana, D I; Quintanilla, R A; Gonzalez-Billault, C; Maccioni, R B

2005-11-01

Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns.

Odorant receptors directly activate phospholipase C/inositol-1,4,5-trisphosphate coupled to calcium influx in Odora cells.

PubMed

Liu, Guang; Badeau, Robert M; Tanimura, Akihiko; Talamo, Barbara R

2006-03-01

Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5-trisphosphate (IP(3)), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP(3) receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system.

Application of natural zeolite for phosphorus and ammonium removal from aqueous solutions.

PubMed

Karapinar, Nuray

2009-10-30

Removal of both nutrients ammonium and phosphorus by natural zeolite has been studied in lab scale by using a mechanically stirred batch system (1000 ml). Zeolite, a mean particle size of 13 microm, was used as an adsorbent for the removal of ammonium and then as a seed material for the precipitation of calcium phosphate. A relationship was established between the uptake of ammonium by zeolite and the ratio of initial ammonium concentration to zeolite dosage. Ammonium uptake of zeolite was almost completed within initial 5 min of adsorption period. There is no pronounced effect of zeolite and ammonium, neither positive nor negative on the amount of calcium phosphate precipitation. The extent of the precipitation of phosphate increased with rising pH. It was also observed that when the system was allowed to relax at constant pH (i.e. under relatively low super saturations), a certain lag time was noted to elapse at the onset of the precipitation. At the pH 7.2, the amount of initial fast precipitation within 5 min and total precipitation within 120 min were around 34% and 93%, respectively. Precipitation of calcium phosphate on to ammonium-loaded zeolite was achieved at low super saturations (< pH 7.5) through secondary nucleation and crystal growth, leading to an increase in particle size.

Silver Nanoparticle-Directed Mast Cell Degranulation Is Mediated through Calcium and PI3K Signaling Independent of the High Affinity IgE Receptor

PubMed Central

Alsaleh, Nasser B.; Persaud, Indushekhar; Brown, Jared M.

2016-01-01

Engineered nanomaterial (ENM)-mediated toxicity often involves triggering immune responses. Mast cells can regulate both innate and adaptive immune responses and are key effectors in allergic diseases and inflammation. Silver nanoparticles (AgNPs) are one of the most prevalent nanomaterials used in consumer products due to their antimicrobial properties. We have previously shown that AgNPs induce mast cell degranulation that was dependent on nanoparticle physicochemical properties. Furthermore, we identified a role for scavenger receptor B1 (SR-B1) in AgNP-mediated mast cell degranulation. However, it is completely unknown how SR-B1 mediates mast cell degranulation and the intracellular signaling pathways involved. In the current study, we hypothesized that SR-B1 interaction with AgNPs directs mast cell degranulation through activation of signal transduction pathways that culminate in an increase in intracellular calcium signal leading to mast cell degranulation. For these studies, we utilized bone marrow-derived mast cells (BMMC) isolated from C57Bl/6 mice and RBL-2H3 cells (rat basophilic leukemia cell line). Our data support our hypothesis and show that AgNP-directed mast cell degranulation involves activation of PI3K, PLCγ and an increase in intracellular calcium levels. Moreover, we found that influx of extracellular calcium is required for the cells to degranulate in response to AgNP exposure and is mediated at least partially via the CRAC channels. Taken together, our results provide new insights into AgNP-induced mast cell activation that are key for designing novel ENMs that are devoid of immune system activation. PMID:27907088