Paclitaxel Induces Apoptosis in Breast Cancer Cells through Different Calcium—Regulating Mechanisms Depending on External Calcium Conditions

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Pan, Zhi; Avila, Andrew; Gollahon, Lauren

2014-01-01

Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis. PMID:24549172

DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

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Takahashi, S; Maecker, H T; Levy, R

1989-10-01

An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

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

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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.

Determination of critical epitope of PcMab-47 against human podocalyxin

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Shunsuke Itai

2018-07-01

Full Text Available Podocalyxin (PODXL is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA, flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

Determination of critical epitope of PcMab-47 against human podocalyxin.

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Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2018-07-01

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

Inhibition of the β-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus.

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Lefebvre, Anne-Laure; Le Moigne, Vincent; Bernut, Audrey; Veckerlé, Carole; Compain, Fabrice; Herrmann, Jean-Louis; Kremer, Laurent; Arthur, Michel; Mainardi, Jean-Luc

2017-04-01

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M abscessus produces the broad-spectrum β-lactamase Bla Mab We determine whether inhibition of Bla Mab by avibactam improves the activity of imipenem against M. abscessus The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of Bla Mab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of Bla Mab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding Bla Mab was induced (20-fold) in the infected macrophages. Inhibition of Bla Mab by avibactam improved the efficacy of imipenem against M. abscessus in vitro , in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of Bla Mab , since the production of the β-lactamase is inducible in macrophages. Copyright © 2017 American Society for Microbiology.

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

International Nuclear Information System (INIS)

Wendling, W.W.; Harakal, C.

1987-01-01

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 ( 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 45 Ca uptake into cerebral artery strips during 5 minutes of 45 Ca loading; for potassium 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 45 Ca uptake but significantly blocked the increase in 45 Ca uptake induced by potassium or serotonin. Potassium, and to a lesser extent serotonin, each induced a brief increase in the rate of 45 Ca efflux into calcium-deficient solutions. Verapamil or nifedipine had no effect on basal or potassium-stimulated 45 Ca efflux. The results demonstrate that verapamil and nifedipine block 45 Ca uptake through both potential-operated (potassium) and receptor-operated (serotonin) channels in bovine middle cerebral arteries

Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance.

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Li, W; Carper, K; Liang, Y; Zheng, X X; Kuhr, C S; Reyes, J D; Perkins, D L; Thomson, A W; Perkins, J D

2006-12-01

Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to naïve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

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Bartlett, Paula J; Antony, Anil Noronha; Agarwal, Amit; Hilly, Mauricette; Prince, Victoria L; Combettes, Laurent; Hoek, Jan B; Gaspers, Lawrence D

2017-05-15

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca 2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca 2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca 2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) production and does not involve changes in the sensitivity of the IP 3 receptor or size of internal Ca 2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca 2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca 2+ ] i ) increases, which can adversely affect mitochondrial Ca 2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca 2+ -mobilizing hormones resulting in more sustained and prolonged [Ca 2+ ] i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca 2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) accumulation and phospholipase C

Electrical stimulation induces calcium-dependent release of NGF from cultured Schwann cells.

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Huang, Jinghui; Ye, Zhengxu; Hu, Xueyu; Lu, Lei; Luo, Zhuojing

2010-04-01

Production of nerve growth factor (NGF) from Schwann cells (SCs) progressively declines in the distal stump, if axonal regeneration is staggered across the suture site after peripheral nerve injuries. This may be an important factor limiting the outcome of nerve injury repair. Thus far, extensive efforts are devoted to modulating NGF production in cultured SCs, but little has been achieved. In the present in vitro study, electrical stimulation (ES) was attempted to stimulate cultured SCs to release NGF. Our data showed that ES was capable of enhancing NGF release from cultured SCs. An electrical field (1 Hz, 5 V/cm) caused a 4.1-fold increase in NGF release from cultured SCs. The ES-induced NGF release is calcium dependent. Depletion of extracellular or/and intracellular calcium partially/ completely abolished the ES-induced NGF release. Further pharmacological interventions showed that ES induces calcium influx through T-type voltage-gated calcium channels and mobilizes calcium from 1, 4, 5-trisphosphate-sensitive stores and caffeine/ryanodine-sensitive stores, both of which contributed to the enhanced NGF release induced by ES. In addition, a calcium-triggered exocytosis mechanism was involved in the ES-induced NGF release from cultured SCs. These findings show the feasibility of using ES in stimulating SCs to release NGF, which holds great potential in promoting nerve regeneration by enhancing survival and outgrowth of damaged nerves, and is of great significance in nerve injury repair and neuronal tissue engineering.

Bighorn sheep × domestic sheep hybrids survive Mannheimia haemolytica challenge in the absence of vaccination.

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Subramaniam, R; Shanthalingam, S; Bavananthasivam, J; Kugadas, A; Raghavan, B; Batra, S A; Herndon, C N; Rodriguez, J; Tibary, A; Nelson, D; Potter, K A; Foreyt, W J; Srikumaran, S

2014-06-04

Bighorn sheep (BHS, Ovis canadensis) are much more susceptible than domestic sheep (DS, Ovis aries) to pneumonia caused by leukotoxin (Lkt)-producing members of the Family Pasteurellaceae, particularly Mannheimia haemolytica and Bibersteinia trehalosi. Leukotoxin is widely accepted as the critical virulence factor of these bacteria since Lkt-negative mutants do not cause death of BHS. Typically, DS carry Lkt-positive M. haemolytica and/or B. trehalosi as commensal bacteria in their nasopharynx. In contrast, most BHS do not carry Lkt-positive M. haemolytica or B. trehalosi, or carry Lkt-negative strains in their nasopharynx. In previous studies, we demonstrated that unimmunized DS resist M. haemolytica challenge while BHS succumb to it. We hypothesized that Lkt-neutralizing antibodies, induced by Lkt-positive M. haemolytica and/or B. trehalosi innately carried by DS in their nasopharynx, render them less susceptible to infection by these bacteria. In this study we developed BHS×DS F1 hybrids by artificial insemination of domestic ewes with BHS semen. F1 hybrids were fertile, and produced F2 hybrids and back-crosses. The F1, F2, and back-crosses were raised together with domestic ewes. All these animals acquired Lkt-positive M. haemolytica and/or B. trehalosi, and developed high titers of Lkt-neutralizing antibodies in the absence of vaccination. Furthermore, all of these animals resisted challenge with lethal dose of M. haemolytica. These results suggest that lack of previous exposure to Lkt is at least partially responsible for fatal pneumonia in BHS when they acquire Lkt-positive M. haemolytica and/or B. trehalosi from DS when the two species commingle. Copyright © 2014 Elsevier B.V. All rights reserved.

Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

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Qin, S; Chock, P B

2001-07-10

Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for

Visualisation of an nsPEF induced calcium wave using the genetically encoded calcium indicator GCaMP in U87 human glioblastoma cells.

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Carr, Lynn; Bardet, Sylvia M; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

2018-02-01

Cytosolic, synthetic chemical calcium indicators are typically used to visualise the rapid increase in intracellular calcium ion concentration that follows nanosecond pulsed electric field (nsPEF) application. This study looks at the application of genetically encoded calcium indicators (GECIs) to investigate the spatiotemporal nature of nsPEF-induced calcium signals using fluorescent live cell imaging. Calcium responses to 44kV/cm, 10ns pulses were observed in U87-MG cells expressing either a plasma membrane targeted GECI (GCaMP5-G), or one cytosolically expressed (GCaMP6-S), and compared to the response of cells loaded with cytosolic or plasma membrane targeted chemical calcium indicators. Application of 100 pulses, to cells containing plasma membrane targeted indicators, revealed a wave of calcium across the cell initiating at the cathode side. A similar spatial wave was not observed with cytosolic indicators with mobile calcium buffering properties. The speed of the wave was related to pulse application frequency and it was not propagated by calcium induced calcium release. Copyright © 2017 Elsevier B.V. All rights reserved.

Calcium mobilization in HeLa cells induced by nitric oxide.

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Huang, Yimei; Zheng, Liqin; Yang, Hongqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

2014-01-01

Nitric oxide (NO) has been proposed to be involved in tumor growth and metastasis. However, the mechanism by which nitric oxide modulates cancer cell growth and metastasis on cellular and molecular level is still not fully understood. This work utilized confocal microscopy and fluorescence microplate reader to investigate the effects of exogenous NO on the mobilization of calcium, which is one of the regulators of cell migration, in HeLa cells. The results show that NO elevates calcium in concentration-dependent manner in HeLa cells. And the elevation of calcium induced by NO is due to calcium influx and calcium release from intracellular calcium stores. Moreover, calcium release from intracellular stores is dominant. Furthermore, calcium release from mitochondria is one of the modulation pathways of NO. These findings would contribute to recognizing the significance of NO in cancer cell proliferation and metastasis. © Wiley Periodicals, Inc.

Radiation-induced radical ions in calcium sulfite

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Bogushevich, S. E.

2006-07-01

We have used EPR to study the effect of γ radiation on calcium sulfite. We have observed and identified the radiation-induced radical ions SO 2 - (iso) with g = 2.0055 and SO 2 - (orth-1) with g1 = 2.0093, g2 = 2.0051, g3 = 2.0020, identical to the initial and thermally induced SO 2 - respectively, SO 3 - (iso) with g = 2.0031 and SO 3 - (axial) with g⊥ = 2.0040, g∥ = 2.0023, identical to mechanically induced SO 3 - . We have established the participation of radiation-induced radical ions SO 3 - in formation of post-radiation SO 2 - .

Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

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Brinton Roberta

2008-12-01

Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that APα-induced

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore.

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Elustondo, Pia A; Negoda, Alexander; Kane, Constance L; Kane, Daniel A; Pavlov, Evgeny V

2015-02-01

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix. Copyright © 2014 Elsevier B.V. All rights reserved.

First experiences with the AMERLEX-MAB FREE T4 assay

International Nuclear Information System (INIS)

Nijhof, W.A.; Penders, T.J.

1989-01-01

The new Amerlex-MAB FT 4 is a quick direct free T 4 assay with good reproducability. The correlation between the Amerlex-MAB FT 4 and the free T 4 of Byk is good. In the non-thyreoidal illness patient group no deviation for the values were found. Amerlex-MAB FT 4 is cheaper, because no total T4 has to be measured. More research has to be done for special patient sera. Disturbing influences as free fatty acids, heparin and auto-antibodies have to be checked. (R.B.). 3 refs.; 3 figs.; 4 tabs

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

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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

In vitro and in vivo comparison of binding of 99m-Tc-labeled anti-CEA MAb F33-104 with 99m-Tc-labeled anti-CEA MAb BW431/26

International Nuclear Information System (INIS)

Watanabe, N.; Gunma Univ. School of Medicine; Oriuchi, N.; Inoue, T.; Sugiyama, S.; Kuroki, M.; Matsuoka, Y.; Tanada, S.; Murata, H.; Sasaki, Y.

1999-01-01

Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tc-labeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 10 7 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tc-activity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer. (orig.) [de

The anti-(+-methamphetamine monoclonal antibody mAb7F9 attenuates acute (+-methamphetamine effects on intracranial self-stimulation in rats.

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Andrew C Harris

Full Text Available Passive immunization with monoclonal antibodies (mAbs against (+-methamphetamine (METH is being evaluated for the treatment of METH addiction. A human/mouse chimeric form of the murine anti-METH mAb7F9 has entered clinical trials. This study examined the effects of murine mAb7F9 on certain addiction-related behavioral effects of METH in rats as measured using intracranial self-stimulation (ICSS. Initial studies indicated that acute METH (0.1-0.56 mg/kg, s.c. lowered the minimal (threshold stimulation intensity that maintained ICSS. METH (0.3 mg/kg, s.c. also blocked elevations in ICSS thresholds (anhedonia-like behavior during spontaneous withdrawal from a chronic METH infusion (10 mg/kg/day x 7 days. In studies examining effects of i.v. pretreatment with mAb7F9 (at 30, 100, or 200 mg/kg, 200 mg/kg blocked the ability of an initial injection of METH (0.3 mg/kg, s.c. to reduce baseline ICSS thresholds, but was less capable of attenuating the effect of subsequent daily injections of METH. MAb7F9 (200 mg/kg also produced a small but significant reduction in the ability of METH (0.3 mg/kg, s.c. to reverse METH withdrawal-induced elevations in ICSS thresholds. These studies demonstrate that mAb7F9 can partially attenuate some addiction-related effects of acute METH in an ICSS model, and provide some support for the therapeutic potential of mAb7F9 for the treatment of METH addiction.

Podocyte changes after induction of acute albuminuria in mice by anti-aminopeptidase A mAb.

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Dijkman, Henry B P M; Gerlofs-Nijland, Miriam E; van der Laak, Jeroen A W M; Wetzels, Jack F M; Groenen, Patricia J T A; Assmann, Karel J M

2003-01-01

Administration of a specific combination of anti-aminopeptidase A (APA) mAb (ASD-37/41) in mice induces an acute albuminuria which is independent of angiotensin II, a well-known substrate of APA. In the present experiments, we examined whether binding of the mAb initiated changes in the podocytic expression of cytoskeleton (-associated), adhesion and slit-diaphragm proteins in relation to the time course of albuminuria. In addition, we measured ultrastructurally the extent of foot process retraction (the number of foot processes per microm GBM) and the width of the slit pore between the podocytes by morphometric methods. An injection of the mAb combination ASD-37/41 induced a massive but transient albuminuria that started at 6 h, and peaked at 8 h, after which it declined. However, even at day 7 after injection of the mAbs some albuminuria was present. Injection of the combination ASD-3/41 or saline did not induce an albuminuria. Notably, we observed changes in the staining of CD2AP and podocin, two slit-pore-associated proteins that coincided with the start of the albuminuria. Nephrin staining was reduced and podocytic actin staining became more granular only at a time albuminuria was declining (24 h). The number of foot processes per microm GBM was already decreased at 4 h with a further reduction thereafter. The width of the slit pore was unchanged at the time of peak albuminuria and gradually decreased thereafter. At day 7, podocytic foot process effacement was even more prominent although albuminuria was only slightly abnormal. Expression of CD2AP was still granular. We observed however a change toward normal in the expression of podocin. Injection of saline or ASD-3/41 had no effect on the expression of podocytic proteins, the number of foot processes or width of the slit pore. Our data show that the onset of albuminuria in the anti-APA model is related to alterations in CD2AP and podocin, proteins that are important for maintaining slit-diaphragm structure

Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand.

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Rajak, Poonam; Vijayalakshmi, M A; Jayaprakash, N S

2013-05-05

Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 10(8) to 10(9)M(-1). An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined. Copyright © 2013 Elsevier B.V. All rights reserved.

A model of propagating calcium-induced calcium release mediated by calcium diffusion

NARCIS (Netherlands)

Backx, P. H.; de Tombe, P. P.; van Deen, J. H.; Mulder, B. J.; ter Keurs, H. E.

1989-01-01

The effect of sudden local fluctuations of the free sarcoplasmic [Ca++]i in cardiac cells on calcium release and calcium uptake by the sarcoplasmic reticulum (SR) was calculated with the aid of a simplified model of SR calcium handling. The model was used to evaluate whether propagation of calcium

Structural and biochemical characterization of the cell fate determining nucleotidyltransferase fold protein MAB21L1.

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de Oliveira Mann, Carina C; Kiefersauer, Reiner; Witte, Gregor; Hopfner, Karl-Peter

2016-06-08

The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.

Calmodulin immunolocalization to cortical microtubules is calcium independent

Energy Technology Data Exchange (ETDEWEB)

Fisher, D.D.; Cyr, R.J.

1992-12-31

Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 {mu}M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 {mu}M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

Calmodulin immunolocalization to cortical microtubules is calcium independent

Energy Technology Data Exchange (ETDEWEB)

Fisher, D.D.; Cyr, R.J.

1992-01-01

Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10), was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical Mats of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tobacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these Mats, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized Mats were lysed in the presence of various concentrations of calcium and examined for the association of Cam with cortical Mats. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical Mats, while at 3.6 [mu]M calcium, this association was completely abolished. The results are discussed in terms of a model in which CaM associates with Mats via two types of interactions; one calcium dependent and one independent.

Angiotensin II induces calcium/calcineurin signaling and podocyte injury by downregulating microRNA-30 family members.

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Zhao, Yue; Wu, Junnan; Zhang, Mingchao; Zhou, Minlin; Xu, Feng; Zhu, Xiaodong; Zhou, Xianguang; Lang, Yue; Yang, Fan; Yun, Shifeng; Shi, Shaolin; Liu, Zhihong

2017-08-01

Angiotensin II (AngII) is capable of inducing calcium/calcineurin signaling and podocyte injury; however, the precise underlying mechanism is not well understood. Because we have previously demonstrated that microRNA-30s (miR-30s) inhibit calcium/calcineurin signaling in podocytes, we hypothesize that AngII may induce podocyte injury by downregulating miR-30s and thereby activating calcium/calcineurin signaling. To test this hypothesis, we used an AngII-induced podocyte injury mouse model. The mice were treated with AngII via infusion for 28 days, which resulted in hypertension, albuminuria, and glomerular damage. AngII treatment also resulted in a significant reduction of miR-30s and upregulation of calcium/calcineurin signaling components, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, which are the known targets of miR-30s in podocytes. The delivery of miR-30a-expressing lentivirus to the podocytes on day 14 of the infusion ameliorated the AngII-induced podocyte and glomerular injury and attenuated the upregulation of the calcium/calcineurin signaling components. Similarly, treatment with losartan, which is an AngII receptor blocker, also prevented AngII-induced podocyte injury and calcium/calcineurin signaling activation. Notably, losartan was found to sustain miR-30 levels during AngII treatment both in vivo and in vitro. In conclusion, the effect of AngII on podocytes is in part mediated by miR-30s through calcium/calcineurin signaling, a novel mechanism underlying AngII-induced podocyte injury. • AngII infusion resulted in downregulation of miR-30s in podocytes. • Exogenous miR-30a delivery mitigated the glomerular and podocyte injuries induced by AngII. • Both miR-30a and losartan prevented AngII-induced activation of calcium-calcineurin signaling.

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

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Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

2014-08-01

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

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Vőfély, Gergő; Berecz, Tünde; Szabó, Eszter; Szebényi, Kornélia; Hathy, Edit; Orbán, Tamás I; Sarkadi, Balázs; Homolya, László; Marchetto, Maria C; Réthelyi, János M; Apáti, Ágota

2018-04-01

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

The UC Davis/NIH NeuroMab Facility

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Federal Laboratory Consortium — The mission of the UC Davis/NIH NeuroMab facility is to generate and distribute high quality, validated mouse monoclonal antibodies against molecular targets found...

Usefulness of cardiotoxicity assessment using calcium transient in human induced pluripotent stem cell-derived cardiomyocytes.

Science.gov (United States)

Watanabe, Hitoshi; Honda, Yayoi; Deguchi, Jiro; Yamada, Toru; Bando, Kiyoko

2017-01-01

Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be an attractive strategy for assessing compounds on cardiac contractility. In addition, as arrhythmogenic compounds are known to induce characteristic waveform changes in hiPSC-CMs, it is expected that calcium transient would allow evaluation of not only compound-induced effects on cardiac contractility, but also compound arrhythmogenic potential. Using a combination of calcium transient in hiPSC-CMs and a fast kinetic fluorescence imaging detection system, we examined in this study changes in calcium transient waveforms induced by a series of 17 compounds that include positive/negative inotropic agents as well as cardiac ion channel activators/inhibitors. We found that all positive inotropic compounds induced an increase in peak frequency and/or peak amplitude. The effects of a negative inotropic compound could clearly be detected in the presence of a β-adrenergic receptor agonist. Furthermore, most arrhythmogenic compounds raised the ratio of peak decay time to peak rise time (D/R ratio) in calcium transient waveforms. Compound concentrations at which these parameters exceeded cutoff values correlated well with systemic exposure levels at which arrhythmias were reported to be evoked. In conclusion, we believe that peak analysis of calcium transient and determination of D/R ratio are reliable methods for assessing compounds' cardiac contractility and arrhythmogenic potential, respectively. Using these approaches would allow selection of compounds with low cardiotoxic potential at the early stage of drug discovery.

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

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Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Drug Release Profile from Calcium-Induced Alginate-Phosphate Composite Gel Beads

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Yoshifumi Murata

2009-01-01

Full Text Available Calcium-induced alginate-phosphate composite gel beads were prepared, and model drug release profiles were investigated in vitro. The formation of calcium phosphate in the alginate gel matrix was observed and did not affect the rheological properties of the hydrogel beads. X-ray diffraction patterns showed that the calcium phosphate does not exist in crystalline form in the matrix. The initial release amount and release rate of a water-soluble drug, diclofenac, from the alginate gel beads could be controlled by modifying the composition of the matrix with calcium phosphate. In contrast, the release profile was not affected by the modification for hydrocortisone, a drug only slightly soluble in water.

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

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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 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.

Analysis of Japanese Municipalities With Geopark, MAB, and GIAHS Certification

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Ryo Kohsaka

2015-11-01

Full Text Available We analyzed the discussions of Japanese municipalities in their process for obtaining certifications for the Geoparks by the United Nations Educational, Scientific and Cultural Organization (UNESCO, the Man and the Biosphere Programme (MAB by the UNESCO, and the Globally Important Agricultural Heritage systems (GIAHS by the Food and Agriculture Organization (FAO of the United Nations. The official records at the municipality diet were analyzed in a quantitative manner from 2011 to 2013. As the first step, we analyzed the eight municipalities of Noto and Sado for the GIAHS, the cities Itoigawa and Hakusan for the Geopark, and Katsuyama Yamanouchi village from Nagano for the MAB. As individual examples, we analyzed City of Suzu with GIAHS, Itoigawa (Geopark, and Yamanouchi town (MAB with the text-mining approach. For the GIAHS, it was clear that the larger municipalities with city status tended to discuss certification issues more frequently than the smaller towns and villages. Terms such as conservation and certification tended to be used with GIAHS at the Suzu City. The term brand was used with GIAHS and MAB but not for the Geopark. The findings using quantitative methods are at initial stage for analysis of municipality strategies and require further future research.

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

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Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

2007-11-01

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

The Effect of Cell Immobilization by Calcium Alginate on Bacterially Induced Calcium Carbonate Precipitation

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Mostafa Seifan

2017-10-01

Full Text Available Microbially induced mineral precipitation is recognized as a widespread phenomenon in nature. A diverse range of minerals including carbonate, sulphides, silicates, and phosphates can be produced through biomineralization. Calcium carbonate (CaCO3 is one of the most common substances used in various industries and is mostly extracted by mining. In recent years, production of CaCO3 by bacteria has drawn much attention because it is an environmentally- and health-friendly pathway. Although CaCO3 can be produced by some genera of bacteria through autotrophic and heterotrophic pathways, the possibility of producing CaCO3 in different environmental conditions has remained a challenge to determine. In this study, calcium alginate was proposed as a protective carrier to increase the bacterial tolerance to extreme environmental conditions. The model showed that the highest concentration of CaCO3 is achieved when the bacterial cells are immobilized in the calcium alginate beads fabricated using 1.38% w/v Na-alginate and 0.13 M CaCl2.

Protein-induced satiation and the calcium-sensing receptor

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Ojha U

2018-03-01

Full Text Available Utkarsh Ojha Faculty of Medicine, Imperial College School of Medicine, Imperial College London, London, UK Abstract: Obesity is a major global health issue. High-protein diets have been shown to be associated with weight loss and satiety. The precise mechanism by which protein-rich diets promote weight loss remains unclear. Evidence suggests amino acids, formed as a consequence of protein digestion, are sensed by specific receptors on L-cells in the gastrointestinal (GI tract. These L-cells respond by secreting gut hormones that subsequently induce satiety. In recent years, the calcium-sensing receptor has been identified in several cells of the GI tract, including L-cells, and suggested to sense specific amino acids. This review evaluates the evidence for protein-rich diets in inducing weight loss and how the calcium-sensing receptor may be implicated in this phenomenon. Commandeering the mechanisms by which elements of a protein-rich diet suppress appetite may provide another successful avenue for developing anti-obesity drugs. Keywords: amino acids, energy regulation, obesity therapy, glucagon-like-peptide-1, peptide YY

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Primary Lymphoid Tumors and Lymph Node Metastasis

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Blanco, Rancés; Blanco, Damián; Quintana, Yisel; Escobar, Xiomara; Rengifo, Charles E.; Osorio, Marta; Gutiérrez, Zailí; Lamadrid, Janet; Cedeño, Mercedes; Frómeta, Milagros; Carr, Adriana; Rengifo, Enrique

2013-01-01

The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10–1000 μg/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule. PMID:24381785

Image-based Modeling of Biofilm-induced Calcium Carbonate Precipitation

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Connolly, J. M.; Rothman, A.; Jackson, B.; Klapper, I.; Cunningham, A. B.; Gerlach, R.

2013-12-01

Pore scale biological processes in the subsurface environment are important to understand in relation to many engineering applications including environmental contaminant remediation, geologic carbon sequestration, and petroleum production. Specifically, biofilm induced calcium carbonate precipitation has been identified as an attractive option to reduce permeability in a lasting way in the subsurface. This technology may be able to replace typical cement-based grouting in some circumstances; however, pore-scale processes must be better understood for it to be applied in a controlled manor. The work presented will focus on efforts to observe biofilm growth and ureolysis-induced mineral precipitation in micro-fabricated flow cells combined with finite element modelling as a tool to predict local chemical gradients of interest (see figure). We have been able to observe this phenomenon over time using a novel model organism that is able to hydrolyse urea and express a fluorescent protein allowing for non-invasive observation over time with confocal microscopy. The results of this study show the likely existence of a wide range of local saturation indices even in a small (1 cm length scale) experimental system. Interestingly, the locations of high predicted index do not correspond to the locations of higher precipitation density, highlighting the need for further understanding. Figure 1 - A micro-fabricated flow cell containing biofilm-induced calcium carbonate precipitation. (A) Experimental results: Active biofilm is in green and dark circles are calcium carbonate crystals. Note the channeling behavior in the top of the image, leaving a large hydraulically inactive area in the biofilm mass. (B) Finite element model: The prediction of relative saturation of calcium carbonate (as calcite). Fluid enters the system at a low saturation state (blue) but areas of high supersaturation (red) are predicted within the hydraulically inactive area in the biofilm. If only effluent

Activation of TRPV1-dependent calcium oscillation exacerbates seawater inhalation-induced acute lung injury.

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Li, Congcong; Bo, Liyan; Liu, Qingqing; Liu, Wei; Chen, Xiangjun; Xu, Dunquan; Jin, Faguang

2016-03-01

Calcium is an important second messenger and it is widely recognized that acute lung injury (ALI) is often caused by oscillations of cytosolic free Ca2+. Previous studies have indicated that the activation of transient receptor potential‑vanilloid (TRPV) channels and subsequent Ca2+ entry initiates an acute calcium‑dependent permeability increase during ALI. However, whether seawater exposure induces such an effect through the activation of TRPV channels remains unknown. In the current study, the effect of calcium, a component of seawater, on the inflammatory reactions that occur during seawater drowning‑induced ALI, was examined. The results demonstrated that a high concentration of calcium ions in seawater increased lung tissue myeloperoxidase activity and the secretion of inflammatory mediators, such as tumor necrosis factor‑α (TNF‑α) and interleukin (IL)‑1β and IL‑6. Further study demonstrated that the seawater challenge elevated cytosolic Ca2+ concentration, indicated by [Ca2+]c, by inducing calcium influx from the extracellular medium via TRPV1 channels. The elevated [Ca2+c] may have resulted in the increased release of TNF‑α and IL‑1β via increased phosphorylation of nuclear factor‑κB (NF‑κB). It was concluded that a high concentration of calcium in seawater exacerbated lung injury, and TRPV1 channels were notable mediators of the calcium increase initiated by the seawater challenge. Calcium influx through TRPV1 may have led to greater phosphorylation of NF‑κB and increased release of TNF‑α and IL‑1β.

ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

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Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

2017-06-01

Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

Mathematical modeling of calcium waves induced by mechanical stimulation in keratinocytes.

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Yasuaki Kobayashi

Full Text Available Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity.

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

2011-04-15

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.

Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation

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Devesh Radhakrishnan

2017-12-01

Full Text Available In order to meet desired drug product quality targets, the glycosylation profile of biotherapeutics such as monoclonal antibodies (mAbs must be maintained consistently during manufacturing. Achieving consistent glycan distribution profiles requires identifying factors that influence glycosylation, and manipulating them appropriately via well-designed control strategies. Now, the cell culture media supplement, MnCl2, is known to alter the glycosylation profile in mAbs generally, but its effect, particularly when introduced at different stages during cell growth, has yet to be investigated and quantified. In this study, we evaluate the effect of time-dependent addition of MnCl2 on the glycan profile quantitatively, using factorial design experiments. Our results show that MnCl2 addition during the lag and exponential phases affects the glycan profile significantly more than stationary phase supplementation does. Also, using a novel computational technique, we identify various combinations of glycan species that are affected by this dynamic media supplementation scheme, and quantify the effects mathematically. Our experiments demonstrate the importance of taking into consideration the time of addition of these trace supplements, not just their concentrations, and our computational analysis provides insight into what supplements to add, when, and how much, in order to induce desired changes.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

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Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Dynamics of immature mAb glycoform secretion during CHO cell culture

DEFF Research Database (Denmark)

Jimenez del Val, Ioscani; Fan, Yuzhou; Weilguny, Dietmar

2016-01-01

Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immu......Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy...

Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells.

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Lecourieux, David; Lamotte, Olivier; Bourque, Stéphane; Wendehenne, David; Mazars, Christian; Ranjeva, Raoul; Pugin, Alain

2005-12-01

We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca2+](nuc)) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the beta-1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca2+](nuc) elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca2+](nuc) elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca2+](nuc) rise depends on free cytosolic calcium, IP3, and active oxygen species (AOS) but is independent of nitric oxide.

Development of at-line assay to monitor charge variants of MAbs during production.

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St Amand, M M; Ogunnaike, B A; Robinson, A S

2014-01-01

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers.

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

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Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

Effects of diphosphonate on kidney calcium content and duodenal absorption of 45calcium

International Nuclear Information System (INIS)

Goulding, A.; Cameron, V.

1978-01-01

In rats the relationships between EHDP-induced changes in serum calcium concentration, kidney calcium content and duodenal transport of 45 calcium were studied. Body weights and kidney weights were similar in all groups. EHDP administration was associated with an increase in serum calcium concentration and kidney calcium content, and a decrease in duodenal 45 calcium transport. In the EHDP-treated rats, there was a significant negative correlation between kidney calcium concentration and duodenal 45 calcium transport but no correlation between either kidney calcium content and serum calcium concentration (r = 0.116) or between serum calcium concentration and duodenal 45 calcium transport (r = 0.02). Further experiments will be needed to determine whether the demonstrated increase in kidney calcium content induced by EHDP administration was the cause of, or was secondary to, inhibition of 1, 25(OH) 2 D 3 synthesis. (orig./AJ) [de

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

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Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

2017-08-01

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Calcium signals and caspase-12 participated in paraoxon-induced apoptosis in EL4 cells.

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Li, Lan; Cao, Zhiheng; Jia, Pengfei; Wang, Ziren

2010-04-01

In order to investigate whether calcium signals participate in paraoxon (POX)-induced apoptosis in EL4 cells, real-time laser scanning confocal microscopy (LSCM) was used to detect Ca(2+) changes during the POX application. Apoptotic rates of EL4 cells and caspase-12 expression were also evaluated. POX (1-10nM) increased intracellular calcium concentration ([Ca(2+)]i) in EL4 cells in a dose-dependent manner at early stage (0-2h) of POX application, and apoptotic rates of EL4 cells after treatment with POX for 16h were also increased in a dose-dependent manner. Pre-treatment with EGTA, heparin or procaine attenuated POX-induced [Ca(2+)]i elevation and apoptosis. Additionally, POX up-regulated caspase-12 expression in a dose-dependent manner, and pre-treatment with EGTA, heparin or procaine significantly inhibited POX-induced increase of caspase-12 expression. Our results suggested that POX induced [Ca(2+)]i elevation in EL4 cells at the early stage of POX-induced apoptosis, which might involve Ca(2+) efflux from the endoplasmic reticulum (ER) and Ca(2+) influx from extracellular medium. Calcium signals and caspase-12 were important upstream messengers in POX-induced apoptosis in EL4 cells. The ER-associated pathway possibly operated in this apoptosis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

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Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

2016-01-01

Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

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Zhong Yao

Full Text Available The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

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Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

Germline-specific MATH-BTB substrate adaptor MAB1 regulates spindle length and nuclei identity in maize.

Science.gov (United States)

Juranič, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanic, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-12-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Maitotoxin-induced liver cell death involving loss of cell ATP following influx of calcium

International Nuclear Information System (INIS)

Kutty, R.K.; Singh, Y.; Santostasi, G.; Krishna, G.

1989-01-01

Maitotoxin, one of the most potent marine toxins known, produced cell death in cultures of rat hepatocytes with a TD50 of 80 pM at 24 hr. The cell death, as indicated by a dose- and time-dependent leakage of lactate dehydrogenase (LDH), was also associated with the leakage of [14C]adenine nucleotides from hepatocytes prelabeled with [14C]-adenine. The toxic effect of maitotoxin was completely abolished by the omission of calcium from the culture medium. The cell death induced by maitotoxin increased with increasing concentrations of calcium in the medium. Treatment of hepatocytes with low concentrations of the toxin (less than 0.5 ng/ml) resulted in increases in 45Ca influx into the cells. At higher concentrations of maitotoxin (greater than 1ng/ml), the initial increase in 45Ca influx was followed by the release of the 45Ca from the cells into the medium. Since the 45Ca release paralleled the LDH leakage, the release of calcium was due to cell death. The 45Ca influx, [14C]adenine nucleotide leakage, and LDH leakage were effectively inhibited by verapamil, a calcium channel blocker. Maitotoxin also induced a time- and dose-dependent loss of ATP from hepatocytes, which preceded the [14C]adenine nucleotide and LDH leakage. Thus, it appears that the cell death resulting from maitotoxin treatment is caused by the elevated intracellular calcium, which in turn inhibits mitochondrial oxidative phosphorylation causing depletion of cell ATP. Loss of cell ATP may be the causative event in the maitotoxin-induced cell death

Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

Science.gov (United States)

Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

2017-02-01

The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG 2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

SPECT scintigraphy with HDP and Mab BW 250/183 of loosened hip endoprothesis

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Predic, P [Hospital Celje, Celje (Sierra Leone); Gregoric, E [Hospital Izola, Izola (Sierra Leone); Dodig, D [Clinical Hospital Centre, Zagreb (Croatia). Dept. of Nuclear Medicine and Radiation Protection

1994-10-01

Main problem of the loosened hip endoprothesis is in distinguishing between the aseptic and septic loosening of endoprothesis. The study involved 27 pts with a loosened hip; 15 pts with aseptic and 12 pts with septic loosening. The patients were injected 550-770 MBq Tc-99m-HDP and underwent SPECT scintigraphy of the hips to repeat then the examination with only 370 MBq Tc-99m-Mab Bw 230/183. HDP application evidenced positive accumulation at the endoprothesis in all patients with a loosened hip while Mab Bw 250/183 only in the patients with septic loosening. Conclusion: SPECT scintigraphy of hip endoprothesis with HDP and Mab BW 250/183 allows differential diagnosing between septic and aseptic hip loosening and hereby a correct therapeutical approach. (author).

Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

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Wen, Xianjie; Xu, Shiyuan; Liu, Hongzhen; Zhang, Quinguo; Liang, Hua; Yang, Chenxiang; Wang, Hanbing

2013-01-01

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation. PMID:23658789

Neurotoxicity induced by bupivacaine via T-type calcium channels in SH-SY5Y cells.

Directory of Open Access Journals (Sweden)

Xianjie Wen

Full Text Available There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca(2+ ([Ca2+]i, cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.

Blockade of store-operated calcium entry alleviates high glucose-induced neurotoxicity via inhibiting apoptosis in rat neurons.

Science.gov (United States)

Xu, Zhenkuan; Xu, Wenzhe; Song, Yan; Zhang, Bin; Li, Feng; Liu, Yuguang

2016-07-25

Altered store-operated calcium entry (SOCE) has been suggested to be involved in many diabetic complications. However, the association of altered SOCE and diabetic neuronal damage remains unclear. This study aimed to investigate the effects of altered SOCE on primary cultured rat neuron injury induced by high glucose. Our data demonstrated that high glucose increased rat neuron injury and upregulated the expression of store-operated calcium channel (SOC). Inhibition of SOCE by a pharmacological inhibitor and siRNA knockdown of stromal interaction molecule 1 weakened the intracellular calcium overload, restored mitochondrial membrane potential, downregulated cytochrome C release and inhibited cell apoptosis. As well, treatment with the calcium chelator BAPTA-AM prevented cell apoptosis by ameliorating the high glucose-increased intracellular calcium level. These findings suggest that SOCE blockade may alleviate high glucose-induced neuronal damage by inhibiting apoptosis. SOCE might be a promising therapeutic target in diabetic neurotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

Science.gov (United States)

Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-01-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

Conservation Compromises: The MAB and the Legacy of the International Biological Program, 1964-1974.

Science.gov (United States)

Schleper, Simone

2017-02-01

This article looks at the International Biological Program (IBP) as the predecessor of UNESCO's well-known and highly successful Man and the Biosphere Programme (MAB). It argues that international conservation efforts of the 1970s, such as the MAB, must in fact be understood as a compound of two opposing attempts to reform international conservation in the 1960s. The scientific framework of the MAB has its origins in disputes between high-level conservationists affiliated with the International Union for the Conservation of Nature and Natural Resources (IUCN) about what the IBP meant for the future of conservation. Their respective visions entailed different ecological philosophies as much as diverging sets of political ideologies regarding the global implementation of conservation. Within the IBP's Conservation Section, one group propagated a universal systems approach to conservation with a centralized, technocratic management of nature and society by an elite group of independent scientific experts. Within IUCN, a second group based their notion of environmental expert roles on a more descriptive and local ecology of resource mapping as practiced by UNESCO. When the IBP came to an end in 1974, both groups' ecological philosophies played into the scientific framework underlying the MAB's World Network or Biosphere Reserves. The article argues that it is impossible to understand the course of conservation within the MAB without studying the dynamics and discourses between the two underlying expert groups and their respective visions for reforming conservation.

Atorvastatin calcium inhibits phenotypic modulation of PDGF-BB-induced VSMCs via down-regulation the Akt signaling pathway.

Science.gov (United States)

Chen, Shuang; Liu, Baoqin; Kong, Dehui; Li, Si; Li, Chao; Wang, Huaqin; Sun, Yingxian

2015-01-01

Plasticity of vascular smooth muscle cells (VSMCs) plays a central role in the onset and progression of proliferative vascular diseases. In adult tissue, VSMCs exist in a physiological contractile-quiescent phenotype, which is defined by lack of the ability of proliferation and migration, while high expression of contractile marker proteins. After injury to the vessel, VSMC shifts from a contractile phenotype to a pathological synthetic phenotype, associated with increased proliferation, migration and matrix secretion. It has been demonstrated that PDGF-BB is a critical mediator of VSMCs phenotypic switch. Atorvastatin calcium, a selective inhibitor of 3-hydroxy-3-methyl-glutaryl l coenzyme A (HMG-CoA) reductase, exhibits various protective effects against VSMCs. In this study, we investigated the effects of atorvastatin calcium on phenotype modulation of PDGF-BB-induced VSMCs and the related intracellular signal transduction pathways. Treatment of VSMCs with atorvastatin calcium showed dose-dependent inhibition of PDGF-BB-induced proliferation. Atorvastatin calcium co-treatment inhibited the phenotype modulation and cytoskeleton rearrangements and improved the expression of contractile phenotype marker proteins such as α-SM actin, SM22α and calponin in comparison with PDGF-BB alone stimulated VSMCs. Although Akt phosphorylation was strongly elicited by PDGF-BB, Akt activation was attenuated when PDGF-BB was co-administrated with atorvastatin calcium. In conclusion, atorvastatin calcium inhibits phenotype modulation of PDGF-BB-induced VSMCs and activation of the Akt signaling pathway, indicating that Akt might play a vital role in the modulation of phenotype.

Smad signaling pathway in pathogenesis of kidney injury induced by calcium oxalate stone in rats

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Fan Zhang

2016-10-01

Full Text Available Objective: To investigate the involvement of Smad signaling pathway in the pathogenesis of kidney injury induced by calcium oxalate stone in rats to provide a reference for clinical treatment. Methods: Clean SD rats were randomly divided into 3 group, namely the control group, model group and pirfenidone group. Ethylene glycol + αhydroxy vitamin D3 was used as a stone-inducing agent to replicate the renal calcium oxalate stone model. Rats in the pirfenidone group were treated with pirfenidone intragastric administration. The serum Cr, BUN and 24-hour oxalate and calcium in renal tissues were assayed. The expressions of Bax/ Bcl2 protein, Caspase3 protein, TGFβ, Smad1, Smad2 and Smad3 proteins were detected by the fluorescent quantitation PCR method. Results: Compared with the rats of the control group, the results showed that the levels of serum BUN, Cr and 24-hour oxalate in rats of the model group were increased greatly, Bax and Caspase3 mRNA also increased while the level of Bcl2 decreased significantly, and the expressions of TGFβ, Smad1, Smad2 and Smad3 proteins increased distinctly as well (P<0.01. These abnormal parameters could be normalized effectively by pirfenidone. Conclusions: Activated TGFβ/Smad signaling pathway is involved in the pathogenesis of kidney injury induced by calcium oxalate stone in rats.

Sequence diversity, cytotoxicity and antigenic similarities of the leukotoxin of isolates of Mannheimia species from mastitis in domestic sheep.

Science.gov (United States)

Omaleki, Lida; Browning, Glenn F; Barber, Stuart R; Allen, Joanne L; Srikumaran, Subramaniam; Markham, Philip F

2014-11-07

Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid insight into heating-induced phase transformations in the solid state of the calcium salt of atorvastatin using multivariate data analysis

DEFF Research Database (Denmark)

Christensen, Niels Peter Aae; Van Eerdenbrugh, Bernard; Kwok, Kaho

2013-01-01

To investigate the heating-induced dehydration and melting behavior of the trihydrate phase of the calcium salt of atorvastatin.......To investigate the heating-induced dehydration and melting behavior of the trihydrate phase of the calcium salt of atorvastatin....

Assessment of fluoride-induced changes on physicochemical and structural properties of bone and the impact of calcium on its control in rabbits.

Science.gov (United States)

Gopalakrishnan, Subarayan Bothi; Viswanathan, Gopalan

2012-03-01

Bone deformities caused by the chronic intake of large quantities of fluoride and the beneficial effect of calcium on its control have been studied for many years, but only limited data are available on the quantitative effect of fluoride intake and the beneficial impact of calcium on fluoride-induced changes in bone at the molecular level. It is necessary to determine the degree of fluoride-induced changes in bone at different levels of fluoride intake to evaluate the optimum safe intake level of fluoride for maintaining bone health and quality. The ameliorative effect of calcium at different dose levels on minimizing fluoride-induced changes in bone is important to quantify the amount of calcium intake necessary for reducing fluoride toxicity. Thirty rabbits, 2 months old, were divided into five groups. Group I animals received 1 mg/l fluoride and 0.11% calcium diet; groups II and III received 10 mg/l fluoride and diet with 0.11% or 2.11% calcium, respectively; and groups IV and V received 150 mg/l fluoride and diet with 2.11% or 0.11% calcium, respectively. Analysis of bone density, ash content, fluoride, calcium, phosphorus, and Ca:P molar ratio levels after 6 months of treatment indicated that animals that received high fluoride with low-calcium diet showed significant detrimental changes in physicochemical properties of bone. Animals that received fluoride with high calcium intake showed notable amelioration of the impact of calcium on fluoride-induced changes in bone. The degree of fluoride-induced characteristic changes in structural properties such as crystalline size, crystallinity, and crystallographic "c"-axis length of bone apatite cells was also assessed by X-ray diffraction and Fourier transform infrared studies. X-ray images showed bone deformity changes such as transverse stress growth lines, soft tissue ossification, and calcification in different parts of bones as a result of high fluoride accumulation and the beneficial role of calcium

Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

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Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

Growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin-induced cachexia.

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Conte, Elena; Camerino, Giulia Maria; Mele, Antonietta; De Bellis, Michela; Pierno, Sabata; Rana, Francesco; Fonzino, Adriano; Caloiero, Roberta; Rizzi, Laura; Bresciani, Elena; Ben Haj Salah, Khoubaib; Fehrentz, Jean-Alain; Martinez, Jean; Giustino, Arcangela; Mariggiò, Maria Addolorata; Coluccia, Mauro; Tricarico, Domenico; Lograno, Marcello Diego; De Luca, Annamaria; Torsello, Antonio; Conte, Diana; Liantonio, Antonella

2017-06-01

Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose-limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium-dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS-R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood. By a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast-twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin-induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention. Cisplatin-treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up-regulation of atrogin1/Murf-1 genes and a down-regulation of Pgc1-a gene, all indexes of muscle atrophy, and by a two-fold increase in resting intracellular calcium, [Ca 2+ ] i , compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store-operated calcium entry were ~50% significantly reduced in cisplatin-treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in

The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans

DEFF Research Database (Denmark)

Jafari, Gholamali; Appleford, Peter J; Seago, Julian

2011-01-01

, an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat...

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen.

Science.gov (United States)

Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Watanabe, Kanako; Suzuki, Osamu

2018-02-01

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was investigated. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB-CHMINACA metabolites from a commercial source. The MAB-CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with β-glucuronidase. Among the six MAB-CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4-monohydroxycyclohexylmethyl metabolite M1 (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide) and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butylhydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB-CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB-CHMINACA metabolites in an authentic human urine specimen. Copyright © 2017 John Wiley & Sons, Ltd.

The transition between monostable and bistable states induced by time delay in intracellular calcium oscillation

International Nuclear Information System (INIS)

Duan, Wei-Long

2013-01-01

The revised role of the time delay of active processes with colored noises of transmission of intracellular Ca 2+ in intracellular calcium oscillation (ICO) is investigated by means of a first-order algorithm based on stochastic simulation. The simulation results indicate that time delay induces a double critical phenomenon and a transition between the monostable and bistable states of the ICO system. In addition, as the time delay increases, for a cytosolic Ca 2+ concentration with weak colored noises there appears a calcium burst, and the Ca 2+ concentration of the calcium store shows nonmonotonic variation. (paper)

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

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Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian; Gadeberg, Ole V; Frank, David A; Petersen, Jørgen; Jurlander, Jesper; Pedersen, Mikkel W

2013-10-01

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies. © 2013 John Wiley & Sons Ltd.

Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing — A bioprocess review

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Tobias Oppermann

2017-07-01

Full Text Available Mannheimia haemolytica leukotoxin (LKT is a known cause of bovine respiratory disease (BRD which results in severe economic losses in the cattle industry (up to USD 1 billion per year in the USA. Vaccines based on LKT offer the most promising measure to contain BRD outbreaks and are already commercially available. However, insufficient LKT yields, predominantly reflecting a lack of knowledge about the LKT expression process, remain a significant engineering problem and further bioprocess optimization is required to increase process efficiency. Most previous investigations have focused on LKT activity and cell growth, but neither of these parameters defines reliable criteria for the improvement of LKT yields. In this article, we review the most important process conditions and operational parameters (temperature, pH, substrate concentration, dissolved oxygen level, medium composition and the presence of metabolites from a bioprocess engineering perspective, in order to maximize LKT yields.

Glycine Receptor Activation Impairs ATP-Induced Calcium Transients in Cultured Cortical Astrocytes

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Tatiana P. Morais

2018-01-01

Full Text Available In central nervous system, glycine receptor (GlyR is mostly expressed in the spinal cord and brainstem, but glycinergic transmission related elements have also been identified in the brain. Astrocytes are active elements at the tripartite synapse, being responsible for the maintenance of brain homeostasis and for the fine-tuning of synaptic activity. These cells communicate, spontaneously or in response to a stimulus, by elevations in their cytosolic calcium (calcium transients, Ca2+T that can be propagated to other cells. How these Ca2+T are negatively modulated is yet poorly understood. In this work, we evaluated GlyR expression and its role on calcium signaling modulation in rat brain astrocytes. We first proved that GlyR, predominantly subunits α2 and β, was expressed in brain astrocytes and its localization was confirmed in the cytoplasm and astrocytic processes by immunohistochemistry assays. Calcium imaging experiments in cultured astrocytes showed that glycine (500 μM, a GlyR agonist, caused a concentration-dependent reduction in ATP-induced Ca2+T, an effect abolished by the GlyR antagonist, strychnine (0.8 μM, as well as by nocodazole (1 μM, known to impair GlyR anchorage to the plasma membrane. This effect was mimicked by activation of GABAAR, another Cl--permeable channel. In summary, we demonstrated that GlyR activation in astrocytes mediates an inhibitory effect upon ATP induced Ca2+T, which most probably involves changes in membrane permeability to Cl- and requires GlyR anchorage at the plasma membrane. GlyR in astrocytes may thus be part of a mechanism to modulate astrocyte-to-neuron communication.

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

2018-03-01

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L 1 Mab-4 (IgG 2b , kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L 1 Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L 1 Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L 1 Mab-4. These results indicate that L 1 Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.

Protocol of a randomized controlled trial of culturally sensitive interventions to improve African Americans' and non-African Americans' early, shared, and informed consideration of live kidney transplantation: the Talking About Live Kidney Donation (TALK) Study.

Science.gov (United States)

Boulware, L Ebony; Hill-Briggs, Felicia; Kraus, Edward S; Melancon, J Keith; McGuire, Raquel; Bonhage, Bobbie; Senga, Mikiko; Ephraim, Patti; Evans, Kira E; Falcone, Brenda; Troll, Misty U; Depasquale, Nicole; Powe, Neil R

2011-07-08

Live kidney transplantation (LKT) is underutilized, particularly among ethnic/racial minorities. The effectiveness of culturally sensitive educational and behavioral interventions to encourage patients' early, shared (with family and health care providers) and informed consideration of LKT and ameliorate disparities in consideration of LKT is unknown. We report the protocol of the Talking About Live Kidney Donation (TALK) Study, a two-phase study utilizing qualitative and quantitative research methods to design and test culturally sensitive interventions to improve patients' shared and informed consideration of LKT. Study Phase 1 involved the evidence-based development of culturally sensitive written and audiovisual educational materials as well as a social worker intervention to encourage patients' engagement in shared and informed consideration of LKT. In Study Phase 2, we are currently conducting a randomized controlled trial in which participants with progressing chronic kidney disease receive: 1) usual care by their nephrologists, 2) usual care plus the educational materials, or 3) usual care plus the educational materials and the social worker intervention. The primary outcome of the randomized controlled trial will include patients' self-reported rates of consideration of LKT (including family discussions of LKT, patient-physician discussions of LKT, and identification of an LKT donor). We will also assess differences in rates of consideration of LKT among African Americans and non-African Americans. The TALK Study rigorously developed and is currently testing the effectiveness of culturally sensitive interventions to improve patients' and families' consideration of LKT. Results from TALK will provide needed evidence on ways to enhance consideration of this optimal treatment for patients with end stage renal disease. ClinicalTrials.gov number, NCT00932334.

Calcium supplementation decreases BCP-induced inflammatory processes in blood cells through the NLRP3 inflammasome down-regulation.

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Lagadec, Patricia; Balaguer, Thierry; Boukhechba, Florian; Michel, Grégory; Bouvet-Gerbettaz, Sébastien; Bouler, Jean-Michel; Scimeca, Jean-Claude; Rochet, Nathalie

2017-07-15

Interaction of host blood with biomaterials is the first event occurring after implantation in a bone defect. This study aimed at investigating the cellular and molecular consequences arising at the interface between whole blood and biphasic calcium phosphate (BCP) particles. We observed that, due to calcium capture, BCP inhibited blood coagulation, and that this inhibition was reversed by calcium supplementation. Therefore, we studied the impact of calcium supplementation on BCP effects on blood cells. Comparative analysis of BCP and calcium supplemented-BCP (BCP/Ca) effects on blood cells showed that BCP as well as BCP/Ca induced monocyte proliferation, as well as a weak but significant hemolysis. Our data showed for the first time that calcium supplementation of BCP microparticles had anti-inflammatory properties compared to BCP alone that induced an inflammatory response in blood cells. Our results strongly suggest that the anti-inflammatory property of calcium supplemented-BCP results from its down-modulating effect on P2X7R gene expression and its capacity to inhibit ATP/P2X7R interactions, decreasing the NLRP3 inflammasome activation. Considering that monocytes have a vast regenerative potential, and since the excessive inflammation often observed after bone substitutes implantation limits their performance, our results might have great implications in terms of understanding the mechanisms leading to an efficient bone reconstruction. Although scaffolds and biomaterials unavoidably come into direct contact with blood during bone defect filling, whole blood-biomaterials interactions have been poorly explored. By studying in 3D the interactions between biphasic calcium phosphate (BCP) in microparticulate form and blood, we showed for the first time that calcium supplementation of BCP microparticles (BCP/Ca) has anti-inflammatory properties compared to BCP-induced inflammation in whole blood cells and provided information related to the molecular mechanisms

Inhibition of Helicobacter pylori CagA-Induced Pathogenesis by Methylantcinate B from Antrodia camphorata

Directory of Open Access Journals (Sweden)

Chun-Jung Lin

2013-01-01

Full Text Available The bacterial pathogen Helicobacter pylori (Hp is the leading risk factor for the development of gastric cancer. Hp virulence factor, cytotoxin-associated gene A (CagA interacted with cholesterol-enriched microdomains and leads to induction of inflammation in gastric epithelial cells (AGS. In this study, we identified a triterpenoid methylantcinate B (MAB from the medicinal mushroom Antrodia camphoratawhich inhibited the translocation and phosphorylation of CagA and caused a reduction in hummingbird phenotype in HP-infected AGS cells. Additionally, MAB suppressed the Hp-induced inflammatory response by attenuation of NF-κB activation, translocation of p65 NF-κB, and phosphorylation of IκB-α, indicating that MAB modulates CagA-mediated signaling pathway. Additionally, MAB also suppressed the IL-8 luciferase activity and its secretion in HP-infected AGS cells. On the other hand, molecular structure simulations revealed that MAB interacts with CagA similarly to that of cholesterol. Moreover, binding of cholesterol to the immobilized CagA was inhibited by increased levels of MAB. Our results demonstrate that MAB is the first natural triterpenoid which competes with cholesterol bound to CagA leading to attenuation of Hp-induced pathogenesis of epithelial cells. Thus, this study indicates that MAB may have a scope to develop as a therapeutic candidate against Hp CagA-induced inflammation.

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4

Directory of Open Access Journals (Sweden)

Shinji Yamada

2018-03-01

Full Text Available Programmed cell death-ligand 1 (PD-L1, which is a ligand of programmed cell death-1 (PD-1, is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1 expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb, L1Mab-4 (IgG2b, kappa, using cell-based immunization and screening (CBIS method and investigated hPD-L1 expression in oral cancers. L1Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 in flow cytometry and stained oral cancers in a membrane-staining pattern. L1Mab-4 stained 106/150 (70.7% of oral squamous cell carcinomas, indicating the very high sensitivity of L1Mab-4. These results indicate that L1Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers. Keywords: Programmed cell death-ligand 1, Monoclonal antibody, Oral cancer

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Acidosis and Urinary Calcium Excretion

DEFF Research Database (Denmark)

Alexander, R Todd; Cordat, Emmanuelle; Chambrey, Régine

2016-01-01

Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and inhibi...

Role of calcium in gonadotropin releasing hormone-induced luteinizing hormone secretion from the bovine pituitary

International Nuclear Information System (INIS)

Kile, J.P.

1986-01-01

The hypothesis was tested that GnRH acts to release LH by increasing calcium uptake by gonadotroph which in turn stimulates calcium-calmodulin activity and results in LH release from bovine pituitary cells as it does in the rat. Pituitary glands of calves (4-10 months of age) were enzymatically dispersed (0.2% collagenase) and grown for 5 days to confluency in multiwell plates (3 x 10 5 /well). Cells treated with GnRH Ca ++ ionophore A23187, and ouabain all produced significant releases of LH release in a pronounced all or none fashion, while thorough washing of the cells with 0.5 mM EGTA in Ca ++ -free media prevented the action of GnRH. GnRH caused a rapid efflux of 45 Ca ++ . Both GnRH-stimulated 45 Ca efflux and LH release could be partially blocked by verapamil GnRH-induced LH release could also be blocked by nifedipine and tetrodotoxin, although these agents did not affect 45 Ca efflux. The calmodulin antagonists calmidazolium and W7 were found to block GnRH induced LH release, as well as LH release induced by theophylline, KC PGE 2 and estradiol. These data indicated that: (1) calcium is required for GnRH action, but extracellular Ca ++ does not regulate LH release; (2) GnRH elevates intracellular Ca ++ by opening both voltage sensitive and receptor mediated Ca ++ channels; (3) activation of calmodulin is one mechanism involved in GnRH-induced LH release

Neutrophils and the calcium-binding protein MRP-14 mediate carrageenan-induced antinociception in mice

Directory of Open Access Journals (Sweden)

Rosana L. Pagano

2002-01-01

Full Text Available Background: We have previously shown that the calcium-binding protein MRP-14 secreted by neutrophils mediates the antinociceptive response in an acute inflammatory model induced by the intraperitoneal injection of glycogen in mice.

Baroreflex deficiency induces additional impairment of vagal tone, diastolic function and calcium handling proteins after myocardial infarction

Science.gov (United States)

Mostarda, Cristiano; Rodrigues, Bruno; Medeiros, Alessandra; Moreira, Edson D; Moraes-Silva, Ivana C; Brum, Patricia C; Angelis, Katia De; Irigoyen, Maria-Cláudia

2014-01-01

Baroreflex dysfunction has been considered an important mortality predictor after myocardial infarction (MI). However, the impact of baroreflex deficiency prior to MI on tonic autonomic control and cardiac function, and on the profile of proteins associated with intracellular calcium handling has not yet been studied. The aim of the present study was to analyze how the impairment of baroreflex induced by sinoaortic denervation (SAD) prior to MI in rats affects the tonic autonomic control, ventricular function and cardiomyocyte calcium handling proteins. After 15 days of following or SAD surgery, rats underwent MI. Echocardiographic, hemodynamic, autonomic and molecular evaluations were performed 90 days after MI. Baroreflex impairment led to additional damage on: left ventricular remodeling, diastolic function, vagal tonus and intrinsic heart rate after MI. The loss of vagal component of the arterial baroreflex and vagal tonus were correlated with changes in the cardiac proteins involved in intracellular calcium homeostasis. Furthermore, additional increase in sodium calcium exchanger expression levels was associated with impaired diastolic function in experimental animals. Our findings strongly suggest that previous arterial baroreflex deficiency may induce additional impairment of vagal tonus, which was associated with calcium handling proteins abnormalities, probably triggering ventricular diastolic dysfunction after MI in rats. PMID:24936224

Pseudomonas, Pantoea and Cupriavidus isolates induce calcium carbonate precipitation for biorestoration of ornamental stone.

Science.gov (United States)

Daskalakis, M I; Magoulas, A; Kotoulas, G; Catsikis, I; Bakolas, A; Karageorgis, A P; Mavridou, A; Doulia, D; Rigas, F

2013-08-01

Bacterially induced calcium carbonate precipitation from various isolates was investigated aiming at developing an environmentally friendly technique for ornamental stone protection and restoration. Micro-organisms isolated from stone samples and identified using 16S rDNA and biochemical tests promoted calcium carbonate precipitation in solid and novel liquid growth media. Biomineral morphology was studied on marble samples with scanning electron microscopy. Most isolates demonstrated specimen weight increase, covering partially or even completely the marble surfaces mainly with vaterite. The conditions under which vaterite precipitated and its stability throughout the experimental runs are presented. A growth medium that facilitated bacterial growth of different species and promoted biomineralization was formulated. Most isolates induced biomineralization of CaCO3 . Micro-organisms may actually be a milestone in the investigation of vaterite formation facilitating our understanding of geomicrobiological interactions. Pseudomonas, Pantoea and Cupriavidus strains could be candidates for bioconsolidation of ornamental stone protection. Characterization of biomineralization capacity of different bacterial species improves understanding of the bacterially induced mineralization processes and enriches the list of candidates for biorestoration applications. Knowledge of biomineral morphology assists in differentiating mineral from biologically induced precipitates. © 2013 The Society for Applied Microbiology.

Physico-chemical Stability of MabThera Drug-product Solution for Subcutaneous Injection under in-use Conditions with Different Administration Materials.

Science.gov (United States)

Mueller, Claudia; Dietel, Elke; Heynen, Severin R; Nalenz, Heiko; Goldbach, Pierre; Mahler, Hanns-Christian; Schmidt, Johannes; Grauschopf, Ulla; Schoenhamnmer, Karin

2015-01-01

MabThera is an essential component of the standard-of-care regimens in the treatment of non-Hodgkin lymphoma and Chronic Lymphatic Leukemia. MabThera for subcutaneous injection is a novel line extension that has been approved by the European Medicines Agency for the treatment of patients with follicular lymphoma and diffuse large B-cell lymphoma. This study aimed to evaluate in-use stability data of MabThera subcutaneous drug-product solution in single-use syringes for subcutaneous administration according to the European Medicines Agency guideline. The drug-product solution was exposed to material contact surfaces of five different administration setups commonly used in subcutaneous drug delivery. MabThera subcutaneous was transferred under aseptic conditions into polypropylene and polycarbonate syringes and stored for 1, 2, and 4 weeks at 2°C to 8°C followed by 24 hours at 30°C. After storage, subcutaneous administration was simulated and MabThera subcutaneous drug-product solution quality attributes were evaluated by using compendial physico-chemical tests, as well as suitable and validated molecule- and formulation-specific analytical methods. MabThera subcutaneous vials were treated and analyzed in parallel. The physico-chemical results of MabThera subcutaneous in the different setups were comparable to the control for all timepoints. No change in drug-product quality after storage and simulated administration was found compared to the control. However, since single-dose products do not contain preservatives, microbial contamination and growth needs to be avoided and product sterility needs to be ensured. The results showed that MabThera subcutaneous remains compatible and stable, from a physico-chemical perspective, for up to 4 weeks at 2°C to 8°C followed by 24 hours at 30°C with the contact materials tested in this study. In order to avoid and minimize microbial growth, MabThera subcutaneous should be used immediately after removal from the original

Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus.

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Aguilar, Jorge L; Varshney, Avanish K; Pechuan, Ximo; Dutta, Kaushik; Nosanchuk, Joshua D; Fries, Bettina C

2017-08-18

Staphylococcus aureus is a leading infectious cause of life-threatening disease in humans, yet there is currently no vaccine to combat this bacterium. The pathogenesis of S. aureus is mediated by a diverse array of protein toxins including a large family of secreted pyrogenic superantigens. Neutralization of superantigens, including SEB and TSST-1, has proven to be protective in several animal models of toxic shock and sepsis. We demonstrate, for the first time, that a far more prevalent staphylococcal superantigen, SEK, can also induce lethal shock in mice. Additionally, we describe monoclonal antibodies (mAbs) that inhibit SEK-induced mitogenicity as well as protect against SEK-induced lethality, and enhance survival from S. aureus septicemia in murine models. MAb-4G3 (IgG2b), mAb-5G2 (IgG1), and mAb-9H2 (IgG1), all inhibit SEK-induced proliferation and cytokine production of human immune cells. We then demonstrate that passive immunization with a combination of mAb-4G3 and mAb-5G4, 2 mAbs that do not compete for epitope(s) on SEK, significantly enhance survival in a murine model of SEK-induced toxic shock (p = 0.006). In the setting of sepsis, passive immunization with this combination of mAbs also significantly enhances survival in mice after challenge with CA-MRSA strain USA300 (p = 0.03). Furthermore, septic mice that received mAb treatment in conjunction with vancomycin exhibit less morbidity than mice treated with vancomycin alone. Taken together, these findings suggest that the contribution of SEK to S. aureus pathogenesis may be greater than previously appreciated, and that adjunctive therapy with passive immunotherapy against SEs may be beneficial.

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

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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

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

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Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

2018-05-01

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

Potassium-induced contraction in the lamb proximal urethra: Involvement of norepinephrine and different calcium entry pathways

International Nuclear Information System (INIS)

Garcia-Pascual, A.; Costa, G.; Isla, M.; Jimenez, E.; Garcia-Sacristan, A.

1991-01-01

The purpose of this work was to investigate the mechanisms involved in the peculiar biphasic response of the lamb urethral smooth muscle to high K+ solutions. The relative amplitude of the phasic and tonic components of the contraction and its reproducibility were dependent on the concentration of K+ used. Only concentrations higher than 80 mM (i.e., 120 mM) showed a tonic component greater in amplitude than the phasic one and manifested a tachyphylactic effect. Phentolamine (10(-6) M), prazosin (10(-6) M) and chemical denervation with 6-hydroxydopamine significantly inhibited the tonic component of the K+ (120 mM)-induced contraction, modifying its morphology. Reproducible contractions to K+ (120 mM) could be obtained in the presence of prazosin (10(-6) M) or cocaine (10(-6) M). The preparations were also shown to accumulate [3H]noradrenaline and release it upon depolarization with K+ (60 and 120 mM). Calcium removal inhibited the K+ (120 mM)-induced contraction. After addition of calcium (0.5-5 mM) the contractile activity was restored. Nifedipine (10(-6) M) and verapamil (10(-6) M) but not sodium nitroprusside (10(-6) M) significantly blocked the contractile response for calcium as well as the phasic component of the K+ contraction in calcium-containing medium. In preparations treated with prazosin (10(-6) M) the tonic component of the K+ (120 mM) contraction was more sensitive to nifedipine and removal of extracellular calcium than the phasic one

Copper-induced activation of TRP channels promotes extracellular calcium entry and activation of CaMs and CDPKs leading to copper entry and membrane depolarization in Ulva compressa

Directory of Open Access Journals (Sweden)

Melissa eGómez

2015-03-01

Full Text Available In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1 and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9 and 12 min of exposure, respectively, and antagonists of TRPC5, A1 and V1 corresponding to SKF-96365 (SKF, HC-030031 (HC and capsazepin (CPZ, respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9 and 12 min which were inhibited by SKF, HC and CPZ, respectively, indicating that copper activate TRPC5, A1 and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require CaMs and CDPKs activation. In addition, copper induced membrane depolarization events at 4, 8 and 11 min and these events were inhibited by SKF, HC, CPZ and bathocuproine, a specific copper chelating agent, indicating copper entry through TRP channels leading to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that CaMs and CDPKs are required in order to activate TRPs to allow copper entry. Thus, light-dependent copper-induced activation TRPC5, A1 and V1 promotes extracellular calcium entry leading to activation of CaMs and CDPKs which, in turn, promotes copper entry through these TRP channels leading to membrane depolarization.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

Science.gov (United States)

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5

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Shinji Yamada

2018-07-01

Full Text Available CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s, which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C44Mab-5 (IgG1, kappa, recognized both CD44s and CD44v3-10. C44Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C44Mab-5 detected 166/182 (91.2% of oral cancers. These results suggest that the C44Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Kaneko, Mika K; Kato, Yukinari

2018-07-01

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C 44 Mab-5 (IgG 1 , kappa), recognized both CD44s and CD44v3-10. C 44 Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C 44 Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C 44 Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

[Changes induced by hypertonic solutions in the transportation of calcium by the cardiac reticular sarcoplasma].

Science.gov (United States)

Sierra, M; Holguín, J A

1979-01-01

In the sarcoplasmic reticulum of the myocardium, celular organell which function is to regulate the cytoplasmic concentration of calcium in contraction and relaxation, we have studied the effect of hypertonic solutions of sucrose between 1 and 6.96 times the normal tonicity in order to observe the behavior of the internal linked or free calcium of this structure, as well as to prove the hypothesis that hypertonic solutions encourage the calcium exit of the sarcoplasmatic reticulum with the resulting signs of contractures. The following results were obtained: 1. The ATP hydrolisis and calcium transport rate are 14% and 90% respectively of the maximum speeds of 10(-5) M in calcium, while for concentrations of 10(-7) M or ess of the said cation, the transport rates and the ATPase do not reach 5% of the maximum values. 2. Between 1 and 2.54 times of the normal tonicity the calcium uptake remains between 400 and 500 nmoles of calcium/mg protein/min, the transported amount of calcium varies between 14 and 16 nmoles/mg protein and the rate of the ATP hydrolysis increases a 37% to 0.4 M in sucrose. 3. Between 0.4 and 1.2 M in sucrose of 2.54 to 6.96 times the isotonicity, the calcium transport rate velocity as well as the ATP hydrolisis are strongly inhibited. The vesicles volume minimizes and the amount of linked calcium remains within the control values, proving that the capacity of linking this cathion is independent from sarcoplasmic reticulum volume. These results show that the sarcoplasmic reticulum is involved in the contractures induced by hypertonic solutions in intact cells, since the osmolarity increase produces changes of volume which results in a decrease of the calcium transportation velocity or in an increase of the exit of said cathion.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Science.gov (United States)

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-01-01

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

Science.gov (United States)

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-05-07

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

[Effect of 2,3-butanedione monoxime on calcium paradox-induced heart injury in rats].

Science.gov (United States)

Kong, Ling-Heng; Gu, Xiao-Ming; Su, Xing-Li; Sun, Na; Wei, Ming; Zhu, Juan-Xia; Chang, Pan; Zhou, Jing-Jun

2016-05-01

To investigate the Effect of 2,3-butanedione monoxime (BDM) on calcium paradox-induced heart injury and its underlying mechanisms. Thirty-two adult male SD rats were randomized into 4 groups, namely the control group, BDM treatment control group, calcium paradox group, and BDM treatment group. Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion and the left ventricular pressure (LVP) and left ventricular end-diastolic pressure (LVEDP) were monitored. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Lactate dehydrogenase (LDH) content in the coronary flow was determined. Triphenyltetrazolium chloride staining was used to measure the infarct size, and myocardial cell apoptosis was tested with TUNEL method. Western blotting was used to determine the expression of cleaved caspase-3 and cytochrome c. Compared with the control group, BDM at 20 mmol/L had no effect on cardiac performance, cell death, apoptotic index or the content of LDH, cleaved caspase-3 and cytochrome c at the end of perfusion under control conditions (P>0.05). Calcium paradox treatment significantly decreased the cardiac function and the level of LVDP and induced a larger infarct size (Pparadox, and markedly down-regulated the levels of LVEDP and LDH (Pparadox, suggesting the value of BDM as an potential drug for myocardial ischemia reperfusion injur.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

Science.gov (United States)

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

TGF-beta Sma/Mab signaling mutations uncouple reproductive aging from somatic aging.

Directory of Open Access Journals (Sweden)

Shijing Luo

2009-12-01

Full Text Available Female reproductive cessation is one of the earliest age-related declines humans experience, occurring in mid-adulthood. Similarly, Caenorhabditis elegans' reproductive span is short relative to its total life span, with reproduction ceasing about a third into its 15-20 day adulthood. All of the known mutations and treatments that extend C. elegans' reproductive period also regulate longevity, suggesting that reproductive span is normally linked to life span. C. elegans has two canonical TGF-beta signaling pathways. We recently found that the TGF-beta Dauer pathway regulates longevity through the Insulin/IGF-1 Signaling (IIS pathway; here we show that this pathway has a moderate effect on reproductive span. By contrast, TGF-beta Sma/Mab signaling mutants exhibit a substantially extended reproductive period, more than doubling reproductive span in some cases. Sma/Mab mutations extend reproductive span disproportionately to life span and act independently of known regulators of somatic aging, such as Insulin/IGF-1 Signaling and Dietary Restriction. This is the first discovery of a pathway that regulates reproductive span independently of longevity and the first identification of the TGF-beta Sma/Mab pathway as a regulator of reproductive aging. Our results suggest that longevity and reproductive span regulation can be uncoupled, although they appear to normally be linked through regulatory pathways.

Calcium channel blocker prevents stress-induced activation of renin and aldosterone in conscious pig

International Nuclear Information System (INIS)

Ceremuzynski, L.K.; Klos, J.; Barcikowski, B.; Herbaczynska-Cedro, K.

1991-01-01

A considerable amount of data suggest the involvement of calcium-mediated processes in the activation of the renin-angiotensin-aldosterone (RAA) cascade. To investigate the effect of calcium-channel inhibition on the RAA system, the authors studied 21 conscious pigs. Blood renin and aldosterone levels increased by subjecting animals to 24 hours of immobilization stress. Renin and aldosterone levels were repeatedly measured by radioimmunoassay in blood samples taken periodically over 24 hours from a chronically implanted arterial cannula. Pretreatment of the animals (N = 11) with nisoldipine, 2 x 20 mg p.o. daily for 2 days before and on the day of immobilization, transiently attenuated the stress-induced increase of plasma renin activity and completely prevented the rise of aldosterone, as compared to nontreated controls (N = 10). The finding that nisoldipine suppresses RAA activation induced by a nonpharmacologic stimulus in the conscious intact animal may have clinical implications

Importance of large conductance calcium-activated potassium channels (BKCa) in interleukin-1b-induced adhesion of monocytes to endothelial cells.

Science.gov (United States)

Burgazli, K M; Venker, C J; Mericliler, M; Atmaca, N; Parahuleva, M; Erdogan, A

2014-01-01

The present study investigated the role of the large conductance calcium-activated potassium channels (BKCa) in interleukin-1b (IL-1b) induced inflammation. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured. Endothelial cell membrane potential measurements were accomplished using the fluorescent dye DiBAC4(3). The role of BKCa was assessed using iberiotoxin, a highly selective BKCa inhibitor. Changes in the calcium intracellular calcium were investigated using Fura-2-AM imaging. Fluorescent dyes DCF-AM and DAF-AM were further used in order to measure the formation of reactive oxygen species (ROS) and nitric oxide (NO) synthesis, respectively. Endothelial cell adhesion tests were conducted with BCECF-AM adhesion assay and tritium thymidine uptake using human monocytic cells (U937). Expression of cellular adhesion molecules (ICAM-1, VCAM-1) was determined by flow cytometer. Interleukin-1b induced a BKCa dependent hyperpolarization of HUVECs. This was followed by an increase in the intracellular calcium concentration. Furthermore, IL-1b significantly increased the synthesis of NO and ROS. The increase of intracellular calcium, radicals and NO resulted in a BKCa dependent adhesion of monocytes to HUVECs. Endothelial cells treated with IL-1b expressed both ICAM-1 and VCAM-1 in significantly higher amounts as when compared to controls. It was further shown that the cellular adhesion molecules ICAM-1 and VCAM-1 were responsible for the BKCa-dependent increase in cellular adhesion. Additionally, inhibition of the NADPH oxidase with DPI led to a significant downregulation of IL-1b-induced expression of ICAM and VCAM, as well as inhibition of eNOS by L-NMMA, and intracellular calcium by BAPTA. Activation of the endothelial BKCa plays an important role in the IL-1b-induced monocyte adhesion to endothelial cells.

Structures and Stability of Metal Amidoboranes (MAB): Density Functional Calculations

International Nuclear Information System (INIS)

Li Cailin; Wu Chaoling; Chen Yungui; Zhou Jingjing; Zheng Xin; Pang Lijuan; Deng Gang

2010-01-01

Molecule geometry structures, frequencies, and energetic stabilities of ammonia borane (AB, NH 3 BH 3 ) and metal amidoboranes (MAB, MNH 2 BH 3 ), formed by substituting H atom in AB with one of main group metal atoms, have been investigated by density-functional theory and optimized at the B3LYP levels with 6-311G++ (3df, 3pd) basic set. Their structural parameters and infrared spectrum characteristic peaks have been predicted, which should be the criterion of a successfully synthesized material. Several parameters such as binding energies, vibrational frequencies, and the energy gaps between the HOMO and the LUMO have been adopted to characterize and evaluate their structure stabilities. It is also found that the binding energies and HOMO-LUMO energy gaps of the MAB obviously change with the substitution of the atoms. MgAB has the lowest binding energy and is easier to decompose than any other substitutional structures under same conditions, while CaAB has the highest chemical activity. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

Feasibility and Acceptability of the TALK Social Worker Intervention to Improve Live Kidney Transplantation

Science.gov (United States)

DePasquale, Nicole; Hill-Briggs, Felicia; Darrell, Linda; Boyer, LaPricia Lewis; Ephraim, Patti; Boulware, L. Ebony

2012-01-01

Live kidney transplantation (LKT) is underused by patients with end-stage renal disease. Easily implementable and effective interventions to improve patients' early consideration of LKT are needed. The Talking About Live Kidney Donation (TALK) social worker intervention (SWI) improved consideration and pursuit of LKT among patients with…

Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

Energy Technology Data Exchange (ETDEWEB)

Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

2007-08-01

Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

Calcium waves.

Science.gov (United States)

Jaffe, Lionel F

2008-04-12

Waves through living systems are best characterized by their speeds at 20 degrees C. These speeds vary from those of calcium action potentials to those of ultraslow ones which move at 1-10 and/or 10-20 nm s(-1). All such waves are known or inferred to be calcium waves. The two classes of calcium waves which include ones with important morphogenetic effects are slow waves that move at 0.2-2 microm s(-1) and ultraslow ones. Both may be propagated by cycles in which the entry of calcium through the plasma membrane induces subsurface contraction. This contraction opens nearby stretch-sensitive calcium channels. Calcium entry through these channels propagates the calcium wave. Many slow waves are seen as waves of indentation. Some are considered to act via cellular peristalsis; for example, those which seem to drive the germ plasm to the vegetal pole of the Xenopus egg. Other good examples of morphogenetic slow waves are ones through fertilizing maize eggs, through developing barnacle eggs and through axolotl embryos during neural induction. Good examples of ultraslow morphogenetic waves are ones during inversion in developing Volvox embryos and across developing Drosophila eye discs. Morphogenetic waves may be best pursued by imaging their calcium with aequorins.

6-OHDA induced calcium influx through N-type calcium channel alters membrane properties via PKA pathway in substantia nigra pars compacta dopaminergic neurons.

Science.gov (United States)

Qu, Liang; Wang, Yuan; Zhang, Hai-Tao; Li, Nan; Wang, Qiang; Yang, Qian; Gao, Guo-Dong; Wang, Xue-Lian

2014-07-11

Voltage gated calcium channels (VGCC) are sensitive to oxidative stress, and their activation or inactivation can impact cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remain controversial. In this study, we assessed 6-hydroxydopamine (6-OHDA) induced transformation of firing pattern and functional changes of calcium channels in SNc dopaminergic neurons. Application of 6-OHDA (0.5-2mM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)]i) rise. In voltage clamp, ω-conotoxin-sensitive Ca(2+) current modulation mediated by 6-OHDA reflected an altered sensitivity. Furthermore, we found that 6-OHDA modulated Ca(2+) currents through PKA pathway. These results provided evidence for the potential role of VGCCs and PKA involved in oxidative stress in degeneration of SNc neurons in Parkinson's disease (PD). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients

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Bading Hilmar

2007-07-01

Full Text Available Abstract Background In hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types. Results Using laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border. Conclusion Thus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.

Phagocytosis-induced /sup 45/calcium efflux in polymorphonuclear leucocytes

Energy Technology Data Exchange (ETDEWEB)

Barthelemy, A; Schell-Frederick, E [Brussels Univ. (Belgium). Institut de Recherche Interdisciplinaire; Paridaens, R [Brussels Univ. (Belgium). Faculte de Medicine

1977-10-15

The role of calcium ions in regulating the structure and function of non-muscle cells is a subject of intense study. Several lines of evidence that calcium may be essential in the function of polymorphonuclear leuocytes (PMNL) and an important control element in the process of phagocytosis. Direct studies of calcium distribution and fluxes have only recently been undertaken. To our knowledge, no report of calcium movements during normal phagocytosis has been published. In the context of an overall study of calcium dynamics in the PMNL, we report here initial studies on /sup 45/Ca efflux in prelabelled guinea pig PMNL. The results demonstrate the energy-dependence of resting calcium efflux and an increased efflux upon addition of phagocytic particles which is not dependent on particle internalization.

Circular patterns of calcium oxalate monohydrate induced by defective Langmuir-Blodgett film on quartz substrates

Energy Technology Data Exchange (ETDEWEB)

He Jieyu [Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China); Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China); Ouyang Jianming [Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China); Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632 (China)], E-mail: toyjm@jnu.edu.cn

2009-01-01

The defective Langmuir-Blodgett (LB) film of dipalmitoylphosphatidylcholine (DPPC) on quartz injured by potassium oxalate (K{sub 2}C{sub 2}O{sub 4}) was used as a model system to induce growth of calcium oxalate crystals. Atomic force microscopy (AFM) indicated that circular defective domains with a diameter of 1-200 {mu}m existed in the LB film. Scanning electron microscopy (SEM) showed circular patterns of aggregated calcium oxalate monohydrate (COM) crystallites were induced by these defective domains. It was ascribed to that the interaction between the negatively-charged oxalate ions and the phosphatidyl groups in DPPC headgroups makes the phospholipid molecules rearranged and exist in an out-of-order state in the LB film, especially at the boundaries of liquid-condensed (LC)/liquid-expanded (LE) phases, which provide much more nucleating sites for COM crystals.

A multivalent Mannheimia-Bibersteinia vaccine protects bighorn sheep against Mannheimia haemolytica challenge.

Science.gov (United States)

Subramaniam, Renuka; Shanthalingam, Sudarvili; Bavananthasivam, Jegarubee; Kugadas, Abirami; Potter, Kathleen A; Foreyt, William J; Hodgins, Douglas C; Shewen, Patricia E; Barrington, George M; Knowles, Donald P; Srikumaran, Subramaniam

2011-10-01

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 10(5) CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia.

A Multivalent Mannheimia-Bibersteinia Vaccine Protects Bighorn Sheep against Mannheimia haemolytica Challenge ▿

Science.gov (United States)

Subramaniam, Renuka; Shanthalingam, Sudarvili; Bavananthasivam, Jegarubee; Kugadas, Abirami; Potter, Kathleen A.; Foreyt, William J.; Hodgins, Douglas C.; Shewen, Patricia E.; Barrington, George M.; Knowles, Donald P.; Srikumaran, Subramaniam

2011-01-01

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 105 CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia. PMID:21832104

Spine Calcium Transients Induced by Synaptically-Evoked Action Potentials Can Predict Synapse Location and Establish Synaptic Democracy

Science.gov (United States)

Meredith, Rhiannon M.; van Ooyen, Arjen

2012-01-01

CA1 pyramidal neurons receive hundreds of synaptic inputs at different distances from the soma. Distance-dependent synaptic scaling enables distal and proximal synapses to influence the somatic membrane equally, a phenomenon called “synaptic democracy”. How this is established is unclear. The backpropagating action potential (BAP) is hypothesised to provide distance-dependent information to synapses, allowing synaptic strengths to scale accordingly. Experimental measurements show that a BAP evoked by current injection at the soma causes calcium currents in the apical shaft whose amplitudes decay with distance from the soma. However, in vivo action potentials are not induced by somatic current injection but by synaptic inputs along the dendrites, which creates a different excitable state of the dendrites. Due to technical limitations, it is not possible to study experimentally whether distance information can also be provided by synaptically-evoked BAPs. Therefore we adapted a realistic morphological and electrophysiological model to measure BAP-induced voltage and calcium signals in spines after Schaffer collateral synapse stimulation. We show that peak calcium concentration is highly correlated with soma-synapse distance under a number of physiologically-realistic suprathreshold stimulation regimes and for a range of dendritic morphologies. Peak calcium levels also predicted the attenuation of the EPSP across the dendritic tree. Furthermore, we show that peak calcium can be used to set up a synaptic democracy in a homeostatic manner, whereby synapses regulate their synaptic strength on the basis of the difference between peak calcium and a uniform target value. We conclude that information derived from synaptically-generated BAPs can indicate synapse location and can subsequently be utilised to implement a synaptic democracy. PMID:22719238

Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc

Directory of Open Access Journals (Sweden)

MP Grant

2016-07-01

Full Text Available The cartilaginous endplates (CEPs are thin layers of hyaline cartilage found adjacent to intervertebral discs (IVDs. In addition to providing structural support, CEPs regulate nutrient and metabolic exchange in the disc. In IVD pathogenesis, CEP undergoes degeneration and calcification, compromising nutrient availability and disc cell metabolism. The mechanism(s underlying the biochemical changes of CEP in disc degeneration are currently unknown. Since calcification is often observed in later stages of IVD degeneration, we hypothesised that elevations in free calcium (Ca2+ impair CEP homeostasis. Indeed, our results demonstrated that the Ca2+ content was consistently higher in human CEP tissue with grade of disc degeneration. Increasing the levels of Ca2+ resulted in decreases in the secretion and accumulation of collagens type I, II and proteoglycan in cultured human CEP cells. Ca2+ exerted its effects on CEP matrix protein synthesis through activation of the extracellular calcium-sensing receptor (CaSR; however, aggrecan content was also affected independent of CaSR activation as increases in Ca2+ directly enhanced the activity of aggrecanases. Finally, supplementing Ca2+ in our IVD organ cultures was sufficient to induce degeneration and increase the mineralisation of CEP, and decrease the diffusion of glucose into the disc. Thus, any attempt to induce anabolic repair of the disc without addressing Ca2+ may be impaired, as the increased metabolic demand of IVD cells would be compromised by decreases in the permeability of the CEP.

Efficacy and Safety of Photon Induced Photoacoustic Streaming for Removal of Calcium Hydroxide in Endodontic Treatment

Directory of Open Access Journals (Sweden)

Markus Laky

2018-01-01

Full Text Available Calcium hydroxide removal from the root canal by photon induced photoacoustic streaming (PIPS compared to needle irrigation and irrigation using sonic activation was investigated. Additionally, safety issues regarding apical extrusion were addressed. In endodontic treatment temporary intracanal medication like calcium hydroxide should be completely removed for long term success. For analysis, 60 artificial teeth were prepared, filled with calcium hydroxide, and divided into four groups. The teeth were assigned to needle irrigation, irrigation using a sonic device, PIPS with a lower energy setting (10 mJ, 15 Hz, or PIPS with a higher energy setting (25 mJ/40 Hz. For comparison the weight of each tooth was measured before and after calcium hydroxide incorporation, as well as after removing calcium hydroxide using the four different methods. Regarding safety issues another 24 samples were filled with stained calcium hydroxide and embedded in 0.4% agarose gel. Color changes in the agarose gel due to apical extrusion were digitally analysed using Photoshop. No significant differences were found for calcium hydroxide removal between the two laser groups. Sonic assisted removal and needle irrigation resulted in significant less calcium hydroxide removal than both laser groups, with significantly more calcium hydroxide removal in the ultrasonic group than in the needle irrigation group. For apical extrusion the higher laser (25 mJ/40 Hz group resulted in significant higher color changes of the periapical gel than all other groups. PIPS with the setting of 10 mJ/15 Hz achieved almost complete removal of calcium hydroxide without increasing apical extrusion of the irrigation solution.

Immuno-PET of undifferentiated thyroid carcinoma with radioiodine-labelled antibody cMAb U36: application to antibody tumour uptake studies

Energy Technology Data Exchange (ETDEWEB)

Fortin, Marc-Andre [Centre Hospitalier Universitaire de Quebec and Laval University, Laboratory for Biomaterials and Bioengineering, Quebec City (Canada); Uppsala University, Biomedical Radiation Sciences, Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Salnikov, Alexei V. [Uppsala University, BMC, Department of Medical Biochemistry and Microbiology, Uppsala (Sweden); German Cancer Research Center, Division of Molecular Immunology, Heidelberg (Germany); Nestor, Marika [Uppsala University, Division of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala (Sweden); Heldin, Nils-Erik [Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala (Sweden); Rubin, Kristofer [Uppsala University, BMC, Department of Medical Biochemistry and Microbiology, Uppsala (Sweden); Lundqvist, Hans [Uppsala University, Biomedical Radiation Sciences, Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden)

2007-09-15

We tested the suitability of the chimeric monoclonal anti-human CD44 splice version 6 antibody (cMAb U36) for targeting and visualising human anaplastic thyroid carcinoma with PET. We also performed experiments aimed at elucidating the relation between tumour interstitial fluid pressure (TIFP) and the tumour uptake of antibodies. The affinity and specificity of the cMAb U36 for KAT-4 cells were evaluated in vitro, as was the Na{sup +}/I{sup -} symporter (NIS) expression. Biodistribution studies were performed on KAT-4 carcinoma-bearing mice injected with {sup 124}I-cMAb U36 or free iodine. Biodistribution studies were also performed in animals treated with the specific TGF-{beta}1 and -{beta}3 inhibitor Fc:T{beta}RII, which lowers TIFP. Treated and non-treated animals were scanned by microPET. Cultured human undifferentiated/anaplastic thyroid carcinoma KAT-4 cells expressed low levels of NIS and uptake of free iodine was insignificant. The cMAb U36 expressed an affinity (K{sub D}) of 11 {+-} 2 nM. Tumour radioactivity uptake reached maximum values 48 h after injection of {sup 124}I-cMAb U36 ({proportional_to}22%IA/g). KAT-4 carcinomas were readily identified in all {sup 124}I-immuno-PET images. Radioactivity tumour uptake in Fc:T{beta}RII-treated animals was significantly lower at 24 and 48 h after injection, and five times higher thyroid uptake was also noted. We successfully used {sup 124}I-cMAb U36 to visualise CD44v6-expressing human anaplastic thyroid carcinoma. Given the lack of NIS expression in KAT-4, tumour visualisation is not due to free iodine uptake. Lowering the TIFP in KAT-4 carcinomas did not increase the uptake of mAbs into tumour tissue. (orig.)

Preparation for gluing of Carbon prototype MAB at INEGI, Porto

CERN Multimedia

Miguel Moreira, Porto, INEGI

2001-01-01

MAB's will assure the alignment of the CMS detector. It is equipped with muon cameras, measuring the position of the barrel muon stations and at the same time linking via the link elements, connecting the barrel muon detectors with the Tracker. In addition there is a connection with the endcap. More details can be found on the muon/alignment homepage on the web

Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways.

Science.gov (United States)

Li, Lan; Du, Yi; Ju, Furong; Ma, Shunxiang; Zhang, Shengxiang

2016-01-01

Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells. EL4 cells were exposed to POX for 0-16 h. EGTA was used to chelate Ca(2+ ) in extracellular medium, and heparin and procaine were used to inhibit Ca(2+ )efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis. POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78. Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.

Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

DEFF Research Database (Denmark)

Busti, Stefano; Mapelli, Valeria; Tripodi, Farida

2016-01-01

respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections...... reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular...

Mean field strategies induce unrealistic nonlinearities in calcium puffs

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Guillermo eSolovey

2011-08-01

Full Text Available Mean field models are often useful approximations to biological systems, but sometimes, they can yield misleading results. In this work, we compare mean field approaches with stochastic models of intracellular calcium release. In particular, we concentrate on calcium signals generated by the concerted opening of several clustered channels (calcium puffs. To this end we simulate calcium puffs numerically and then try to reproduce features of the resulting calcium distribution using mean field models were all the channels open and close simultaneously. We show that an unrealistic nonlinear relationship between the current and the number of open channels is needed to reproduce the simulated puffs. Furthermore, a single channel current which is five times smaller than the one of the stochastic simulations is also needed. Our study sheds light on the importance of the stochastic kinetics of the calcium release channel activity to estimate the release fluxes.

Voltage-gated calcium flux mediates Escherichia coli mechanosensation.

Science.gov (United States)

Bruni, Giancarlo N; Weekley, R Andrew; Dodd, Benjamin J T; Kralj, Joel M

2017-08-29

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli , including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando

1999-01-01

Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99m Tc at high yield. The resulting 99m Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

Science.gov (United States)

Samigullin, Dmitry; Fatikhov, Nijaz; Khaziev, Eduard; Skorinkin, Andrey; Nikolsky, Eugeny; Bukharaeva, Ellya

2014-01-01

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers-which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal-has hitherto been technically impossible. With the aim of quantifying both Ca(2+) currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

Science.gov (United States)

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C; Finkelman, Fred D

2013-06-01

Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

Science.gov (United States)

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects.

LENUS (Irish Health Repository)

Lyng, F M

2006-04-01

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of

Changes in calcium and iron levels in the brains of rats during kainate induced epilepsy

Science.gov (United States)

Ren, Min-Qin; Ong, Wei-Yi; Makjanic, Jagoda; Watt, Frank

1999-10-01

Epilepsy is a recurrent disorder of cerebral function characterised by sudden brief attacks of altered consciousness, motor activity or sensory phenomena, and affects approximately 1% of the population. Kainic acid injection induces neuronal degeneration in rats, is associated with glial hypertrophy and proliferation in the CA3-CA4 fields of hippocampal complex, and is a model for temporal lobe epilepsy. In this study we have applied Nuclear Microscopy to the investigation of the elemental changes within the hippocampus and the cortex areas of the rat brain following kainate injection. Analyses of unstained freeze dried tissue sections taken at 1 day and 1, 2, 3 and 4 weeks following injection were carried out using the Nuclear Microscopy facility at the Research Centre for Nuclear Microscopy, National University of Singapore. Quantitative analysis and elemental mapping indicates that there are significant changes in the calcium levels and distributions in the hippocampus as early as 1 day following injection. Preliminary results indicate a rapid increase in cellular calcium. High levels of calcium can activate calcium dependent proteins and phospholipases. Activation of phospholipase A 2 can be harmful to surrounding neurons through free radical damage. In addition to observed increases in calcium, there was evidence of increases in iron levels. This is consistent with measurements in other degenerative brain disorders, and may signal a late surge in free radical production.

Changes in calcium and iron levels in the brains of rats during kainate induced epilepsy

International Nuclear Information System (INIS)

Ren, M.-Q.; Ong, W.-Y.; Makjanic, Jagoda; Watt, Frank

1999-01-01

Epilepsy is a recurrent disorder of cerebral function characterised by sudden brief attacks of altered consciousness, motor activity or sensory phenomena, and affects approximately 1% of the population. Kainic acid injection induces neuronal degeneration in rats, is associated with glial hypertrophy and proliferation in the CA3-CA4 fields of hippocampal complex, and is a model for temporal lobe epilepsy. In this study we have applied Nuclear Microscopy to the investigation of the elemental changes within the hippocampus and the cortex areas of the rat brain following kainate injection. Analyses of unstained freeze dried tissue sections taken at 1 day and 1, 2, 3 and 4 weeks following injection were carried out using the Nuclear Microscopy facility at the Research Centre for Nuclear Microscopy, National University of Singapore. Quantitative analysis and elemental mapping indicates that there are significant changes in the calcium levels and distributions in the hippocampus as early as 1 day following injection. Preliminary results indicate a rapid increase in cellular calcium. High levels of calcium can activate calcium dependent proteins and phospholipases. Activation of phospholipase A 2 can be harmful to surrounding neurons through free radical damage. In addition to observed increases in calcium, there was evidence of increases in iron levels. This is consistent with measurements in other degenerative brain disorders, and may signal a late surge in free radical production

Ketamine alleviates bradykinin-induced disruption of the mouse cerebrovascular endothelial cell-constructed tight junction barrier via a calcium-mediated redistribution of occludin polymerization

International Nuclear Information System (INIS)

Chen, Jui-Tai; Lin, Yi-Ling; Chen, Ta-Liang; Tai, Yu-Ting; Chen, Cheng-Yu; Chen, Ruei-Ming

2016-01-01

Highlights: • Ketamine could suppress bradykinin-induced intracellular calcium mobilization. • Ketamine induced B1R protein and mRNA expressions but did not change B2R protein levels. • Ketamine attenuated bradykinin-induced redistribution of occludin tight junctions. • Ketamine prevented bradykinin-induced breakage of the MCEC-constructed tight junction barrier. - Abstract: Following brain injury, a sequence of mechanisms leads to disruption of the blood-brain barrier (BBB) and subsequent cerebral edema, which is thought to begin with activation of bradykinin. Our previous studies showed that ketamine, a widely used intravenous anesthetic agent, can suppress bradykinin-induced cell dysfunction. This study further aimed to evaluate the protective effects of ketamine against bradykinin-induced disruption of the mouse cerebrovascular endothelial cell (MCEC)-constructed tight junction barrier and the possible mechanisms. Exposure of MCECs to bradykinin increased intracellular calcium (Ca 2+ ) concentrations in a time-dependent manner. However, pretreatment of MCECs with ketamine time- and concentration-dependently lowered the bradykinin-induced calcium influx. As to the mechanisms, although exposure of MCECs to ketamine induced bradykinin R1 receptor protein and mRNA expression, this anesthetic did not change levels of the bradykinin R2 receptor, a major receptor that responds to bradykinin stimulation. Bradykinin increased amounts of soluble occludin in MCECs, but pretreatment with ketamine alleviated this disturbance in occludin polymerization. Consequently, exposure to bradykinin decreased the transendothelial electronic resistance in the MCEC-constructed tight junction barrier. However, pretreatment with ketamine attenuated the bradykinin-induced disruption of the tight junction barrier. Taken together, this study shows that ketamine at a therapeutic concentration can protect against bradykinin-induced breakage of the BBB via suppressing calcium

Live Imaging of Calcium Dynamics during Axon Degeneration Reveals Two Functionally Distinct Phases of Calcium Influx

Science.gov (United States)

Yamagishi, Yuya; Tessier-Lavigne, Marc

2015-01-01

Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program. SIGNIFICANCE STATEMENT Axon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

Directory of Open Access Journals (Sweden)

Dmitry eSamigullin

2015-01-01

Full Text Available At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 рА and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 µM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Aree Protette del Po e della Collina Torinese: studi propedeutici alla Candidatura MAB - Man and the Biosphere

Directory of Open Access Journals (Sweden)

Cimnaghi Elisabetta

2014-03-01

Full Text Available Il programma MAB fu lanciato dall’UNESCO negli anni ’70 al fine di migliorare il rapporto tra uomo e ambiente. Negli anni il programma ha portato al riconoscimento di Riserve della Biosfera che gli Stati Membri si impegnano a gestire nella prospettiva della conservazione delle risorse. L’Organismo Gestore dell’Area Protetta, partendo, da un lato, dalla consapevolezza della ricchezza ambientale che caratterizza il suo territorio e, dall’altro, del contesto di sviluppo antropico elevato in cui essa è situata, ha riconosciuto nel programma MAB un’opportunità per incrementare le sue attività di protezione e potenziamento delle risorse. Questo articolo descrive i primi risultati ottenuti negli studi preliminari per la Designazione al MAB, con particolare attenzione sia alle opportunità di implementazione di progetti legati al marchio “Collina Po”, creato dall’Organo di Gestione del Parco, si ain termini di scambio di ‘best practices’ con altre Riserve di Biosfera.

Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

OpenAIRE

Szentesi, Péter; Szappanos, Henrietta; Szegedi, Csaba; Gönczi, Monika; Jona, István; Cseri, Julianna; Kovács, László; Csernoch, László

2004-01-01

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine bind...

Freeze-dried formulation for direct {sup 99m}Tc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

Energy Technology Data Exchange (ETDEWEB)

Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando E-mail: normando@ict.sld.cu

1999-08-01

Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with {sup 99m}Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with {sup 99m}Tc at high yield. The resulting {sup 99m}Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

Calcium hydroxide induced apexification with apical root development: a clinical case report.

Science.gov (United States)

Soares, J; Santos, S; César, C; Silva, P; Sá, M; Silveira, F; Nunes, E

2008-08-01

To report the induction of apical root development by calcium hydroxide in teeth with pulp necrosis and periapical radiolucency. A 10-year-old male patient was admitted to the clinic complaining of an intense pain and oedema on the anterior facial region, compatible with an acute dentoalveolar abscess. There was a previous history of dental trauma; only tooth 11 was negative to pulp sensitivity tests. Radiographically, tooth 11 exhibited incomplete root formation, characterized by a wide root canal, thin and fragile dentinal walls, and an extensive, divergent foraminal opening associated with an apical radiolucency. The first appointment focused on urgent local and systemic treatment. Apexification treatment commenced at the second session after 7 days, by means of chemo-mechanical debridement throughout the entire root canal, using K-files and irrigation with a 2.5% sodium hypochlorite solution. Subsequently, a calcium hydroxide paste was applied and changed four times over 8 months, when radiographic examination revealed complete closure of the foraminal opening, resulting in resolution of the periapical radiolucency and associated with 5 mm of additional root development. The root canal was filled by thermomechanical compaction of gutta-percha and sealer. A 3-year follow-up revealed normal periapical tissues and the absence of symptoms. * In young patients, dental trauma may cause pulp necrosis and arrest of root formation. * Under certain circumstances, chemo-mechanical debridement, including the use of a calcium hydroxide paste, is a valid alternative to mineral trioxide aggregate and or surgery for root-end closure. * In teeth with incompletely formed roots associated with periapical lesions, calcium hydroxide can induce periapical repair through the closure of the foramen and apical root development.

Therapeutic role of calcium and vitamin K3 in chemically induced hepatocarcinogenesis - new tools for cancer treatment.

Science.gov (United States)

Anwar, Firoz; Khan, Ruqaiyah; Sachan, Richa; Kazmi, Imran; Rawat, Alisha; Sabih, Abdullah; Singh, Rajbala; Afzal, Muhammad; Ahmad, Aftab; Al-Orab, Abdulaziz S; Al-Abbasi, F A; Bhatt, Prakash Chandra; Kumar, Vikas

2018-04-17

HCC has been reported to be immensely occurring carcinoma worldwide. Recent days the mortality occurred due to liver cancer has also been found to be increased at an alarming speed affecting mostly the young patients. The aim of the current study was to decipher the role of calcium and vitamin K3 in the treatment of chemically induced hepatocarcinogenesis in the male Wistar rats. Liver cancer was induced via a subnecrogenic dose of 160 mg/kg body weight, diethylnitrosamine (DENA) when associated with fasting/refeeding in male Wistar rats. It elevated the serum glutamate oxaloacetate (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), bilirubin, total cholesterol (CH), triglycerides (TG), alfa-fetoprotein (AFP) and reduced high-density lipoprotein (HDL). Histopathological examination of liver tissue showed marked carcinogenicity of the chemical carcinogen. Food, water intake and animal weights were also assessed, respectively. The animals exposed to DENA showed a significant decrease in the body weight. The elevated levels of serum SGOT, SGPT, ALP, AFP, TC and TG were restored by administration of calcium and Vit K (ad libitum) combination at higher dose than the normal dietary requirement (3 mg/kg) daily for 12 weeks p.o. Physiological and biochemical analysis showed the beneficial effects of calcium and vitamin K3 combination in the animals exposed to DENA. The results deciphered the beneficial effects of calcium and vitamin K3 in combination.

Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells.

Science.gov (United States)

Vatsa, Parul; Chiltz, Annick; Luini, Estelle; Vandelle, Elodie; Pugin, Alain; Roblin, Gabriel

2011-07-01

The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca²⁺](cyt) which intensity depends on ergosterol concentration in the range 0.002-10 μM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterol-induced pH and [Ca²⁺](cyt) changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl₃, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca²⁺](cyt) increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calcium-independent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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Le Geng

2018-06-01

Full Text Available Background/Aims: Heart failure induced by tachycardia, the most common arrhythmia, is frequently observed in clinical practice. This study was designed to investigate the underlying mechanisms. Methods: Rapid electrical stimulation (RES at a frequency of 3 Hz was applied on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs for 7 days, with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia, respectively. Age-matched hiPSC-CMs without electrical stimulation or with slow electrical stimulation (1 Hz were set as no electrical stimulation (NES control or low-frequency electrical stimulation (LES control. Following stimulation, JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions. Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide (AV/PI staining flow cytometry analysis. Calcium transients and L-type calcium currents were recorded to evaluate calcium homeostasis. Western blotting and qPCR were performed to evaluate the protein and mRNA expression levels of apoptosis-related genes and calcium homeostasis-regulated genes. Results: Compared to the controls, hiPSC-CMs following RES presented mitochondrial dysfunction and an increased apoptotic percentage. Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES. Molecular analysis demonstrated upregulated expression of Caspase3 and increased Bax/Bcl-2 ratio. Genes related to calcium re-sequence were downregulated, while phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII was significantly upregulated following RES. There was no significant difference between the NES control and LES control groups in these aspects. Inhibition of CaMKII with 1 µM KN93 partly reversed these adverse effects of RES. Conclusion: RES on hiPSC-CMs disturbed calcium homeostasis, which led to mitochondrial stress, promoted cell apoptosis and

Anti-GD2 mAb and Vorinostat synergize in the treatment of neuroblastoma

NARCIS (Netherlands)

Kroesen, M.; Bull, C.; Gielen, P.R.; Brok, I.C.; Armandari, I.; Wassink, M.; Looman, M.W.G.; Boon, L.; Brok, M.H.M.G.M. den; Hoogerbrugge, P.M.; Adema, G.J.

2016-01-01

Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical

Experimental Treatment of Bladder Cancer with Bi-213-anti-EGFR MAb

International Nuclear Information System (INIS)

Seidl, Christof; Pfost, Birgit; Müller, Felix

2013-01-01

Therapy of non-muscle-invasive bladder cancer (carcinoma in situ) comprises transurethral resection of the tumour and subsequent instillation of the chemotherapeutic drug mitomycin C in order to eradicate remaining tumour cells. Yet 15 – 40% of treated patients relapse within 5 years. Therefore, new therapeutic strategies to combat tumour recurrence are needed. Alpha-particle emitting radionuclides efficiently kill single tumour cells or small tumour cell clusters. Because the epidermal growth factor receptor (EGFR) is overexpressed on bladder cancer cells, conjugates composed of the alpha-emitter Bi-213 and the anti-EGFR antibody matuzumab should provide a powerful drug to eliminate disseminated bladder cancer cells. Therefore, the aims of our study were (i) to analyse the cytotoxic effects of Bi-213-anti-EGFR radioimmunoconjugates at the cellular level, (ii) to evaluate therapeutic efficacy of intravesically applied Bi-213- anti-EGFR-Mab in a nude mouse model with intravesical human bladder cancer xenografts, (iii) to compare Bi- 213-anti-EGFR-Mab efficacy with chemotherapy using mitomycin C and (iv) to demonstrate that radioimmunotherapy is not toxic to cells of the bladder wall and of the kidneys

Effect of oral calcium and calcium + fluoride treatments on mouse bone properties during suspension

Science.gov (United States)

Simske, S. J.; Luttges, M. W.; Allen, K. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The bone effects of oral dosages of calcium chloride with or without supplementary sodium fluoride were assessed in antiorthostatically suspended mice. Two calcium dosages were used to replace half (3.1 mM) or all(6.3 mM) of the dietary calcium lost due to reduced food intake by the suspended mice. Two groups of 6.3 mM CaCl2-treated mice were additionally treated with 0.25 or 2.5 mM NaF. The results indicate that supplementation of the mouse drinking water with calcium salts prevents bone changes induced by short-term suspension, while calcium salts in combination with fluoride are less effective as fluoride dosage increases. However, the calcium supplements change the relationship between the femur mechanical properties and the mineral composition of the bone. Because of this, it appears that oral calcium supplements are effective through a mechanism other than simple dietary supplementation and may indicate a dependence of bone consistency on systemic and local fluid conditions.

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

Directory of Open Access Journals (Sweden)

Li H

2015-07-01

Full Text Available Hua-Fei Li,1–3,* Cong Wu,4,* Ting Chen,5,* Ge Zhang,1 He Zhao,1 Chang-Hong Ke,1 Zheng Xu21International Joint Cancer Institute, Translation Medicine Institute, 2Planning Division, Scientific Research Department, 3Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, 4Department of Laboratory Diagnosis, Changhai Hospital, 5Department of Cardiology, Changhai Hospital, the Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The CD20-directed monoclonal antibody rituximab (RTX established a new era in the treatment of non-Hodgkin lymphoma (NHL; however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD, they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer–RTX–tositumomab [PPRT nanocomb] was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced “off-rate” to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, “cross-cell link”-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With

Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

International Nuclear Information System (INIS)

Zaffino, Paolo; Spadea, Maria Francesca; Raudaschl, Patrik; Fritscher, Karl; Sharp, Gregory C.

2016-01-01

Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, where a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against

Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

Energy Technology Data Exchange (ETDEWEB)

Zaffino, Paolo; Spadea, Maria Francesca [Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro 88100 (Italy); Raudaschl, Patrik; Fritscher, Karl [Institute for Biomedical Image Analysis, Private University of Health Sciences, Medical Informatics and Technology, Hall in Tirol 6060 (Austria); Sharp, Gregory C. [Department for Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts 02114 (United States)

2016-09-15

Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, where a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against

Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

International Nuclear Information System (INIS)

Hillmann, Petra; Koese, Meryem; Soehl, Kristina; Mueller, Christa E.

2008-01-01

High blood levels of ammonium/ammonia (NH 4 + /NH 3 ) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca 2+ ] i ) in astrocytoma (EC 50 = 6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca 2+ ] i was due to an intracellular effect of NH 4 + /NH 3 and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by G q protein-coupled receptors (H 1 and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH 3 /NH 4 + from neuronal cells. Cellular uptake of NH 4 + /NH 3 was mainly by passive diffusion of NH 3 . Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia

Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries.

Science.gov (United States)

Dassanayake, Rohana P; Shanthalingam, Sudarvili; Herndon, Caroline N; Lawrence, Paulraj K; Frances Cassirer, E; Potter, Kathleen A; Foreyt, William J; Clinkenbeard, Kenneth D; Srikumaran, Subramaniam

2009-02-02

Mannheimia haemolytica causes pneumonia in both bighorn sheep (BHS, Ovis canadensis) and domestic sheep (DS, Ovis aries). Under experimental conditions, co-pasturing of BHS and DS results in fatal pneumonia in BHS. It is conceivable that certain serotypes of M. haemolytica carried by DS are non-pathogenic to them, but lethal for BHS. M. haemolytica serotypes A1 and A2 are carried by DS in the nasopharynx. However, it is the serotype A2 that predominantly causes pneumonia in DS. The objectives of this study were to determine whether serotype A1 exhibits differential pathogenicity to BHS and DS, and to determine whether leukotoxin (Lkt) secreted by this organism is its primary virulence factor. Three groups each of BHS and DS were intra-tracheally administered either 1 x 10(9)cfu of serotype A1 wild-type (lktA-Wt group), Lkt-deletion mutant of serotype A1-(lktA-Mt group), or saline (control group), respectively. In the lktA-Wt groups, all four BHS died within 48h while none of the DS died during the 2-week study period. In the lktA-Mt groups, none of the BHS or DS died. In the control groups, one DS died due to an unrelated cause. Necropsy and histopathological findings revealed that death of BHS in the lktA-Wt group was due to bilateral, fibrinohemorrhagic pneumonia. Although the A1-Mt-inoculated BHS were clinically normal, on necropsy, lungs of two BHS showed varying degrees of mild chronic pneumonia. These results indicate that M. haemolytica serotype A1 is non-pathogenic to DS, but highly lethal to BHS, and that Lkt is the primary virulence factor of M. haemolytica.

Preparation of monoclonal antibodies against radiation-induced protein

International Nuclear Information System (INIS)

Nozawa, R.; Tanaka, A.; Watanabe, H.; Kitayama, S.

1992-01-01

We obtained the 6 monoclonal antibodies against gamma-induced proteins of Deinococcus radiodurans, and these antibodies were designated as Mab-3F, 4B, 4D, 4F, 4G and 12G. Using these antibodies, we investigated the relations between gamma-induced proteins and other stress protein in strain R1, and the induction of proteins were compared among strain R1, resistant mutant (rec1) and radiosensitive mutant (rec30). We found new 6 proteins recognized by these monoclonal antibodies which were induced after gamma-irradiation especially in strain R1 and rec 1, but not induced in strain rec30. We suppose that these proteins participate in repair of DNA damages including double strand breaks caused by gamma-irradiation. One of them was around 46kDa protein band recognized by Mab-12G, and this protein was so induced in a large quantity after irradiation that the protein could detect by gold staining. In addition to this observation, we found some proteins which were induced in R1 and rec 1 by gamma-irradiation and other stress, but not in strain rec30, such as 31kDa protein band recognized by Mab-3F, 4B and 4G, and other 11 proteins which were especially induced in irradiated strain R1. The latter proteins might be reinforcement factor to radioresistance such as GroE and DnaK, or participant in repair of damage by gamma-irradiation in strain R1. (author)

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

Science.gov (United States)

Mehrotra nee Tandon, P; Lind, D S; Bear, H D; Susskind, B M

1992-08-01

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.

Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

DEFF Research Database (Denmark)

Timmermann, D B; Lund, Trine Meldgaard; Belhage, B

2001-01-01

The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured...... channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively...... most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise...

A new synthetic granular calcium phosphate compound induces new bone in a sinus lift rabbit model.

Science.gov (United States)

Trbakovic, Amela; Hedenqvist, Patricia; Mellgren, Torbjörn; Ley, Cecilia; Hilborn, Jöns; Ossipov, Dmitri; Ekman, Stina; Johansson, Carina B; Jensen-Waern, Marianne; Thor, Andreas

2018-03-01

The aim of this study was to investigate if a synthetic granular calcium phosphate compound (CPC) and a composite bisphosphonate-linked hyaluronic acid-calcium phosphate hydrogel (HABP·CaP) induced similar or more amount of bone as bovine mineral in a modified sinus lift rabbit model. Eighteen adult male New Zeeland White rabbits, received randomly one of the two test materials on a random side of the face, and bovine mineral as control on the contralateral side. In a sinus lift, the sinus mucosa was elevated and a titanium mini-implant was placed in the alveolar bone. Augmentation material (CPC, HABP·CaP or bovine bone) was applied in the space around the implant. The rabbits were euthanized three months after surgery and qualitative and histomorphometric evaluation were conducted. Histomorphometric evaluation included three different regions of interest (ROIs) and the bone to implant contact on each installed implant. Qualitative assessment (p = <.05), histomorphometric evaluations (p = < .01), and implant incorporation (p = <.05) showed that CPC and bovine mineral induced similar amount of bone and more than the HABP·CaP hydrogel. CPC induced similar amount of bone as bovine mineral and both materials induced more bone than HABP·CaP hydrogel. The CPC is suggested as a synthetic alternative for augmentations in the maxillofacial area. Copyright © 2017 Elsevier Ltd. All rights reserved.

One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons.

Science.gov (United States)

Yu, Yan; Oberlaender, Kristin; Bengtson, C Peter; Bading, Hilmar

2017-07-01

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.4±5.1 action potentials during a 6.7±1.9s depolarization of 19.5±2.0mV causing a 9.3±0.9s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5-8 consecutive burst-induced nuclear calcium transients at 0.05-0.15Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased. Copyright © 2017 Elsevier Ltd. All rights reserved.

HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

NARCIS (Netherlands)

STEGE, GJJ; WIERENGA, PK; KAMPINGA, HH; KONINGS, AWT

1993-01-01

It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free

Susceptibility of different subsets of immature thymocytes to apoptosis induced by anti-TCRmAb

International Nuclear Information System (INIS)

Li Hongmei; Zhong Renqian; Yu Jiaping; Kong Xiantao; Chen Weifeng

2003-01-01

To analysis the susceptibility of different subsets of immature mice thymocytes to apoptosis induced by anti-TCRmAbs in vitro apoptosis was induced in unfractionated mice thymocytes by anti-TCRmAb. In Vivo apoptosis was induced in BALB/c mice by anti-TCR mAb, and thymocytes were examined by FACS. Results showed that CD4 + CD8 + DP thymocytes and CD4 - CD8 + CD3 - thymocytes were equally sensitive to apoptosis after treatment with the anti-TCR mAb. In sharp contrast, the early migrants or precursor containing thymocytes which are CD4 - CD8 - CD3 - TN have a lower spontaneous apoptosis rate and were relatively resistant to the anti-TCR mAb. The findings showed a breakpoint in thymocyte sensitivity to apoptosis which occurs after the onset of CD4 - CD8 + CD3 expression, suggesting that susceptibility of thymocytes to apoptosis is developmentally regulated

Red meat and colon cancer : dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium

NARCIS (Netherlands)

Sesink, ALA; Termont, DSML; Kleibeuker, JH; Van der Meer, R

2001-01-01

High intake of red meat is associated with increased colon cancer risk. We have shown earlier that this may be due to the high haem content of red meat, because dietary haem increased cytolytic activity of faecal water and colonic epithelial proliferation. Dietary calcium inhibits diet-induced

Effect of Topical Calcium Channel Blockers on Intraocular Pressure in Steroid-induced Glaucoma.

Science.gov (United States)

Ganekal, Sunil; Dorairaj, Syril; Jhanji, Vishal; Kudlu, Krishnaprasad

2014-01-01

To evaluate the effect of 0.125% verapamil and 0.5% diltiazem eye drops on intraocular pressure (IOP) in steroid-induced glaucoma in rabbit eyes. A total of 18 rabbits with steroid-induced glaucoma were divided into three groups (A, B and C; n = 6 each). Right eyes in groups A, B and C received 0.5% diltiazem, 0.125% verapamil and 0.5% timolol eye drops twice daily for 12 days, respectively; whereas, left eyes received distilled water. IOP was measured with Tono-pen XL at baseline, day 4, day 8, and day 12 of treatment. Both 0.5% diltiazem and 0.125% verapamil eye drops significantly reduced IOP compared to control eyes (p cite this article: Ganekal S, Dorairaj S, Jhanji V, Kudlu K. Effect of Topical Calcium Channel Blockers on Intraocular Pressure in Steroid-induced Glaucoma. J Current Glau Prac 2014;8(1):15-19.

Plasma Calcium, Inorganic Phosphate and Magnesium During Hypocalcaemia Induced by a Standardized EDTA Infusion in Cows

Directory of Open Access Journals (Sweden)

Enemark JMD

2001-06-01

Full Text Available The intravenous Na2EDTA infusion technique allows effective specific chelation of circulating Ca2+ leading to a progressive hypocalcaemia. Methods previously used were not described in detail and results obtained by monitoring total and free ionic calcium were not comparable due to differences in sampling and analysis. This paper describes a standardized EDTA infusion technique that allowed comparison of the response of calcium, phosphorus and magnesium between 2 groups of experimental cows. The concentration of the Na2EDTA solution was 0.134 mol/l and the flow rate was standardized at 1.2 ml/kg per hour. Involuntary recumbency occurred when ionised calcium dropped to 0.39 – 0.52 mmol/l due to chelation. An initial fast drop of ionized calcium was observed during the first 20 min of infusion followed by a fluctuation leading to a further drop until recumbency. Pre-infusion [Ca2+] between tests does not correlate with the amount of EDTA required to induce involuntary recumbence. Total calcium concentration measured by atomic absorption remained almost constant during the first 100 min of infusion but declined gradually when the infusion was prolonged. The concentration of inorganic phosphate declined gradually in a fluctuating manner until recumbency. Magnesium concentration remained constant during infusion. Such electrolyte responses during infusion were comparable to those in spontaneous milk fever. The standardized infusion technique might be useful in future experimental studies.

Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

Science.gov (United States)

Safina, Dina R; Surin, Alexander M; Pinelis, Vsevolod G; Kostrov, Sergey V

2015-12-01

Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ±  0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period. © 2015 Wiley Periodicals, Inc.

Kokumi Substances, Enhancers of Basic Tastes, Induce Responses in Calcium-Sensing Receptor Expressing Taste Cells

Science.gov (United States)

Maruyama, Yutaka; Yasuda, Reiko; Kuroda, Motonaka; Eto, Yuzuru

2012-01-01

Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami. PMID:22511946

[The anti-tumour effect of Wuxing soup and its mechanism in inducing apoptosis of tumour cells mediated by calcium].

Science.gov (United States)

Mo, Fei; Hu, Jing-Ying; Gan, Yu; Zhao, Yang-Xing; Zhao, Xin-Tai

2008-09-01

To confirm the anti-cancer effect and mechanism of Wuxing soup. Inhibition of cellular growth under Wuxing soup treatment was observed by MTT; Apoptosis was detected by gel electrophoresis, transmission electron microscopy and FACS; The concentration of calcium was measured by fluorescence probe. After SGC-7901 cell being treated by Wuxing soup, it showed that: 1) Wuxing soup could specifically inhibit cancer cells proliferation in a time and dose dependent manner; 2) Typical apoptotic morphological changes and DNA ladder of SGC-7901 cells were observed; 3) calcium inhibitor Bapta AM could reduce the apoptotic rate and protect SGC-7901 cells in a dose dependent manner. Wuxing soup has an effective inhibition on cancer cells, and can induce SGC-7901 cells to apoptosis by calcium.

Calcium movements and the cellular basis of gravitropism

Science.gov (United States)

Roux, S. J.; Biro, R. L.; Hale, C. C.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.

Role of oxidative stress, mitochondrial membrane potential, and calcium homeostasis in human lymphocyte death induced by nickel carbonate hydroxide in vitro

Energy Technology Data Exchange (ETDEWEB)

M' Bemba-Meka, Prosper [Faculty of Medicine, Universite de Montreal, QC (Canada); University of Louisville, Department of Pharmacology and Toxicology, Center for Genetics and Molecular Medicine, Louisville, KY (United States); Lemieux, Nicole [Universite de Montreal, Department of Pathology and Cellular Biology, Main Station, P.O. Box 6128, Montreal, QC (Canada); Chakrabarti, Saroj K. [Faculty of Medicine, Universite de Montreal, QC (Canada)

2006-07-15

When isolated human lymphocytes were treated in vitro with various concentrations of soluble form of nickel carbonate hydroxide (NiCH) (0-1 mM), at 37 C for 4 h, both concentration- and time-dependent effects of NiCH on lymphocyte death were observed. Increased generation of hydrogen peroxide (H{sub 2}O{sub 2}), superoxide anion (O{sub 2} {sup -}), depletion of both no protein (NP-) and protein (P-) sulfhydryl (SH) contents and lipid peroxidation (LPO) were induced by NiCH. Pretreatment of lymphocytes with either catalase (H{sub 2}O{sub 2} scavenger), or deferoxamine (DFO) (iron chelator), or excess glutathione (GSH) (an antioxidant) not only significantly reduced the NiCH-induced generation of H{sub 2}O{sub 2} and LPO, but also increased the NP-SH and P-SH contents initially reduced by NiCH. NiCH-induced generation of excess O{sub 2} {sup -} but not excess LPO was significantly reduced by pretreatment with superoxide dismutase (SOD). NiCH-induced lymphocyte death was significantly prevented by pre-treatment with either catalase, or dimethylthiourea/mannitol (hydroxyl radical scavengers), or DFO, or excess GSH/N-acetylcysteine. NiCH-induced lymphocyte death was also significantly prevented by pretreatment with excess SOD. Thus, various types of oxidative stresses play an important role in NiCH-induced lymphocyte death. Cotreatment with cyclosporin A, a specific inhibitor of alteration in mitochondrial membrane potential ({delta}{psi}{sub m}), not only inhibited NiCH-induced alteration in {delta}{psi}{sub m}, but also significantly prevented Ni-compound-induced lymphocyte death. Furthermore, NiCH-induced destabilization of cellular calcium homeostasis. As such, NiCH-induced lymphocyte death was significantly prevented by modulating intracellular calcium fluxes such as Ca{sup 2+} channel blockers and intracellular Ca{sup 2+} antagonist. Thus, the mechanism of NiCH (soluble form)-induced activation of lymphocyte death signalling pathways involves not only the excess

The Marine Guanidine Alkaloid Crambescidin 816 Induces Calcium Influx and Cytotoxicity in Primary Cultures of Cortical Neurons through Glutamate Receptors.

Science.gov (United States)

Mendez, Aida G; Juncal, Andrea Boente; Silva, Siguara B L; Thomas, Olivier P; Martín Vázquez, Víctor; Alfonso, Amparo; Vieytes, Mercedes R; Vale, Carmen; Botana, Luís M

2017-07-19

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

The effect of calcium on auxin depletion-induced tomato ...

African Journals Online (AJOL)

Indole-3-acetic acid (IAA) and calcium are the most important factors that instigate plant organ abscission. This study aimed to elucidate the mechanisms that underlie the effects of IAA and calcium on delayed abscission in tomato. The results showed a clear trend towards reduced abscission rates with increased ...

Dopamine Induces LTP Differentially in Apical and Basal Dendrites through BDNF and Voltage-Dependent Calcium Channels

Science.gov (United States)

Navakkode, Sheeja; Sajikumar, Sreedharan; Korte, Martin; Soong, Tuck Wah

2012-01-01

The dopaminergic modulation of long-term potentiation (LTP) has been studied well, but the mechanism by which dopamine induces LTP (DA-LTP) in CA1 pyramidal neurons is unknown. Here, we report that DA-LTP in basal dendrites is dependent while in apical dendrites it is independent of activation of L-type voltage-gated calcium channels (VDCC).…

Effect of calcium intake on urinary oxalate excretion in calcium stone-forming patients

Directory of Open Access Journals (Sweden)

Nishiura J.L.

2002-01-01

Full Text Available Dietary calcium lowers the risk of nephrolithiasis due to a decreased absorption of dietary oxalate that is bound by intestinal calcium. The aim of the present study was to evaluate oxaluria in normocalciuric and hypercalciuric lithiasic patients under different calcium intake. Fifty patients (26 females and 24 males, 41 ± 10 years old, whose 4-day dietary records revealed a regular low calcium intake (<=500 mg/day, received an oral calcium load (1 g/day for 7 days. A 24-h urine was obtained before and after load and according to the calciuria under both diets, patients were considered as normocalciuric (NC, N = 15, diet-dependent hypercalciuric (DDHC, N = 9 or diet-independent hypercalciuric (DIHC, N = 26. On regular diet, mean oxaluria was 30 ± 14 mg/24 h for all patients. The 7-day calcium load induced a significant decrease in mean oxaluria compared to the regular diet in NC and DIHC (20 ± 12 vs 26 ± 7 and 27 ± 18 vs 32 ± 15 mg/24 h, respectively, P<0.05 but not in DDHC patients (22 ± 10 vs 23 ± 5 mg/24 h. The lack of an oxalate decrease among DDHC patients after the calcium load might have been due to higher calcium absorption under higher calcium supply, with a consequent lower amount of calcium left in the intestine to bind with oxalate. These data suggest that a long-lasting regular calcium consumption <500 mg was not associated with high oxaluria and that a subpopulation of hypercalciuric patients who presented a higher intestinal calcium absorption (DDHC tended to hyperabsorb oxalate as well, so that oxaluria did not change under different calcium intake.

Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

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Rami Shinnawi

2015-10-01

Full Text Available The advent of the human-induced pluripotent stem cell (hiPSC technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs. To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight and calcium (GCaMP5G fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing.

Pressure induced reactions amongst calcium aluminate hydrate phases

KAUST Repository

Moon, Ju-hyuk

2011-06-01

The compressibilities of two AFm phases (strätlingite and calcium hemicarboaluminate hydrate) and hydrogarnet were obtained up to 5 GPa by using synchrotron high-pressure X-ray powder diffraction with a diamond anvil cell. The AFm phases show abrupt volume contraction regardless of the molecular size of the pressure-transmitting media. This volume discontinuity could be associated to a structural transition or to the movement of the weakly bound interlayer water molecules in the AFm structure. The experimental results seem to indicate that the pressure-induced dehydration is the dominant mechanism especially with hygroscopic pressure medium. The Birch-Murnaghan equation of state was used to compute the bulk modulus of the minerals. Due to the discontinuity in the pressure-volume diagram, a two stage bulk modulus of each AFm phase was calculated. The abnormal volume compressibility for the AFm phases caused a significant change to their bulk modulus. The reliability of this experiment is verified by comparing the bulk modulus of hydrogarnet with previous studies. © 2011 Elsevier Ltd. All rights reserved.

Intracellular sphingosine releases calcium from lysosomes.

Science.gov (United States)

Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

2015-11-27

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family.

Science.gov (United States)

Kaufman, I; Luchinsky, D G; Tindjong, R; McClintock, P V E; Eisenberg, R S

2013-11-01

We use Brownian dynamics (BD) simulations to study the ionic conduction and valence selectivity of a generic electrostatic model of a biological ion channel as functions of the fixed charge Q(f) at its selectivity filter. We are thus able to reconcile the discrete calcium conduction bands recently revealed in our BD simulations, M0 (Q(f)=1e), M1 (3e), M2 (5e), with a set of sodium conduction bands L0 (0.5e), L1 (1.5e), thereby obtaining a completed pattern of conduction and selectivity bands vs Q(f) for the sodium-calcium channels family. An increase of Q(f) leads to an increase of calcium selectivity: L0 (sodium-selective, nonblocking channel) → M0 (nonselective channel) → L1 (sodium-selective channel with divalent block) → M1 (calcium-selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L0 band is putatively identified with the eukaryotic sodium channel The scheme created is able to account for the experimentally observed mutation-induced transformations between nonselective channels, sodium-selective channels, and calcium-selective channels, which we interpret as transitions between different rows of the identification table. By considering the potential energy changes during permeation, we show explicitly that the multi-ion conduction bands of calcium and sodium channels arise as the result of resonant barrierless conduction. The pattern of periodic conduction bands is explained on the basis of sequential neutralization taking account of self-energy, as Q(f)(z,i)=ze(1/2+i), where i is the order of the band and z is the valence of the ion. Our results confirm the crucial influence of electrostatic interactions on conduction and on the Ca(2+)/Na(+) valence selectivity of calcium and sodium ion channels. The model and results could be also applicable to biomimetic nanopores with charged walls.

Micropore-induced capillarity enhances bone distribution in vivo in biphasic calcium phosphate scaffolds.

Science.gov (United States)

Rustom, Laurence E; Boudou, Thomas; Lou, Siyu; Pignot-Paintrand, Isabelle; Nemke, Brett W; Lu, Yan; Markel, Mark D; Picart, Catherine; Wagoner Johnson, Amy J

2016-10-15

The increasing demand for bone repair solutions calls for the development of efficacious bone scaffolds. Biphasic calcium phosphate (BCP) scaffolds with both macropores and micropores (MP) have improved healing compared to those with macropores and no micropores (NMP), but the role of micropores is unclear. Here, we evaluate capillarity induced by micropores as a mechanism that can affect bone growth in vivo. Three groups of cylindrical scaffolds were implanted in pig mandibles for three weeks: MP were implanted either dry (MP-Dry), or after submersion in phosphate buffered saline, which fills pores with fluid and therefore suppresses micropore-induced capillarity (MP-Wet); NMP were implanted dry. The amount and distribution of bone in the scaffolds were quantified using micro-computed tomography. MP-Dry had a more homogeneous bone distribution than MP-Wet, although the average bone volume fraction, BVF‾, was not significantly different for these two groups (0.45±0.03 and 0.37±0.03, respectively). There was no significant difference in the radial bone distribution of NMP and MP-Wet, but the BVF‾, of NMP was significantly lower among the three groups (0.25±0.02). These results suggest that micropore-induced capillarity enhances bone regeneration by improving the homogeneity of bone distribution in BCP scaffolds. The explicit design and use of capillarity in bone scaffolds may lead to more effective treatments of large and complex bone defects. The increasing demand for bone repair calls for more efficacious bone scaffolds and calcium phosphate-based materials are considered suitable for this application. Macropores (>100μm) are necessary for bone ingrowth and vascularization. However, studies have shown that microporosity (micropore-induced capillarity had the potential to enhance bone growth in vivo. This work illustrates the positive effects of capillarity on bone regeneration in vivo; it demonstrates that micropore-induced capillarity significantly

Calcium regulation and Alzheimer’s disease

Directory of Open Access Journals (Sweden)

Deepthi Rapaka

2014-09-01

Full Text Available Activation of the neuron induces transient fluctuations in [Ca2+]i. This transient rise in [Ca2+]i is dependent on calcium entry via calcium channels and release of calcium from intracellular stores, finally resulting in increase in calcium levels, which activates calcium regulatory proteins to restore the resting calcium levels by binding to the calcium-binding proteins, sequestration into the endoplasmic reticulum and the mitochondria, and finally extrusion of calcium spike potential from the cell by adenosine triphosphate-driven Ca2+ pumps and the Na+/Ca2+ exchanger. Improper regulation of calcium signaling, sequentially, likely contributes to synaptic dysfunction and excitotoxic and/or apoptotic death of the vulnerable neuronal populations. The cognitive decline associated with normal aging is not only due to neuronal loss, but is fairly the result of synaptic connectivity. Many evidences support that Ca2+ dyshomeostasis is implicated in normal brain aging. Thus the chief factor associated with Alzheimer’s disease was found to be increase in the levels of free intracellular calcium, demonstrating that the excessive levels might lead to cell death, which provides a key target for the calcium channel blockers might be used as the neuroprotective agents in Alzheimer’s disease.

Analysis of calcium-induced conformational changes in calcium-binding allergens and quantitative determination of their IgE binding properties.

Science.gov (United States)

Parody, Nuria; Fuertes, Miguel Angel; Alonso, Carlos; Pico de Coaña, Yago

2013-01-01

The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

Science.gov (United States)

Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

2014-11-14

We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb(P) CO17-1A × BR55-treated. The mAb(P) CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb(P) CO17-1A × BR55. In addition, the mAb(P) CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells

DEFF Research Database (Denmark)

Müller, B K; Kabos, P; Belhage, B

1996-01-01

Indirect evidence supports a protective role of some EF-hand calcium-binding proteins against calcium-induced neurotoxicity. Little is known about how these proteins influence cytosolic calcium levels. After cloning the parvalbumin cDNA into an expression vector, teratocarcinoma cells (PCC7) were...

Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

Science.gov (United States)

Lee, J. S.; Mulkey, T. J.; Evans, M. L.

1984-01-01

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.

Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium.

Science.gov (United States)

Wang, Hongbo; Wang, Xiaolong; Li, Yan; Yu, Hao; Wang, Changliang; Feng, Chunmei; Xu, Guohui; Chen, Jiajun; You, Jiabin; Wang, Pengfei; Wu, Xu; Zhao, Rui; Zhang, Guohua

2018-04-01

It has been identified that chronic ethanol exposure damages the nervous system, particularly neurons. There is scientific evidence suggesting that neuronal loss caused by chronic ethanol exposure has an association with neuron apoptosis and intracellular calcium oscillation is one of the primary inducers of apoptosis. Therefore, the present study aimed to investigate the inductive effects of intracellular calcium oscillation on apoptosis in SK-N-SH human neuroblastoma cells and the protective effects of the N-methyl-D-aspartic acid receptor (NMDAR) antagonist, memantine, on SK-N-SH cell apoptosis caused by chronic ethanol exposure. SK-N-SH cells were treated with 100 mM ethanol and memantine (4 µM) for 2 days. Protein expression of NR1 was downregulated by RNA interference (RNAi). Apoptosis was detected by Annexin V/propidium iodide (PI) double-staining and flow cytometry and cell viability was detected using an MTS kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration and the levels of NR1 and caspase-3 were detected using western blotting. NR1 mRNA levels were also detected using qPCR. It was found that chronic ethanol exposure reduced neuronal cell viability and caused apoptosis of SK-N-SH cells, and the extent of damage in SK-N-SH cells was associated with ethanol exposure concentration and time. In addition, chronic ethanol exposure increased the concentration of intracellular calcium in SK-N-SH cells by inducing the expression of NMDAR, resulting in apoptosis, and memantine treatment reduced ethanol-induced cell apoptosis. The results of the present study indicate that the application of memantine may provide a novel strategy for the treatment of alcoholic dementia.

Can atorvastatin calcium cause asymptomatic hypercalcemia?

Science.gov (United States)

Ipekçi, Süleyman Hilmi; Baldane, Süleyman; Sözen, Mehmet; Kebapçılar, Levent

2014-10-01

The use of statins may have unnatural effects. A 54-year-old woman was admitted to the hospital with an incidental finding of hypercalcemia (10.8 mg/dL). There was no disease other than hyperlipidemia, and the patient had been on a course of atorvastatin calcium 10 mg for 1.5 years. A workup investigation to diagnose the cause of hypercalcemia was completed. The investigation did not reveal any pathological diseases that may have caused the hypercalcemia. The hypercalcemia resolved after atorvastatin-calcium was stopped, and the patient developed hypercalcemia shortly after the initiation of the atorvastatin calcium. Here, we report a clinical case of recurrent hypercalcemia possibly induced by atorvastatin calcium administration.

Regulation of intracellular calcium in resting and stimulated rat basophilic leukemia cells

International Nuclear Information System (INIS)

Mohr, F.C.

1988-01-01

Intracellular calcium regulation was studied in a cell line of mast cells, the rat basophilic leukemia (RBL) cells with the purpose of determining (1) The properties of the plasma membrane calcium permeability pathway and (2) The role of intracellular calcium stores. The first set of experiments showed that depolarization did not induce calcium entry or secretion in resting cells and did inhibit antigen-stimulated calcium uptake and secretion. In the second set of experiments the ionic basis of antigen-induced depolarization was studied using the fluorescent potential-sensitive probe bis-oxonol. The properties of the calcium entry pathway were more consistent with a calcium channel than a calcium transport mechanism such as Na:Ca exchange. The third set of experiments examined the effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on RBL cells. CCCP inhibited antigen-stimulated 45 Ca uptake and secretion by depolarizing the plasma membrane

64Cu-DOTA-Anti-CTLA-4 mAb Enabled PET Visualization of CTLA-4 on the T-Cell Infiltrating Tumor Tissues

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Higashikawa, Kei; Yagi, Katsuharu; Watanabe, Keiko; Kamino, Shinichiro; Ueda, Masashi; Hiromura, Makoto; Enomoto, Shuichi

2014-01-01

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) targeted therapy by anti-CTLA-4 monoclonal antibody (mAb) is highly effective in cancer patients. However, it is extremely expensive and potentially produces autoimmune-related adverse effects. Therefore, the development of a method to evaluate CTLA-4 expression prior to CTLA-4-targeted therapy is expected to open doors to evidence-based and cost-efficient medical care and to avoid adverse effects brought about by ineffective therapy. In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, we examined CTLA-4 expression in normal colon tissues, cultured CT26 cells, and CT26 tumor tissues from tumor-bearing BALB/c mice and BALB/c nude mice by reverse transcription polymerase chain reaction (RT-PCR) analysis and confirmed whether CTLA-4 is strongly expressed in CT26 tumor tissues. Second, we newly synthesized 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-anti-mouse CTLA-4 mAb (64Cu-DOTA-anti-CTLA-4 mAb) and evaluated its usefulness in positron emission tomography (PET) and ex-vivo biodistribution analysis in CT26-bearing BALB/c mice. High CTLA-4 expression was confirmed in the CT26 tumor tissues of tumor-bearing BALB/c mice. However, CTLA-4 expression was extremely low in the cultured CT26 cells and the CT26 tumor tissues of tumor-bearing BALB/c nude mice. The results suggested that T cells were responsible for the high CTLA-4 expression. Furthermore, 64Cu-DOTA-anti-CTLA-4 mAb displayed significantly high accumulation in the CT26 tumor, thereby realizing non-invasive CTLA-4 visualization in the tumor. Together, the results indicate that 64Cu-DOTA-anti-CTLA-4 mAb would be useful for the evaluation of CTLA-4 expression in tumor. PMID:25365349

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

Science.gov (United States)

Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

2011-06-01

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

Science.gov (United States)

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

2013-12-01

Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

Diagram of Calcium Movement in the Human Body

Science.gov (United States)

2002-01-01

This diagram shows the normal pathways of calcium movement in the body and indicates changes (green arrows) seen during preliminary space flight experiments. Calcium plays a central role because 1) it gives strength and structure to bone and 2) all types of cells require it to function normally. To better understand how and why weightlessness induces bone loss, astronauts have participated in a study of calcium kinetics -- that is, the movement of calcium through the body, including absorption from food, and its role in the formation and breakdown of bone.

Calcium induces long-term legacy effects in a subalpine ecosystem.

Directory of Open Access Journals (Sweden)

Urs Schaffner

Full Text Available Human activities have transformed a significant proportion of the world's land surface, with profound effects on ecosystem processes. Soil applications of macronutrients such as nitrate, phosphorus, potassium or calcium are routinely used in the management of croplands, grasslands and forests to improve plant health or increase productivity. However, while the effects of continuous fertilization and liming on terrestrial ecosystems are well documented, remarkably little is known about the legacy effect of historical fertilization and liming events in terrestrial ecosystems and of the mechanisms involved. Here, we show that more than 70 years after the last application of lime on a subalpine grassland, all major soil and plant calcium pools were still significantly larger in limed than in unlimed plots, and that the resulting shift in the soil calcium/aluminium ratio continues to affect ecosystem services such as primary production. The difference in the calcium content of the vegetation and the topmost 10 cm of the soil in limed vs. unlimed plots amounts to approximately 19.5 g m(-2, equivalent to 16.3% of the amount that was added to the plots some 70 years ago. In contrast, plots that were treated with nitrogen-phosphorus-potassium fertilizer in the 1930s did not differ from unfertilized plots in any of the soil and vegetation characteristics measured. Our findings suggest that the long-term legacy effect of historical liming is due to long-term storage of added calcium in stable soil pools, rather than a general increase in nutrient availability. Our results demonstrate that single applications of calcium in its carbonated form can profoundly and persistently alter ecosystem processes and services in mountain ecosystems.

Electrophoretic mobility shift in native gels indicates calcium-dependent structural changes of neuronal calcium sensor proteins.

Science.gov (United States)

Viviano, Jeffrey; Krishnan, Anuradha; Wu, Hao; Venkataraman, Venkat

2016-02-01

In proteins of the neuronal calcium sensor (NCS) family, changes in structure as well as function are brought about by the binding of calcium. In this article, we demonstrate that these structural changes, solely due to calcium binding, can be assessed through electrophoresis in native gels. The results demonstrate that the NCS proteins undergo ligand-dependent conformational changes that are detectable in native gels as a gradual decrease in mobility with increasing calcium but not other tested divalent cations such as magnesium, strontium, and barium. Surprisingly, such a gradual change over the entire tested range is exhibited only by the NCS proteins but not by other tested calcium-binding proteins such as calmodulin and S100B, indicating that the change in mobility may be linked to a unique NCS family feature--the calcium-myristoyl switch. Even within the NCS family, the changes in mobility are characteristic of the protein, indicating that the technique is sensitive to the individual features of the protein. Thus, electrophoretic mobility on native gels provides a simple and elegant method to investigate calcium (small ligand)-induced structural changes at least in the superfamily of NCS proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Polymer-induced liquid precursor (PILP) phases of calcium carbonate formed in the presence of synthetic acidic polypeptides - relevance to biomineralization

NARCIS (Netherlands)

Schenk, A.S.; Zope, H.; Kim, Y.; Kros, A.; Sommerdijk, N.A.J.M.; Meldrum, F.C.

2012-01-01

Polymer-induced liquid precursor (PILP) phases of calcium carbonate have attracted significant interest due to possible applications in materials synthesis, and their resemblance to intermediates seen in biogenic mineralisation processes. Further, these PILP phases have been formed in vitro using

Tetany: quantitative interrelationships between calcium and alkalosis.

Science.gov (United States)

Edmondson, J W; Brashear, R E; Li, T K

1975-04-01

Tetany occurs with hypocalcemia and alkalosis or both. The interrelationship of calcium and acid-base balance necessary for inducing tetany, the role of the central nervous system, and the rate of development of hypocalcemia have been investigated. Tetany occurred in less than 50 percent of one group of dogs made alkalotic by hyperventilation or made hypocalcemic by infusion of ethylene glycol-bis(beta-amino ethyl ether) N, N'-tetraacetate. In contrast, hypocalcemia combined with hypocapnic alkalosis always produced tetany. Slowly evolving hypocalcemia was achieved inanother group of dogs by thyroparathyroidectomy, and tetany was induced postoperatively by hypocapnic alkalosis. An identical relationship between serum calcium ion concentration and arterial pH or CO2 tension was found in both groups. Tetany could not be related to the cerebrospinal fluid (CSF) calcium ion content in either group. Hypocalcemia and alkalosis are therefore coparticipants in the development of tetany and are independent of the rate of development of hypocalcemia and of CSF calcium ion concentration. The importance of alkalosis in tetany with hypoparathyroidism is emphasized.

Oral Exposure to Atrazine Induces Oxidative Stress and Calcium Homeostasis Disruption in Spleen of Mice

Directory of Open Access Journals (Sweden)

Shuying Gao

2016-01-01

Full Text Available The widely used herbicide atrazine (ATR can cause many adverse effects including immunotoxicity, but the underlying mechanisms are not fully understood. The current study investigated the role of oxidative stress and calcium homeostasis in ATR-induced immunotoxicity in mice. ATR at doses of 0, 100, 200, or 400 mg/kg body weight was administered to Balb/c mice daily for 21 days by oral gavage. The studies performed 24 hr after the final exposure showed that ATR could induce the generation of reactive oxygen species in the spleen of the mice, increase the level of advanced oxidation protein product (AOPP in the host serum, and cause the depletion of reduced glutathione in the serum, each in a dose-related manner. In addition, DNA damage was observed in isolated splenocytes as evidenced by increase in DNA comet tail formation. ATR exposure also caused increases in intracellular Ca2+ within splenocytes. Moreover, ATR treatment led to increased expression of genes for some antioxidant enzymes, such as HO-1 and Gpx1, as well as increased expression of NF-κB and Ref-1 proteins in the spleen. In conclusion, it appears that oxidative stress and disruptions in calcium homeostasis might play an important role in the induction of immunotoxicity in mice by ATR.

Hyperthyroidism enhances 5-HT-induced contraction of the rat pulmonary artery: role of calcium-activated chloride channel activation.

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Oriowo, Mabayoje A; Oommen, Elsie; Khan, Islam

2011-11-01

Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD(2) values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT(2B)-receptor agonist) nor CP 93129 (5-HT(1B)-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10(-4)M. There was no significant difference in the level of expression of 5-HT(2A)-receptor protein between the two groups. Ketanserin (3 × 10(-8)M) produced a rightward shift of the concentration-response curve to 5-HT in both groups with equal potency (-logK(B) values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10(-6)M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10(-4)M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels. Copyright © 2011 Elsevier B.V. All rights reserved.

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

Science.gov (United States)

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.

Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells.

Science.gov (United States)

Misonou, Yoshiko; Takahashi, Motoko; Park, Yong Seek; Asahi, Michio; Miyamoto, Yasuhide; Sakiyama, Haruhiko; Cheng, Xinyao; Taniguchi, Naoyuki

2005-05-01

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.

Stretch induced endothelin-1 secretion by adult rat astrocytes involves calcium influx via stretch-activated ion channels (SACs)

International Nuclear Information System (INIS)

Ostrow, Lyle W.; Suchyna, Thomas M.; Sachs, Frederick

2011-01-01

Highlights: → Endothelin-1 expression by adult rat astrocytes correlates with cell proliferation. → Stretch-induced ET-1 is inhibited by GsMtx-4, a specific inhibitor of Ca 2+ permeant SACs. → The less specific SAC inhibitor streptomycin also inhibits ET-1 secretion. → Stretch-induced ET-1 production depends on a calcium influx. → SAC pharmacology may provide a new class of therapeutic agents for CNS pathology. -- Abstract: The expression of endothelins (ETs) and ET-receptors is often upregulated in brain pathology. ET-1, a potent vasoconstrictor, also inhibits the expression of astrocyte glutamate transporters and is mitogenic for astrocytes, glioma cells, neurons, and brain capillary endothelia. We have previously shown that mechanical stress stimulates ET-1 production by adult rat astrocytes. We now show in adult astrocytes that ET-1 production is driven by calcium influx through stretch-activated ion channels (SACs) and the ET-1 production correlates with cell proliferation. Mechanical stimulation using biaxial stretch ( 2+ threshold. This coupling of mechanical stress to the astrocyte endothelin system through SACs has treatment implications, since all pathology deforms the surrounding parenchyma.

A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

OpenAIRE

Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

2016-01-01

Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab cou...

Analysis of calcium-induced effects on the conformation of fengycin.

Science.gov (United States)

Nasir, Mehmet Nail; Laurent, Pascal; Flore, Christelle; Lins, Laurence; Ongena, Marc; Deleu, Magali

2013-06-01

Fengycin is a natural lipopeptide with antifungal and eliciting properties and able to inhibit the activity of phospholipase A2. A combination of CD, FT-IR, NMR and fluorescence spectroscopic techniques was applied to elucidate its conformation in a membrane-mimicking environment and to investigate the effect of calcium ions on it. We mainly observed that fengycin adopts a turn conformation. Our results showed that calcium ions are bound by the two charged glutamates. The calcium binding has an influence on the fengycin conformation and more particularly, on the environment of the tyrosine residues. The modulation of the fengycin conformation by the environmental conditions may influence its biological properties. Copyright © 2013 Elsevier B.V. All rights reserved.

Kit W-sh Mutation Prevents Cancellous Bone Loss during Calcium Deprivation.

Science.gov (United States)

Lotinun, Sutada; Suwanwela, Jaijam; Poolthong, Suchit; Baron, Roland

2018-01-01

Calcium is essential for normal bone growth and development. Inadequate calcium intake increases the risk of osteoporosis and fractures. Kit ligand/c-Kit signaling plays an important role in regulating bone homeostasis. Mice with c-Kit mutations are osteopenic. The present study aimed to investigate whether impairment of or reduction in c-Kit signaling affects bone turnover during calcium deprivation. Three-week-old male WBB6F1/J-Kit W /Kit W-v /J (W/W v ) mice with c-Kit point mutation, Kit W-sh /HNihrJaeBsmJ (W sh /W sh ) mice with an inversion mutation in the regulatory elements upstream of the c-Kit promoter region, and their wild-type controls (WT) were fed either a normal (0.6% calcium) or a low calcium diet (0.02% calcium) for 3 weeks. μCT analysis indicated that both mutants fed normal calcium diet had significantly decreased cortical thickness and cancellous bone volume compared to WT. The low calcium diet resulted in a comparable reduction in cortical bone volume and cortical thickness in the W/W v and W sh /W sh mice, and their corresponding controls. As expected, the low calcium diet induced cancellous bone loss in the W/W v mice. In contrast, W sh /W sh cancellous bone did not respond to this diet. This c-Kit mutation prevented cancellous bone loss by antagonizing the low calcium diet-induced increase in osteoblast and osteoclast numbers in the W sh /W sh mice. Gene expression profiling showed that calcium deficiency increased Osx, Ocn, Alp, type I collagen, c-Fms, M-CSF, and RANKL/OPG mRNA expression in controls; however, the W sh mutation suppressed these effects. Our findings indicate that although calcium restriction increased bone turnover, leading to osteopenia, the decreased c-Kit expression levels in the W sh /W sh mice prevented the low calcium diet-induced increase in cancellous bone turnover and bone loss but not the cortical bone loss.

Polymerization of calcium caseinates solutions induced by gamma irradiation

International Nuclear Information System (INIS)

Lacroix, M.; Jobin, M.; Mezgheni, E.; Srour, M.; Boileau, S.

1998-01-01

Solutions of calcium caseinate (5%) combined with propylene glycol (PG) or triethylene glycol(TEG) (0, 2.5% and 5%) and used for the development of edible films and coatings, were irradiated at doses between 0 to 128 kGy. Solutions were chromatographed through toyopearl HW 55F resin to observe the effect of irradiation on cross-link reactions. In unirradiated calcium caseinate solutions, two peaks could be observed (fractions 30 and 37) while samples irradiated at 64 kGy and 128 kGy showed one shifted peak at fraction 32 and 29 respectively. No effect of the plasticizers was observed. According to proteins standards of knowed molecular weights, the molecular weight of calcium caseinate increased approximately 10 times when irradiated at 128 kGy and 5 times when irradiated at 64 kGy. The physico-chemical properties of bio-films prepared with the irradiated solutions, demonstrated that tensile strength at break increased with increase of irradiation dose. A maximum dose was obtained at 16 kGy

Calcium as a cardiovascular toxin in CKD-MBD.

Science.gov (United States)

Moe, Sharon M

2017-07-01

Disordered calcium balance and homeostasis are common in patients with chronic kidney disease. Such alterations are commonly associated with abnormal bone remodeling, directly and indirectly. Similarly, positive calcium balance may also be a factor in the pathogenesis of extra skeletal soft tissue and arterial calcification. Calcium may directly affect cardiac structure and function through direct effects to alter cell signaling due to abnormal intracellular calcium homeostasis 2) extra-skeletal deposition of calcium and phosphate in the myocardium and small cardiac arterioles, 3) inducing cardiomyocyte hypertrophy through calcium and hormone activation of NFAT signaling mechanisms, and 4) increased aorta calcification resulting in chronic increased afterload leading to hypertrophy. Similarly, calcium may alter vascular smooth muscle cell function and affect cell signaling which may predispose to a proliferative phenotype important in arteriosclerosis and arterial calcification. Thus, disorders of calcium balance and homeostasis due to CKD-MBD may play a role in the high cardiovascular burden observed in patients with CKD. Published by Elsevier Inc.

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle.

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Szentesi, Péter; Szappanos, Henrietta; Szegedi, Csaba; Gönczi, Monika; Jona, István; Cseri, Julianna; Kovács, László; Csernoch, László

2004-03-01

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

Space flight calcium: implications for astronaut health, spacecraft operations, and Earth.

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Smith, Scott M; McCoy, Torin; Gazda, Daniel; Morgan, Jennifer L L; Heer, Martina; Zwart, Sara R

2012-12-18

The space flight environment is known to induce bone loss and, subsequently, calcium loss. The longer the mission, generally the more bone and calcium are lost. This review provides a history of bone and calcium studies related to space flight and highlights issues related to calcium excretion that the space program must consider so that urine can be recycled. It also discusses a novel technique using natural stable isotopes of calcium that will be helpful in the future to determine calcium and bone balance during space flight.

Inhibition of 4NQO-Induced Oral Carcinogenesis by Dietary Oyster Shell Calcium.

Science.gov (United States)

Chen, Ying; Jiang, Yi; Liao, Liyan; Zhu, Xiaoxin; Tang, Shengan; Yang, Qing; Sun, Lihua; Li, Yujie; Gao, Shuangrong; Xie, Zhongjian

2016-03-01

Oyster has gained much attention recently for its anticancer activity but it is unclear whether calcium, the major antitumor ingredient in oyster shell, is responsible for the anticarcinogenic role of the oyster. To address this issue, C57BL/6 mice were fed with the carcinogen 4-nitroquinoline-1-oxide (4NQO, 50 µg/mL) and normal diet or a diet containing oyster powder, oyster calcium, or calcium depleted oyster powder. The tongue tissue specimens isolated from these mice were histologically evaluated for hyperplasia, dysplasia, and papillary lesions, and then analyzed for proliferation and differentiation markers by immunohistochemistry. The results showed that mice on the diet containing oyster calcium significantly reduced rates of tumors in the tongue and proliferation and enhanced differentiation in the oral epithelium compared with the diet containing calcium depleted oyster powder. These results suggest that calcium in oyster plays a critical role in suppressing formation of oral squamous cell carcinoma and proliferation and promoting differentiation of the oral epithelium. © The Author(s) 2015.

Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells

OpenAIRE

Ma, Liwei; Wang, Hongjun; Wang, Chunyan; Su, Jing; Xie, Qi; Xu, Lu; Yu, Yang; Liu, Shibing; Li, Songyan; Xu, Ye; Li, Zhixin

2016-01-01

Cisplatin is a commonly used chemotherapeutic drug, used for the treatment of malignant ovarian cancer, but acquired resistance limits its application. There is therefore an overwhelming need to understand the mechanism of cisplatin resistance in ovarian cancer, that is, ovarian cancer cells are insensitive to cisplatin treatment. Here, we show that failure of elevating calcium and oxidative stress tolerance play key roles in cisplatin resistance in ovarian cancer cell lines. Cisplatin induce...

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Epithelial Malignant Tumors from Digestive System

OpenAIRE

Blanco, Rancés; Rengifo, Enrique; Cedeño, Mercedes; Rengifo, Charles E.; Alonso, Daniel F.; Carr, Adriana

2011-01-01

The limited expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in human normal tissues, as well as its presence in melanoma and breast carcinoma using 14F7 Mab (anti-NeuGcGM3), has been previously reported. In this work we evaluated for the first time the 14F7 Mab immunorecognition in some digestive system tumors. Immunohistochemical assays were made with 14F7, followed by anti-mouse biotinylated antibody and ABC/HRP system in normal and pathological human tissues were made. No immunoreactio...

Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

2012-01-01

in vivo. Calcium electroporation elicited dramatic antitumor responses in which 89% of treated tumors were eliminated. Histologic analyses indicated complete tumor necrosis. Mechanistically, calcium electroporation caused acute ATP depletion likely due to a combination of increased cellular use of ATP......, decreased production of ATP due to effects on the mitochondria, as well as loss of ATP through the permeabilized cell membrane. Taken together, our findings offer a preclinical proof of concept for the use of electroporation to load cancer cells with calcium as an efficient anticancer treatment...

The Role of Calcium in Osteoporosis

Science.gov (United States)

Arnaud, C. D.; Sanchez, S. D.

1991-01-01

Calcium requirements may vary throughout the lifespan. During the growth years and up to age 25 to 30, it is important to maximize dietary intake of calcium to maintain positive calcium balance and achieve peak bone mass, thereby possibly decreasing the risk of fracture when bone is subsequently lost. Calcium intake need not be greater than 800 mg/day during the relatively short period of time between the end of bone building and the onset of bone loss (30 to 40 years). Starting at age 40 to 50, both men and women lose bone slowly, but women lose bone more rapidly around the menopause and for about 10 years after. Intestinal calcium absorption and the ability to adapt to low calcium diets are impaired in many postmenopausal women and elderly persons owing to a suspected functional or absolute decrease in the ability of the kidney to produce 1,25(OH)2D2. The bones then become more and more a source of calcium to maintain critical extracellular fluid calcium levels. Excessive dietary intake of protein and fiber may induce significant negative calcium balance and thus increase dietary calcium requirements. Generally, the strongest risk factors for osteoporosis are uncontrollable (e.g., sex, age, and race) or less controllable (e.g., disease and medications). However, several factors such as diet, physical activity, cigarette smoking, and alcohol use are lifestyle related and can be modified to help reduce the risk of osteoporosis.

Effects of Calcium Source on Biochemical Properties of Microbial CaCO3 Precipitation.

Science.gov (United States)

Xu, Jing; Du, Yali; Jiang, Zhengwu; She, Anming

2015-01-01

The biochemical properties of CaCO3 precipitation induced by Sporosarcina pasteurii, an ureolytic type microorganism, were investigated. Effects of calcium source on the precipitation process were examined, since calcium source plays a key role in microbiologically induced mineralization. Regardless of the calcium source type, three distinct stages in the precipitation process were identified by Ca(2+), NH4 (+), pH and cell density monitoring. Compared with stage 1 and 3, stage 2 was considered as the most critical part since biotic CaCO3 precipitation occurs during this stage. Kinetics studies showed that the microbial CaCO3 precipitation rate for calcium lactate was over twice of that for calcium nitrate, indicating that calcium lactate is more beneficial for the cell activity, which in turn determines urease production and CaCO3 precipitation. X-ray diffraction analysis confirmed the CaCO3 crystal as calcite, although scanning electron microscopy revealed a difference in crystal size and morphology if calcium source was different. The findings of this paper further suggest a promising application of microbiologically induced CaCO3 precipitation in remediation of surface and cracks of porous media, e.g., cement-based composites, particularly by using organic source of calcium lactate.

Effect of orally administered dipterinyl calcium pentahydrate on oral glucose tolerance in diet-induced obese mice

Directory of Open Access Journals (Sweden)

Fuchs D

2012-02-01

Full Text Available Svetlana E Nikoulina1, Dietmar Fuchs2, Phillip Moheno11SanRx Pharmaceuticals, Inc, La Jolla, CA, USA; 2Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, AustriaAbstract: Calcium pterins have been shown to be significant immunotherapeutic agents in models of breast cancer, hepatitis B, and tuberculosis (Bacillus Calmette-Guérin mycobacteria. These compunds modulate the immuno-enzyme indoleamine 2,3-dioxygenase (IDO and the blood levels of several identified inflammatory cytokines. Recent research into the pathology of diabetes implicates inflammatory factors in the progression of the disease, leading the authors to study its possible control by one of the calcium pterins, dipterinyl calcium pentahydrate (DCP.The investigators tested DCP as a novel therapeutic for type 2 diabetes. Female C57BL/6 J mice with diet-induced obesity were fed a high-fat diet and were administered DCP in 0.4% carboxymethylcellulose for 21 days. Blood glucose was followed during the dosing period, and an oral glucose tolerance test (OGTT was carried out on day 21. Measurements of plasma indoleamine 2,3-dioxygenase metabolites (tryptophan and kynurenine and certain cytokines and chemokines were also taken. DCP 7 mg/kg/day reduced OGTT area under the curve (OGTT/AUC by 50% (P < 0.05. A significant multivariate regression (P = 0.013; R2 = 0.571 of OGTT/AUC was derived from DCP dosage and plasma Trp. Elevated plasma Trp concentration, likely from heterogeneity in diet and/or indoleamine 2,3-dioxygenase activity, was found to correlate with higher OGTT/AUC diabetic measures, possibly via inhibition of histamine degradation. In conclusion, an optimum dose of DCP 7 mg/kg/day significantly improved the OGTT diabetic state in these female diet-induced obese mice.Keywords: diabetes, immunotherapy, oral glucose tolerance test, tryptophan, kynurenine

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum.

Science.gov (United States)

Dixit, Ajay Kumar; Jayabaskaran, Chelliah

2015-05-01

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca(2+) ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 μM and 120 μM indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 μM and 1.7 μM. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a α-helical rich protein. Calcium binding further increased the α-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. Copyright © 2015 Elsevier GmbH. All rights reserved.

Calcium-induced stabilization of -amylase against guanidine ...

African Journals Online (AJOL)

Guanidine hydrochloride (GdnHCl) denaturation of native and Ca- depleted Bacillus licheniformis α-amylase (BLA) was investigated both in the absence and presence of 2 mM calcium chloride (CaCl2) using circular dichroism, fluorescence spectroscopy and biological activity. In both states (Cadepleted and native form), ...

The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

Directory of Open Access Journals (Sweden)

Yajin Liao

2017-02-01

Full Text Available 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.

Hydrogen-enriched water restoration of impaired calcium propagation by arsenic in primary keratinocytes

Science.gov (United States)

Yu, Wei-Tai; Chiu, Yi-Ching; Lee, Chih-Hung; Yoshioka, Tohru; Yu, Hsin-Su

2013-11-01

Endemic contamination of artesian water for drinking by arsenic is known to cause several human cancers, including cancers of the skin, bladder, and lungs. In skin, multiple arsenic-induced Bowen's disease (As-BD) can develop into invasive cancers after decades of arsenic exposure. The characteristic histological features of As-BD include full-layer epidermal dysplasia, apoptosis, and abnormal proliferation. Calcium propagation is an essential cellular event contributing to keratinocyte differentiation, proliferation, and apoptosis, all of which occur in As-BD. This study investigated how arsenic interferes calcium propagation of skin keratinocytes through ROS production and whether hydrogen-enriched water would restore arsenic-impaired calcium propagation. Arsenic was found to induce oxidative stress and inhibit ATP- and thapsigaragin-induced calcium propagation. Pretreatment of arsenic-treated keratinocytes by hydrogen-enriched water or beta-mercaptoethanol with potent anti-oxidative effects partially restored the propagation of calcium by ATP and by thapsigaragin. It was concluded that arsenic may impair calcium propagation, likely through oxidative stress and interactions with thiol groups in membrane proteins.

Exopolysaccharides regulate calcium flow in cariogenic biofilms

Science.gov (United States)

Varenganayil, Muth M.; Decho, Alan W.

2017-01-01

Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries. PMID:29023506

Exopolysaccharides regulate calcium flow in cariogenic biofilms.

Directory of Open Access Journals (Sweden)

Monika Astasov-Frauenhoffer

Full Text Available Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya's agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC. Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries.

Contracture of Slow Striated Muscle during Calcium Deprivation

Science.gov (United States)

Irwin, Richard L.; Hein, Manfred M.

1963-01-01

When deprived of calcium the slow striated muscle fibers of the frog develop reversible contractures in either hypertonic or isotonic solutions. While calcium deprivation continues because of a flowing calcium-free solution the muscles relax slowly and completely. Restoration of calcium during contracture relaxes the muscle promptly to initial tension. When relaxed during calcium lack the return of calcium does not change tension and the muscle stays relaxed. When contractures are induced by solutions containing small amounts of calcium relaxation does not occur or requires several hours. The rate of tension development depends upon the rate at which calcium moves outward since the contractures develop slower in low concentrations of calcium and are absent or greatly slowed in a stagnant calcium-free solution. Withdrawal of calcium prevents the contractile responses to ACh, KCl, or electrical stimulation through the nerve. Muscles return to their original excitability after calcium is restored. Origin of the contractures is unrelated to nerve activity since they are maximal during transmission failure from calcium lack, occur in denervated muscles, and are not blocked by high concentrations of d-tubocurarine, procaine, or atropine. The experiments also indicate that the contractures do not originate from repetitive activity of muscle membranes. The findings are most simply explained by relating the outward movement of calcium as a link for initiating contraction in slow type striated muscle. PMID:14065284

T-type calcium channel antagonism decreases motivation for nicotine and blocks nicotine- and cue-induced reinstatement for a response previously reinforced with nicotine.

Science.gov (United States)

Uslaner, Jason M; Vardigan, Joshua D; Drott, Jason M; Uebele, Victor N; Renger, John J; Lee, Ariel; Li, Zhaoxia; Lê, A D; Hutson, Pete H

2010-10-15

Recent evidence suggests an involvement of T-type calcium channels in the effects of drugs of abuse. We examined the influence of the novel, potent, and selective T-type calcium channel antagonist [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[2,2,2-trifluoroethyl]oxo}pyridine-2-yl)ethyl]acetamide] (TTA-A2) (.3, 1, or 3 mg/kg) on motivation for nicotine, as measured by nicotine self-administration on a progressive ratio (PR) schedule, and nicotine- and cue-induced reinstatement for a response previously reinforced with nicotine delivery (n = 11 or 12 Long Evans rats/group). Furthermore, we examined the specificity of the TTA-A2 effects by characterizing its influence on PR responding for food (in the absence or presence of nicotine-potentiated responding), food- versus nicotine-induced cue-potentiated reinstatement for a response previously reinforced by food administration (n = 11 or 12 Wistar Hannover rats/group), and its ability to induce a conditioned place aversion. TTA-A2 dose-dependently decreased self-administration of nicotine on a PR schedule and the ability of both nicotine and a cue paired with nicotine to reinstate responding. The effects were specific for nicotine's incentive motivational properties, as TTA-A2 did not influence responding for food on a PR schedule but did attenuate the ability of nicotine to potentiate responding for food. Likewise, TTA-A2 did not alter food-induced cue-potentiated reinstatement for a response previously reinforced by food but did decrease nicotine-induced cue-potentiated reinstatement. Finally, TTA-A2 did not produce an aversive state, as indicated by a lack of ability to induce conditioned place aversion. These data suggest that T-type calcium channel antagonists have potential for alleviating nicotine addiction by selectively decreasing the incentive motivational properties of nicotine. Copyright © 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Multiparameter imaging of calcium and abscisic acid and high-resolution quantitative calcium measurements using R-GECO1-mTurquoise in Arabidopsis.

Science.gov (United States)

Waadt, Rainer; Krebs, Melanie; Kudla, Jörg; Schumacher, Karin

2017-10-01

Calcium signals occur in specific spatio-temporal patterns in response to various stimuli and are coordinated with, for example, hormonal signals, for physiological and developmental adaptations. Quantification of calcium together with other signalling molecules is required for correlative analyses and to decipher downstream calcium-decoding mechanisms. Simultaneous in vivo imaging of calcium and abscisic acid has been performed here to investigate the interdependence of the respective signalling processes in Arabidopsis thaliana roots. Advanced ratiometric genetically encoded calcium indicators have been generated and in vivo calcium calibration protocols were established to determine absolute calcium concentration changes in response to auxin and ATP. In roots, abscisic acid induced long-term basal calcium concentration increases, while auxin triggered rapid signals in the elongation zone. The advanced ratiometric calcium indicator R-GECO1-mTurquoise exhibited an increased calcium signal resolution compared to commonly used Förster resonance energy transfer-based indicators. Quantitative calcium measurements in Arabidopsis root tips using R-GECO1-mTurquoise revealed detailed maps of absolute calcium concentration changes in response to auxin and ATP. Calcium calibration protocols using R-GECO1-mTurquoise enabled high-resolution quantitative imaging of resting cytosolic calcium concentrations and their dynamic changes that revealed distinct hormonal and ATP responses in roots. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Kinetics of platelets in dogs with thrombocytopenia induced by antiglycoprotein IIb/IIIa receptor monoclonal antibody

International Nuclear Information System (INIS)

Hosono, Makoto; Sone, Naoaki; Endo, Keigo; Saga, Tsuneo; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Yasunaga, Kojiro; Konishi, Junji

1995-01-01

To experimentally assess the kinetics of platelets in thrombocytopenia, we constructed a canine model using 111 In-oxine labeled autologous platelets and an intact antiplatelet monoclonal antibody (MAb) NNKY2-11 (IgG2a). With the infusion of radiolabeled autologous platelets into dogs, the peripheral platelet count and blood radioactivity level were examined, and the radioactivity in the liver, spleen and heart was determined with scintigraphic analysis. Thereafter, i.v. injection of 100 μg/kg of NNKY2-11 had no effect on platelet counts or the biodistribution of radiolabeled platelets. However, 200 and 300 μg/kg of MAb reduced the platelets, and the radioactivity of the liver and spleen augmented clearly after injection of MAb. Platelet radioactivity in serum, which had decreased after MAb infusion, did not recover, even when peripheral platelet counts returned to the normal levels, indicating that these new platelets might be derived from the platelet-storage pool or new thrombocytogenesis. This model of antiplatelet MAb induced thrombocytopenia seems to be useful for analyzing the kinetics of platelets in thrombocytopenia

Letrozole induced low estrogen levels affected the expressions of duodenal and renal calcium-processing gene in laying hens.

Science.gov (United States)

Li, Qiao; Zhao, Xingkai; Wang, Shujie; Zhou, Zhenlei

2018-01-01

Estrogen regulates the calcium homeostasis in hens, but the mechanisms involved are still unclear fully. In this study, we investigated whether letrozole (LZ) induced low estrogen levels affected the calcium absorption and transport in layers. In the duodenum, we observed a significant decrease of mRNA expressions of Calbindin-28k (CaBP-28k) and plasma membrane Ca 2+ -ATPase (PMCA 1b) while CaBP-28k protein expression was declined in birds with LZ treatment, and the mRNA levels of duodenal transient receptor potential vanilloid 6 (TRPV6) and Na + /Ca 2+ exchanger 1 (NCX1) were not affected. Interestingly, we observed the different changes in the kidney. The renal mRNA expressions of TRPV6 and NCX1 were unregulated while the PMCA1b was down-regulated in low estrogen layers, however, the CaBP-28k gene and protein expressions were no changed in the kidney. Furthermore, it showed that the duodenal estradiol receptor 2 (ESR2) transcripts rather than parathyroid hormone 1 receptor (PTH1R) and calcitonin receptor (CALCR) played key roles to down-regulate calcium transport in LZ-treated birds. In conclusion, CaBP-28k, PMCA 1b and ESR2 genes in the duodenum may be primary targets for estrogen regulation in order to control calcium homeostasis in hens. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of soaking and acidification on physicochemical properties of calcium-fortified rice.

Science.gov (United States)

Sirisoontaralak, Porntip; Limboon, Pailin; Jatuwong, Sujitra; Chavanalikit, Arusa

2016-06-01

Calcium-fortified rice was prepared by soaking milled rice in calcium lactate solution, steaming and drying, and physicochemical properties were determined to evaluate effects of calcium concentration (0, 30, 50 g L(-1) ), soaking temperature (ambient temperature, 40 °C, 60 °C) and acidification. Calcium-fortified rice had less lightness. More total solid loss was observed, especially at high soaking temperature. Harder texture was detected with increased calcium concentration. Calcium fortification lowered pasting viscosity of milled rice. Panelists accepted all fortified rice; however, only rice soaked at 50 g L(-1) concentration could be claimed as a good source of calcium. Increasing of soaking temperature induced more penetration of calcium to rice kernels but calcium was lost more easily after washing. With addition of acetic acid to the soaking solution, enriched calcium content was comparable to that of high soaking temperature but with better retention after washing and calcium solubility was improved. Acid induced reduction of lightness and cooked rice hardness but increased total solid loss and pasting viscosity. Although the taste of acetic acid remained, panelists still accepted the fortified rice. Calcium-fortified rice (190.47-194.3 mg 100 g(-1) ) could be successfully produced by soaking milled rice in 50 g L(-1) calcium lactate solution at 40 °C or at ambient temperature with acidification. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

A review on the kinetics of microbially induced calcium carbonate precipitation by urea hydrolysis

Science.gov (United States)

van Paassen, L. A.

2017-12-01

In this study the kinetics of calcium carbonate precipitation induced by the ureolytic bacteria are reviewed based on experiments and mathematical modelling. The study shows how urea hydrolysis rate depends on the amount of bacteria and the conditions during growth, storage, hydrolysis and precipitation. The dynamics of Microbially Induced Carbonate Precipitation has been monitored in non-seeded liquid batch experiments. Results show that particulary for a fast hydrolysis of urea (>1 M-urea day-1) in a highly concentrated equimolar solution with calcium chloride (>0.25 M) the solubility product of CaCO3 is exceeded within a short period (less than 30 minutes), the supersaturation remains high for an exended period, resulting in prolonged periods of nucleation and crystal growth and extended growth of metastable precursor mineral phases. The pH, being a result of the speciation, quickly rises until critical supersaturation is reached and precipitation is initiated. Then pH drops (sometimes showing oscillating behaviour) to about neutral where it stays until all substrates are depleted. Higher hydrolysis rates lead to higher supersaturation and pH and relatively many small crystals, whereas higher concentrations of urea and calcium chloride mainly lead to lower pH values. The conversion can be reasonably monitored by electrical conductivity and reasonably predicted, using a simplified model based on a single reaction as long as the urea hydrolysis rate is known. Complex geochemical models, which include chemical speciciation through acid-base equilibria and kinetic equations to describe mineral precipitation, do not show significant difference from the simplified model regarding the bulk chemistry and the total amount of precipitates. However, experiments show that ureolytic MICP can result in a highly variable crystal morphologies with large variation in the affected hydraulic properties when applied in a porous medium. In order to calculate the number, size and

Biophysical characterization and functional studies on calbindin-D28K: A vitamin D-induced calcium-binding protein

International Nuclear Information System (INIS)

Leathers, V.L.

1989-01-01

Vitamin D dependent calcium binding protein, or calbindin-D, is the principal protein induced in the intestine in response to the steroid hormone 1,25(OH) 2 -vitamin D 3 . A definitive role for calbindin-D in vitamin D 3 mediated biological responses remains unclear. Biophysical and functional studies on chick intestinal calbindin-D 28K (CaBP) were initiated so that some insight might be gained into its relevance to the process of intestinal calcium transport. Calbindin-D belongs to a class of high affinity calcium binding proteins which includes calmodulin, parvalbumin and troponin C. The Ca 2+ binding stoichiometry and binding constants for calbindin-D 28K were quantitated by Quin 2 titration analysis. The protein was found to bind 5-6 Ca 2+ ions with a K D on the order of 10 -8 , in agreement with the 6 domains identified from the amino acid sequence. A slow Ca 2+ exchange rate (80 s -1 ) as assessed by 43 Ca NMR and extensive calcium dependent conformational changes in 1 H NMR spectra were also observed. Functional studies on chick intestinal CaBP were carried out by two different methods. Interactions between CaBP and intestinal cellular components were assessed via photoaffinity labeling techniques. Specific calcium dependent complexes for CaBP were identified with bovine intestinal alkaline phosphatase and brush border membrane proteins of 60 and 150 kD. CaBP was also found to co-migrate with the alkaline phosphatase activity of chick intestinal brush border membranes as evaluated by gel filtration chromatography. The second procedure for evaluating CaBP functionality has involved the quantitation of CaBP association with vesicular transport components as assessed by ELISA. CaBP, immunoreactivity was observed in purified lysosomes, microsomes and microtubules

Tissue Reactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Tumors of Neuroectodermal, Mesodermal, and Epithelial Origin

Science.gov (United States)

Blanco, Rancés; Quintana, Yisel; Blanco, Damián; Cedeño, Mercedes; Rengifo, Charles E.; Frómeta, Milagros; Ríos, Martha; Rengifo, Enrique; Carr, Adriana

2013-01-01

The expression of N-glycolylneuraminic acid forming the structure of gangliosides and/or other glycoconjugates (Hanganutziu-Deicher antigen) in human has been considered as a tumor-associated antigen. Specifically, some reports of 14F7 Mab (a highly specific Mab raised against N-glycolyl GM3 ganglioside) reactivity in human tumors have been recently published. Nevertheless, tumors of epithelial origin have been mostly evaluated. The goal of the present paper was to evaluate the immunohistochemical recognition of 14F7 Mab in different human tumors of neuroectodermal, mesodermal, and epithelial origins using an immunoperoxidase staining method. Samples of fetal, normal, and reactive astrocytosis of the brain were also included in the study. In general, nontumoral tissues, as well as, low-grade brain tumors showed no or a limited immunoreaction with 14F7 Mab. Nevertheless, high-grade astrocytomas (III-IV) and neuroblastomas, as well as, sarcomas and thyroid carcinomas were mostly reactive with 14F7. No reaction was evidenced in medulloblastomas and ependymoblastomas. Our data suggest that the expression of N-glycolyl GM3 ganglioside could be related to the aggressive behavior of malignant cells, without depending on the tumor origin. Our data could also support the possible use of N-glycolyl GM3 as a target for both active and passive immunotherapies of malignancies expressing this molecule. PMID:26317019

The Acid Test: Calcium Signaling in the Skeletogenic Layer of Reef-Building Coral

Science.gov (United States)

Florn, A. M.

2016-02-01

Since the Industrial Revolution, carbon dioxide (CO2) emissions have increased more than 40%. This increased atmospheric CO2 drives ocean acidification and has potentially serious consequences for all marine life, especially calcifying organisms. The specific goal of this study was to examine calcium homeostasis and signaling dynamics within the skeletogenic tissue layers (calicodermal cells) of two coral species (Pavona maldivensis and Porites rus) at three pH treatments corresponding to present-future ocean acidification levels. Confocal microscopy techniques were used to analyze in vivo calcium dynamics of the calicodermal cells in Pavona maldivensis and Porites rus. The results show biological variation between the two reef-building coral species and their response to ocean acidification. Pavona maldivensis showed a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments, but not among the microcolonies. Porites rus did not show a significant difference (p < 0.01) in the ionomycin-induced calcium response among the pH treatments or the microcolonies. Upon comparing the calcium response curves, the ionomycin-induced calcium response exhibited by Pavona maldivensis is phenomenologically similar to a calcium response that is commonly found in vertebrates. This well-studied phenomenon in vertebrate biology is known as store-operated calcium entry (SOCE) and is closely associated with the endoplasmic reticulum (ER) and mitochondria-associated endoplasmic reticulum (MAM) calcium stores. This study provides insight into the preliminary steps needed to understand in vivo calcium signaling in the calicodermis of reef-building coral and the associated consequences of ocean acidification.

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

DEFF Research Database (Denmark)

Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

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 Cx...

Spatiotemporal multiple coherence resonances and calcium waves in a coupled hepatocyte system

International Nuclear Information System (INIS)

Bao-Hua, Wang; Qi-Shao, Lu; Shu-Juan, Lü; Xiu-Feng, Lang

2009-01-01

Spatiotemporal multiple coherence resonances for calcium activities induced by weak Gaussian white noise in coupled hepatocytes are studied. It is shown that bi-resonances in hepatocytes are induced by the interplay and competition between noise and coupling of cells, in other words, the cell in network can be excited either by noise or by its neighbour via gap junction which can transfer calcium ions between cells. Furthermore, the intercellular annular calcium waves induced by noise are observed, in which the wave length decreases with noise intensity augmenting but increases monotonically with coupling strength increasing. And for a fixed noise level, there is an optimal coupling strength that makes the coherence resonance reach maximum. (general)

Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

DEFF Research Database (Denmark)

Kennedy, Arion; Martinez, Kristina; Chung, Soonkyu

2010-01-01

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor kappaB (NFkappaB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated...... that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated......, and suppression of peroxisome proliferator activated receptor gamma protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca2+]i levels, particularly calcium from the ER....

Evaluation of anti-IL-6 monoclonal antibody therapy using murine type II collagen-induced arthritis

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Shealy David

2009-04-01

Full Text Available Abstract Interleukin-6 is a multifunctional cytokine that is critical for T/B-cell differentiation and maturation, immunoglobulin secretion, acute-phase protein production, and macrophage/monocyte functions. Extensive research into the biology of IL-6 has implicated IL-6 in the pathophysiology and pathogenesis of RA. An anti-murine IL-6 mAb that neutralizes mouse IL-6 activities was tested in animal model of collagen-induced arthritis. Prophylactic treatment with anti-IL-6 mAb significantly reduced the incidence and severity of arthritis compared to control mAb treated mice. The mitogenic response of B and T cells isolated from the lymph nodes of anti-IL-6 treated mice was significantly reduced compared to cells isolated from control mAb treated mice. The overall histopathology score for paws from the anti-IL-6 treated mice was significantly reduced when compared to paws from mice treated with control mAb, including both inflammatory (synovitis and pannus and erosive (erosions and architecture parameters. Reduced loss of cartilage matrix components was also observed in the anti-IL-6 treated mice. Collectively, these data suggest that IL-6 plays a major role in the pathophysiology of rheumatoid arthritis, and thus support the potential benefit of anti-IL-6 mAb treatment in rheumatoid arthritis patients.

Russian biosphere reserves at the youth MAB-2017 Forum in Italy

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Elena A. Shuyskaya

2018-02-01

Full Text Available Russia was represented by 9 participants from Biosphere Reserves at the MAB Youth Forum in Italy. The main question of the Forum was «How to involve the young in the work of biosphere reserves?» The debate resulted in a Declaration, elaborated by participants from around the world (282 delegates from 85 countries. Measures to improve scientific cooperation, data exchange in the sphere of educational tourism and administrative management, development of joint projects in environmental education were formulated. The article contains a number of recommendations for the network of biosphere reserves in Russia, based on Seville strategy and Lima Action Plan.

Anti-osteopontin monoclonal antibody prevents ovariectomy-induced osteoporosis in mice by promotion of osteoclast apoptosis

Energy Technology Data Exchange (ETDEWEB)

Zhang, Bo [International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433 (China); PLA General Hospital Cancer Center and PLA Cancer Research Institute, PLA Postgraduate School of Medicine, 28 Fuxing Road, Beijing (China); Dai, Jianxin [International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433 (China); PLA General Hospital Cancer Center and PLA Cancer Research Institute, PLA Postgraduate School of Medicine, 28 Fuxing Road, Beijing (China); National Engineering Research Center for Antibody Medicine and Shanghai Key Lab. of Cell Engineering and Antibody, 399 Libing Road, Shanghai 201203 (China); Wang, Huaqing [International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433 (China); Wei, Huafeng [International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433 (China); PLA General Hospital Cancer Center and PLA Cancer Research Institute, PLA Postgraduate School of Medicine, 28 Fuxing Road, Beijing (China); Zhao, Jian [International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433 (China); National Engineering Research Center for Antibody Medicine and Shanghai Key Lab. of Cell Engineering and Antibody, 399 Libing Road, Shanghai 201203 (China); Guo, Yajun, E-mail: yguo_smmu@163.com [International Joint Cancer Institute, The Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433 (China); PLA General Hospital Cancer Center and PLA Cancer Research Institute, PLA Postgraduate School of Medicine, 28 Fuxing Road, Beijing (China); National Engineering Research Center for Antibody Medicine and Shanghai Key Lab. of Cell Engineering and Antibody, 399 Libing Road, Shanghai 201203 (China); and others

2014-09-26

Highlight: • We first report that anti-osteopontin mAb could protect osteoporosis in mice. • Anti-osteopontin mAb could promote the osteoclast apoptosis. • Targeting osteopontin might have therapeutic potentials for osteoporosis. - Abstract: Osteopontin (OPN) is abundant in mineralized tissues and has long been implicated in bone remodeling. However, the therapeutic effect of targeting OPN in bone loss diseases and the underlying molecular mechanism remain largely unknown. Here, we reported that anti-OPN mAb (23C3) could protect against ovariectomy-induced osteoporosis in mice, demonstrated by microcomputed tomography analysis and histopathology evaluation. In vitro assay showed that 23C3 mAb reduced osteoclasts (OCs)-mediated bone resorption through promotion of mature OC apoptosis. Thus, the study has important implications for understanding the role of OPN in OC bone resorption and survival, and OPN antagonists may have therapeutic potential for osteoporosis and other osteopenic diseases.

Anti-osteopontin monoclonal antibody prevents ovariectomy-induced osteoporosis in mice by promotion of osteoclast apoptosis

International Nuclear Information System (INIS)

Zhang, Bo; Dai, Jianxin; Wang, Huaqing; Wei, Huafeng; Zhao, Jian; Guo, Yajun

2014-01-01

Highlight: • We first report that anti-osteopontin mAb could protect osteoporosis in mice. • Anti-osteopontin mAb could promote the osteoclast apoptosis. • Targeting osteopontin might have therapeutic potentials for osteoporosis. - Abstract: Osteopontin (OPN) is abundant in mineralized tissues and has long been implicated in bone remodeling. However, the therapeutic effect of targeting OPN in bone loss diseases and the underlying molecular mechanism remain largely unknown. Here, we reported that anti-OPN mAb (23C3) could protect against ovariectomy-induced osteoporosis in mice, demonstrated by microcomputed tomography analysis and histopathology evaluation. In vitro assay showed that 23C3 mAb reduced osteoclasts (OCs)-mediated bone resorption through promotion of mature OC apoptosis. Thus, the study has important implications for understanding the role of OPN in OC bone resorption and survival, and OPN antagonists may have therapeutic potential for osteoporosis and other osteopenic diseases

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

Science.gov (United States)

Krawczyk, Adalbert; Dirks, Miriam; Kasper, Maren; Buch, Anna; Dittmer, Ulf; Giebel, Bernd; Wildschütz, Lena; Busch, Martin; Goergens, Andre; Schneweis, Karl E.; Eis-Hübinger, Anna M.; Sodeik, Beate; Heiligenhaus, Arnd; Roggendorf, Michael; Bauer, Dirk

2015-01-01

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. PMID:25587898

Prevention of herpes simplex virus induced stromal keratitis by a glycoprotein B-specific monoclonal antibody.

Directory of Open Access Journals (Sweden)

Adalbert Krawczyk

Full Text Available The increasing incidence of acyclovir (ACV and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis or 24, 40, and 56 hours after infection (post-exposure immunotherapy. Topical treatment was performed by periodical inoculations (5 times per day of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.

Science.gov (United States)

Aquino-Martínez, Rubén; Angelo, Alcira P; Pujol, Francesc Ventura

2017-11-16

Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca 2+ -containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO 4 ) on MSC migration. In addition, to evaluate the influence of CaSO 4 on MSC differentiation and the potential molecular mechanisms involved. A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO 4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO 4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO 4 treatment was also evaluated by qPCR. CaSO 4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO 4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO 4 -containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO 4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO 4 effects on MSC migration. Specific CaSO 4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO 4 regulates BMP-2-induced MSC migration by differentially activating the PI3

Role of polyhydroxybutyrate in mitochondrial calcium uptake

Science.gov (United States)

Smithen, Matthew; Elustondo, Pia A.; Winkfein, Robert; Zakharian, Eleonora; Abramov, Andrey Y.; Pavlov, Evgeny

2013-01-01

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to the class of polyesters and is ubiquitously present in all living organisms. Mammalian mitochondrial membranes contain PHB consisting of up to 120 hydroxybutyrate residues. Roles played by PHB in mammalian mitochondria remain obscure. It was previously demonstrated that PHB of the size similar to one found in mitochondria mediates calcium transport in lipid bilayer membranes. We hypothesized that the presence of PHB in mitochondrial membrane might play a significant role in mitochondrial calcium transport. To test this, we investigated how the induction of PHB hydrolysis affects mitochondrial calcium transport. Mitochondrial PHB was altered enzymatically by targeted expression of bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. The expression of PhaZ7 induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential in HepG2 but not in U87 and HeLa cells. Furthermore, it significantly inhibited mitochondrial calcium uptake in intact HepG2, U87 and HeLa cells stimulated by the ATP or by the application of increased concentrations of calcium to the digitonin permeabilized cells. Calcium uptake in PhaZ7 expressing cells was restored by mimicking calcium uniporter properties with natural electrogenic calcium ionophore - ferutinin. We propose that PHB is a previously unrecognized important component of the mitochondrial calcium uptake system. PMID:23702223

A literatura como resistência política: traços neorrealistas na produção literária do Movimento dos Atingidos por Barragens (MAB

Directory of Open Access Journals (Sweden)

Carolina Alves Pereira

2018-04-01

Full Text Available Este trabalho busca analisar as relações existentes entre as produções literárias do inicio do século XX, com o surgimento da corrente neo-realista, e a literatura produzida pelo Movimento dos Atingidos por Barragens (MAB, como manifestação de resistência à cultura elitista, no que tange ao espaço das criações artísticas. Para tanto, discute-se a característica do neo-realismo como expressão literária definidora dos aspectos sociais e políticos representados nas obras da época. Analisa-se, também, o conceito de resistência como determinante para a definição da literatura marginalizada do movimento campesino (MAB. Por fim, a seguinte pesquisa ratifica a importância da literatura e das artes produzida para além das universidades e dos cânones habituais. Palavras-chave: Literatura de resistência, Neorrealismo, Movimento dos Atingidos por Barragens (MAB. _________________________ This paper analyzes the relationship between the literary productions of the early twentieth century, with the rise of neo-realist current, and the literature produced by the Brazilian social group called Movimento do Atingidos por Barragens (MAB as a manifestation of resistance to elitist culture in terms the space of artistic creations. To this aim, I discuss the characteristic of neorealism as the defining literary expression of social and political represented in the works of the time aspects. In addition, I analyze the concept of resistance as a determinant for defining the literature of marginalized campesino movement (MAB. Finally, the following research confirms the importance of literature and the arts produced in excess of the usual canons and universities. Keywords: Literature of Resistance, Neorealism, Movimento dos Atingidos por Barragens (MAB.

Impaired Compensation for Salt-Induced Urinary Calcium Loss in a Space Flight Model

Science.gov (United States)

Navidi, Meena; Harper, J. S.; Evans, J.; Fung, P.; Wolinsky, I.; Arnaud, S. B.; Wade, Charles E. (Technical Monitor)

1994-01-01

The loss of urinary calcium (UCa) induced by high sodium (HiNa) diets is compensated for by an increase in net intestinal Ca absorption (abs.). To determine the capacity of the intestine to absorb Ca in a space flight model in which the formation of 1,25-dihydroxyvitamin D (1,25-D) is suppressed, we induced Ca loss with HiNa diets (8%) and restricted dietary Ca (0.2%). In 200 g rats with hind limbs unloaded by tail suspension (S), we examined intestinal Ca abs. by direct measurement in the duodenum (everted gut sac or S/M), vitamin D receptors (VDR) and Ca balance. We also measured serum ionized calcium (ICa), pH, parathyroid hormone (PTH) and 1,25D. PTH was related to ICa (r = -0.44, p is less than 0.02), pH (r = -0.47, p is less than 0.02) and %Ca abs. (r = -0.40, p is less than 0.05). 1,25-D was related to %Ca abs. (r = 0.60, p is less than 0.001) but not VDR or S/M. Effects of the model were lower serum 1,25-D (110 +/- 59 vs. 199 +/- 80 pg/ml, p is less than 0.005), %Ca abs. (83 +/- 6.9 vs. 93 +/- 3.2, p is less than 0.03) and Ca balance (27 +/- 0.2 vs. 30 +/- 0.3 mg/d, p is less than 0.001) in S than controls (C). The HiNa diet increased UCa excretion from 2 to 13% of dietary Ca. Responses to HiNa diets, compared to normal Na, revealed no differences in 1,25-D, Ca abs. or VDR. Ca balances were lower in HiNa (27 +/- 0.3 vs. 30 +/- 0.4 mg/d, p is less than 0.001) in spite of higher Ca intakes. The failure of S rats fed HiNa diets to increase Ca abs. in response to Na-induced Ca loss appears to be related to suppressed 1,25-D in the space flight model, the cause of which remains obscure.

Role of calcium-enriched mixture in endodontics

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Pradeep Kabbinale

2015-01-01

Full Text Available Calcium-enriched mixture (CEM has been recently introduced as a hydrophilic tooth-colored cement. The CEM cement powder is composed of calcium oxide, calcium sulfate, phosphorus oxide, and silica as major elements. CEM is alkaline cement (pH~11 that releases calcium hydroxide (CH during and after setting. The physical properties of CEM, such as flow, film thickness, and primary setting time are favorable. This cement is biocompatible and induces formation of cementum, dentin, bone and periodontal tissues. This novel cement has an antibacterial effect comparable to CH and superior to mineral trioxide aggregate (MTA and sealing ability similar to MTA. Its clinical applications include pulp capping, pulpotomy, root-end filling and perforation repair. This review describes the composition, properties and clinical applications of CEM in endodontics.

Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(+)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices.

Science.gov (United States)

Valentijn, K; Tranchand Bunel, D; Vaudry, H

1992-07-01

The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps

Pressure induced reactions amongst calcium aluminate hydrate phases

KAUST Repository

Moon, Ju-hyuk; Oh, Jae Eun; Balonis, Magdalena; Glasser, Fredrik P.; Clark, Simon M.; Monteiro, Paulo J.M.

2011-01-01

The compressibilities of two AFm phases (strätlingite and calcium hemicarboaluminate hydrate) and hydrogarnet were obtained up to 5 GPa by using synchrotron high-pressure X-ray powder diffraction with a diamond anvil cell. The AFm phases show abrupt

Monoclonal antibodies (MAb) made against insect-derived metacyclic trypomastigotes (IMT) of Trypanosoma cruzi (TC) cross-react with other parasite forms

International Nuclear Information System (INIS)

Kirchhoff, L.V.; Gilliam, F.C.

1986-01-01

Considerable information has been generated in recent years about stage-specific surface membrane antigens of a number of protozoa, and this phenomenon has been observed among several stages of TC as well. However, little is known about the surface antigens of IMT, the true infective stage of TC, because of the difficulty of obtaining sufficient numbers of these organisms for analysis. The Tulahuen strain of TC was maintained in the reduviid vector Dipetalogaster maximus by repeated feeding on mice with high parasitemias. IMT collected with insect urine were irradiated (150 krad) and used to immunize a BALB/c mouse for hybridoma production. Supernatants were screened by immunofluorescence assay for the presence of IgG MAb that react with methanol-fixed IMT, epimastogotes (EPI) and culture-derived metacyclic trypomastigoes (CMT). Of 41 MAb obtained, 40 reacted with IMT, 37 with EPI and 38 with CMT. Four MAb immunoprecipitated radioiodinated proteins or protein conjugates of M/sub r/ 80, 72, 45 and 45 from lysates of 125 I surface-labeled EPI. These results indicate that, at least at the epitopic level, there is considerable overlap among IMT, EPI and CMT surface antigens. This finding suggests that analysis of surface proteins of the latter 2 parasite forms may lead to identification of molecules useful for vaccine development

Calcium paradox and calcium entry blockers

NARCIS (Netherlands)

Ruigrok, T.J.C.; Slade, A.M.; Nayler, W.G.; Meijler, F.L.

1984-01-01

Reperfusion of isolated hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). This phenomenon is characterized by an excessive influx of calcium into the cells, the rapid onset of myocardial contracture,

Characteristics of calcium signaling in astrocytes induced by photostimulation with femtosecond laser

Science.gov (United States)

Zhao, Yuan; Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zeng, Shaoqun; Luo, Qingming

2010-05-01

Astrocytes have been identified to actively contribute to brain functions through Ca2+ signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca2+) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca2+ signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca2+ wave throughout the network with reduced speed is induced, and intracellular Ca2+ oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca2+ elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca2+ signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca2+ signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.

Calcium and α-tocopherol suppress cured-meat promotion of chemically induced colon carcinogenesis in rats and reduce associated biomarkers in human volunteers123

Science.gov (United States)

Martin, Océane CB; Santarelli, Raphaelle L; Taché, Sylviane; Naud, Nathalie; Guéraud, Françoise; Audebert, Marc; Dupuy, Jacques; Meunier, Nathalie; Attaix, Didier; Vendeuvre, Jean-Luc; Mirvish, Sidney S; Kuhnle, Gunter CG; Cano, Noel; Corpet, Denis E

2013-01-01

Background: Processed meat intake has been associated with increased colorectal cancer risk. We have shown that cured meat promotes carcinogen-induced preneoplastic lesions and increases specific biomarkers in the colon of rats. Objectives: We investigated whether cured meat modulates biomarkers of cancer risk in human volunteers and whether specific agents can suppress cured meat–induced preneoplastic lesions in rats and associated biomarkers in rats and humans. Design: Six additives (calcium carbonate, inulin, rutin, carnosol, α-tocopherol, and trisodium pyrophosphate) were added to cured meat given to groups of rats for 14 d, and fecal biomarkers were measured. On the basis of these results, calcium and tocopherol were kept for the following additional experiments: cured meat, with or without calcium or tocopherol, was given to dimethylhydrazine-initiated rats (47% meat diet for 100 d) and to human volunteers in a crossover study (180 g/d for 4 d). Rat colons were scored for mucin-depleted foci, putative precancer lesions. Biomarkers of nitrosation, lipoperoxidation, and cytotoxicity were measured in the urine and feces of rats and volunteers. Results: Cured meat increased nitroso compounds and lipoperoxidation in human stools (both P meat (P = 0.01). Conclusion: Data suggest that the addition of calcium carbonate to the diet or α-tocopherol to cured meat may reduce colorectal cancer risk associated with cured-meat intake. This trial was registered at clinicaltrials.gov as NCT00994526. PMID:24025632

Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells.

Science.gov (United States)

Narula, Shifa; Tandon, Simran; Singh, Shrawan Kumar; Tandon, Chanderdeep

2016-11-01

Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human population worldwide. Proteins are the major constituents of human kidney stone's organic matrix and considered to play critical role in the pathogenesis of disease but their mechanism of modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated cellular injury. The renal epithelial cells (MDCK) were exposed to 200μg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM injured cells. The alterations in cell-crystal interactions were examined by phase contrast, polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis. Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Unilateral vestibular deafferentation-induced changes in calcium signaling-related molecules in the rat vestibular nuclear complex.

Science.gov (United States)

Masumura, Chisako; Horii, Arata; Mitani, Kenji; Kitahara, Tadashi; Uno, Atsuhiko; Kubo, Takeshi

2007-03-23

Inquiries into the neurochemical mechanisms of vestibular compensation, a model of lesion-induced neuronal plasticity, reveal the involvement of both voltage-gated Ca(2+) channels (VGCC) and intracellular Ca(2+) signaling. Indeed, our previous microarray analysis showed an up-regulation of some calcium signaling-related genes such as the alpha2 subunit of L-type calcium channels, calcineurin, and plasma membrane Ca(2+) ATPase 1 (PMCA1) in the ipsilateral vestibular nuclear complex (VNC) following unilateral vestibular deafferentation (UVD). To further elucidate the role of calcium signaling-related molecules in vestibular compensation, we used a quantitative real-time polymerase chain reaction (PCR) method to confirm the microarray results and investigated changes in expression of these molecules at various stages of compensation (6 h to 2 weeks after UVD). We also investigated the changes in gene expression during Bechterew's phenomenon and the effects of a calcineurin inhibitor on vestibular compensation. Real-time PCR showed that genes for the alpha2 subunit of VGCC, PMCA2, and calcineurin were transiently up-regulated 6 h after UVD in ipsilateral VNC. A subsequent UVD, which induced Bechterew's phenomenon, reproduced a complete mirror image of the changes in gene expressions of PMCA2 and calcineurin seen in the initial UVD, while the alpha2 subunit of VGCC gene had a trend to increase in VNC ipsilateral to the second lesion. Pre-treatment by FK506, a calcineurin inhibitor, decelerated the vestibular compensation in a dose-dependent manner. Although it is still uncertain whether these changes in gene expression are causally related to the molecular mechanisms of vestibular compensation, this observation suggests that after increasing the Ca(2+) influx into the ipsilateral VNC neurons via up-regulated VGCC, calcineurin may be involved in their synaptic plasticity. Conversely, an up-regulation of PMCA2, a brain-specific Ca(2+) pump, would increase an efflux of Ca

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

Science.gov (United States)

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Role of Calcium Sensing Receptor in Streptozotocin-Induced Diabetic Rats Exposed to Renal Ischemia Reperfusion Injury

Directory of Open Access Journals (Sweden)

Bo Hu

2018-02-01

Full Text Available Background/Aims: Renal ischemia/reperfusion (I/R injury (RI/RI is a common complication of diabetes, and it may be involved in altering intracellular calcium concentrations at its onset, which can result in inflammation, abnormal lipid metabolism, the production of reactive oxygen species (ROS, and nitroso-redox imbalance. The calcium-sensing receptor (CaSR is a G-protein coupled receptor, however, the functional involvement of CaSR in diabetic RI/ RI remains unclear. The present study was intended to investigate the role of CaSR on RI/RI in diabetes mellitus (DM. Methods: The bilateral renal arteries and veins of streptozotocin (STZ-induced diabetic rats were subjected to 45-min ischemia followed by 2-h reperfusion with or without R-568 (agonist of CaSR and NPS-2143 (antagonist of CaSR at the beginning of I/R procedure. DM without renal I/R rats served as control group. The expressions of CaSR, calmodulin (CaM, and p47phox in the renal tissue were analyzed by qRT-PCR and Western blot. The renal pathomorphology, renal function, oxidative stress, inflammatory response, and calcium disorder were evaluated by detection of a series of indices by hematoxylin-eosin (HE staining, transmission electron microscope (TEM, commercial kits, enzyme-linked immunosorbent assay (ELISA, and spectrophotofluorometry, respectively. Results: Results showed that the expressions of CaSR, CaM, and p47phox in I/R group were significantly up-regulated as compared with those in DM group, which were accompanied by renal tissue injury, increased calcium, oxidative stress, inflammation, and nitroso-redox imbalance. Conclusion: These results suggest that activation of CaSR is involved in the induction of damage of renal tubular epithelial cell during diabetic RI/RI, resulting in lipid peroxidation, inflammatory response, nitroso-redox imbalance, and apoptosis.

Clarification of serotonin-induced effects in peripheral artery disease observed through the femoral artery response in models of diabetes and vascular occlusion: The role of calcium ions.

Science.gov (United States)

Stojanović, Marko; Prostran, Milica; Janković, Radmila; Radenković, Miroslav

2017-07-01

Recent findings have demonstrated that serotonin is an important participant in the development and progression of peripheral artery diseases. Taking this into consideration, the goals of this study were to investigate the effects of serotonin on isolated Wistar rat femoral arteries in both healthy and diabetic animals, with and without artery occlusion, with a particular focus on determining the role of calcium in this process. Contraction experiments with serotonin on intact and denuded femoral artery rings, in the presence or absence of nifedipine and ouabain (both separately, or in combination), as well as Ca 2+ -free Krebs-Ringer bicarbonate solution were performed. The serotonin-induced results were concentration dependent, but only in healthy animals. The endothelium-dependent contraction of the femoral artery was assessed. In healthy animals, the endothelium-reliant part of contraction was dependent on the extracellular calcium, while the smooth muscle-related part was instead dependent on the intracellular calcium. In diabetic animals, both nifedipine and ouabain influenced serotonin-induced vascular effects by blocking intracellular calcium pathways. However, this was diminished after the simultaneous administration of both blockers. © 2017 John Wiley & Sons Australia, Ltd.

IL-2/anti-IL-2 mAb immunocomplexes: A renascence of IL-2 in cancer immunotherapy?

Czech Academy of Sciences Publication Activity Database

Tomala, Jakub; Kovář, Marek

2016-01-01

Roč. 5, č. 3 (2016), e1102829 ISSN 2162-402X R&D Projects: GA ČR GA13-12885S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Anti-IL-2 mAb * cancer immunotherapy * IL-2 Subject RIV: EE - Microbiology, Virology Impact factor: 7.719, year: 2016

Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole-cell oscillations in smooth muscle cells

DEFF Research Database (Denmark)

Jacobsen, Jens Christian; Aalkjær, Christian; Nilsson, Holger

2007-01-01

approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes uniform opening of L-type calcium...... channels on the cell surface stimulating synchronized release of SR-calcium and inducing the shift from waves to whole-cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated...

Effect of cadmium on myocardial contractility and calcium fluxes

International Nuclear Information System (INIS)

Pilati, C.F.

1979-01-01

The effect of cadmium on myocardial mechanical performance and calcium fluxes was studied in kitten isometric papillary muscles and in isovolumic Langendorff-perfused rabbit hearts. Therefore, it is concluded that cadmium-induced decreases in contractility are not primarily the result of cadmium interference with ATP metabolic processes. Furthermore, these results imply that cadmium causes no structural alterations of the contractile proteins. These data suggest that cadmium may be competing with the calcium needed for excitation-contraction coupling. During experiments using radioisotopic calcium, a statistically significant cellular influx of calcium was observed following the onset of 100 μM Cd ++ perfusion of isolated, Langendorff-prepared rabbit hearts

Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals.

Science.gov (United States)

Dai, Mengyao; Wang, Yao; Fang, Lu; Irwin, David M; Zhu, Tengteng; Zhang, Junpeng; Zhang, Shuyi; Wang, Zhe

2014-01-01

Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5' end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.

Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

International Nuclear Information System (INIS)

Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S.

2005-01-01

Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKCα-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis

Inhibition Mechanism of Uranyl Reduction Induced by Calcium-Carbonato Complexes

Science.gov (United States)

Jones, M. E.; Bargar, J.; Fendorf, S. E.

2015-12-01

Uranium mobility in the subsurface is controlled by the redox state and chemical speciation, generally as minimally soluble U(IV) or soluble U(VI) species. In the presence of even low carbonate concentrations the uranyl-carbonato complex quickly becomes the dominant aqueous species; they are, in fact, the primary aqueous species in most groundwaters. Calcium in groundwater leads to ternary calcium-uranyl-carbonato complexes that limit the rate and extent of U(VI) reduction. This decrease in reduction rate has been attributed to surface processes, thermodynamic limitations, and kinetic factors. Here we present a new mechanism for the inhibition of ferrous iron reduction of uranyl-carbonato species in the presence of calcium. A series of experiments under variable Ca conditions were preformed to determine the role of Ca in the inhibition of U reduction by ferrous iron. Calcium ions in the Ca2UO2(CO3)3 complex sterically prevent the interaction of Fe(II) with U(VI), in turn preventing the Fe(II)-U(VI) distance required for electron transfer. The mechanism described here helps to predict U redox transformations in suboxic environments and clarifies the role of Ca in the fate and mobility of U. Electrochemical measurements further show the decrease of the U(VI) to U(V) redox potential of the uranyl-carbonato complex with decreasing pH suggesting the first electron transfer is critical determining the rate and extent of uranium reduction.

N-acetyl-l-cysteine and Mn2+ attenuate Cd2+-induced disturbance of the intracellular free calcium homeostasis in cultured cerebellar granule neurons.

Science.gov (United States)

Isaev, Nickolay K; Avilkina, Svetlana; Golyshev, Sergey A; Genrikhs, Elisaveta E; Alexandrova, Olga P; Kapkaeva, Marina R; Stelmashook, Elena V

2018-01-15

Cadmium is a highly toxic heavy metal that is capable of accumulating in the body via direct exposure or through the alimentary and respiratory tract, leading to neurodegeneration. In this article, we show that the application of CdCl 2 (0.001-0.005mM) for 48h induced high dose-dependent death rate of cultured cerebellar granule neurons (CGNs). Unlike Trolox or vitamin E, antioxidant N-acetyl-l-cysteine (NAC, 1mM) and Mn 2+ (0.0025-0.005mM) significantly protected CGNs from this toxic effect. Using Fluo-4 AM, measurements of intracellular calcium ions demonstrated that 24h-exposure to Cd 2+ induced intensive increase of Fluo-4 fluorescence in neurons accompanied by mitochondria swelling. These data imply that the cadmium-induced Ca 2+ increase is an important element in the death of neurons due to toxic effect of cadmium and the mechanism of protective action of manganese and NAC is mediated by the prevention of increase in calcium levels. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibition of large conductance calcium-dependent potassium ...

African Journals Online (AJOL)

conductance, calcium and voltage- dependent potassium (BKCa) channels thereby promoting vasoconstriction. Our results show that the Rho-kinase inhibitor, Y-27632, induced concentration-dependent relaxation in rat mesenteric artery.

High calcium concentration in bones promotes bone metastasis in renal cell carcinomas expressing calcium-sensing receptor.

Science.gov (United States)

Joeckel, Elke; Haber, Tobias; Prawitt, Dirk; Junker, Kerstin; Hampel, Christian; Thüroff, Joachim W; Roos, Frederik C; Brenner, Walburgis

2014-02-28

The prognosis for renal cell carcinoma (RCC) is related to a high rate of metastasis, including 30% of bone metastasis. Characteristic for bone tissue is a high concentration of calcium ions. In this study, we show a promoting effect of an enhanced extracellular calcium concentration on mechanisms of bone metastasis via the calcium-sensing receptor (CaSR) and its downstream signaling molecules. Our analyses were performed using 33 (11/category) matched specimens of normal and tumor tissue and 9 (3/category) primary cells derived from RCC patients of the 3 categories: non-metastasized, metastasized into the lung and metastasized into bones during a five-year period after nephrectomy. Expression of CaSR was determined by RT-PCR, Western blot analyses and flow cytometry, respectively. Cells were treated by calcium and the CaSR inhibitor NPS 2143. Cell migration was measured in a Boyden chamber with calcium (10 μM) as chemotaxin and proliferation by BrdU incorporation. The activity of intracellular signaling mediators was quantified by a phospho-kinase array and Western blot. The expression of CaSR was highest in specimens and cells of patients with bone metastases. Calcium treatment induced an increased migration (19-fold) and proliferation (2.3-fold) exclusively in RCC cells from patients with bone metastases. The CaSR inhibitor NPS 2143 elucidated the role of CaSR on the calcium-dependent effects. After treatment with calcium, the activity of AKT, PLCγ-1, p38α and JNK was clearly enhanced and PTEN expression was almost completely abolished in bone metastasizing RCC cells. Our results indicate a promoting effect of extracellular calcium on cell migration and proliferation of bone metastasizing RCC cells via highly expressed CaSR and its downstream signaling pathways. Consequently, CaSR may be regarded as a new prognostic marker predicting RCC bone metastasis.

Comparison between anti-CEA and anti-HER2 212Pb-labeled mAbs during α-RIT of small volume peritoneal carcinomatosis - Role of activity distribution on therapeutic efficacy and toxicity?

International Nuclear Information System (INIS)

Elgqvist, J.; Boudousq, V.; Bobyk, L.; Busson, M.; Lozza, C.; Navarro-Teulon, I.; Pouget, J.P.; Maquaire, P.; Torgue, J.

2015-01-01

Full text of publication follows. Objectives: we investigated the role of internalizing/non-internalizing monoclonal antibodies (mAbs) on the final outcome (efficacy/toxicity) of mice treated with alpha radioimmunotherapy (α- RIT) using 212 Pb-labeled mAbs. The relationship between distribution of radioactivity at the tissue level and biological parameters was also assessed. Methods: nude mice bearing 2-3 mm peritoneal nodules obtained by xenograft of A-431 tumor cells, expressing low and high level of HER2 and CEA receptors, respectively, were intraperitoneally (i.p.) injected with increasing activities (370-1480 kBq; 37 MBq/mg) of either 35A7 (non-internalising anti-CEA), Trastuzumab (internalizing anti-HER2) or PX (non-specific) 212 Pb-labeled mAbs. Control groups were injected with corresponding amount of unlabeled mAbs or with NaCl. Tumor growth was followed by bioluminescence and median survival (MS) of control and treated mice was determined. 212 Pb-35A7 and 212 Pb-Trastuzumab biodistribution was used to determine the cumulative uptake of radioactivity (UOR) in organs and tumors. Mean absorbed doses were calculated using the MIRD formalism. Haematological, liver and kidney toxicities were also assessed. Distribution of radioactivity at the tissue level was determined by digital micro-autoradiography and the relationship with biological markers of tissue damage was investigated using immunohistochemistry. Results: a mild and transient haematological toxicity in groups treated with the highest amount of activity was observed. MS of the groups treated either with internalizing or non-internalizing 212 Pb-labeled mAbs was significantly improved compared to those treated with non-specific 212 Pb-PX or those only given unlabeled mAbs or just NaCl. MS ranged from 42 days to 94 days using various activity levels of anti-CEA 212 Pb-35A7 while MS was not reached over the follow-up period of 130 days for mice treated with anti-HER2 212 Pb-Trastuzumab. However, UOR and

Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation

Energy Technology Data Exchange (ETDEWEB)

Grasso, P.; Santa-Coloma, T.A.; Reichert, L.E. Jr. (Department of Biochemistry, Albany Medical College, New York, NY (USA))

1991-06-01

We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

Protein kinase C interaction with calcium: a phospholipid-dependent process.

LENUS (Irish Health Repository)

Bazzi, M D

1990-08-21

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism.

Science.gov (United States)

Yang, Po-Min; Huang, Yu-Ting; Zhang, Yu-Qi; Hsieh, Chia-Wen; Wung, Being-Sun

2016-12-01

The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25μM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca 2+ level. Furthermore, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca 2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca 2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser 1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25μM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser 1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca 2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization. Copyright © 2016 Elsevier Inc. All rights reserved.

The Role of Interleukin-6 in Lipopolysaccharide-Induced Weight Loss, Hypoglycemia and Fibrinogen Production, in Vivo

Science.gov (United States)

1994-07-01

result in chronic wasting or cachexia. In neoplastic severe weight loss in syngeneic hosts.6 In this model, diseases, the presence of wasting of muscle...48 h post-endotoxin). Anti-TNF MAb tribution of TNF and IL-6 in several metabolic changes reduced by c. 50% the LPS-induced weight loss, but...similar results. Also, the addi- hypertriglyceridemia , hypoglycemia as well as stimu- tion of fresh 20F3 MAb to diluted serum sample from late the

An attempt of application of short lived 44K activity induced in the 44Ca(n,p)44K reaction using 14 MeV neutrons for total body calcium assessment in human subject

International Nuclear Information System (INIS)

Haratym, Z.; Kempisty, T.; Mikolajewski, S.; Rurarz, E.

1999-01-01

The status of in vivo neutron activation analysis techniques for the measurement of total body calcium in human subject is reviewed. Relevant data on the nuclear characteristics of calcium isotopes during interaction with neutrons ranging from slow up to 14 MeV neutrons are presented. Physical aspects of the measurement of in vivo total body calcium (TBCa) using 44 K activity induced in the 44 Ca(n,p) 44 K(T 1/2 =22.3 min) reaction by 14 MeV neutrons are discussed. The measurement of delayed γ-ray emitted during decay of activities induced in enriched 44 Ca, nat Ca, phantom filled with water solution of natural calcium and skeletal arm are considered. Results of measurements on the phantom and skeletal arm indicate a possibility to measure the TBCa using the 44 K activity. (author)

Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

Science.gov (United States)

Valliere-Douglass, John F; Lewis, Patsy; Salas-Solano, Oscar; Jiang, Shan

2015-02-01

We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Chemical changes demonstrated in cartilage by synchrotron infrared microspectroscopy in an antibody-induced murine model of rheumatoid arthritis

Science.gov (United States)

Croxford, Allyson M.; Selva Nandakumar, Kutty; Holmdahl, Rikard; Tobin, Mark J.; McNaughton, Don; Rowley, Merrill J.

2011-06-01

Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-μm sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM). None of the mice injected with mAb showed visual or histological evidence of inflammation but there were histological changes in the articular cartilage including loss of proteoglycan and altered chondrocyte morphology. Findings using FTIRM at high lateral resolution revealed loss of collagen and the appearance of a new peak at 1635 cm-1 at the surface of the cartilage interpreted as cellular activation. Thus, we demonstrate the utility of synchrotron FTIRM for examining chemical changes in diseased cartilage at the microscopic level and establish that arthritogenic mAbs to CII do cause cartilage damage in vivo in the absence of inflammation.

Acute Cocaine Exposure elicits rises in calcium in Arousal Related Laterodorsal Tegmental Neurons

DEFF Research Database (Denmark)

Lambert, Mads; Ipsen, Theis; Kohlmeier, Kristi Anne

2017-01-01

Cocaine has strong reinforcing properties, which underlie its high addiction potential. Reinforcement of use of addictive drugs is associated with rises in dopamine (DA) in mesoaccumbal circuitry. Excitatory afferent input to mesoaccumbal circuitry sources from the laterodorsal tegmental nucleus...... (LDT). Chronic, systemic cocaine exposure has been shown to have cellular effects on LDT cells, but acute actions of local application have never been demonstrated. Using calcium imaging, we show that acute application of cocaine to mouse brain slices induces calcium spiking in cells of the LDT....... Spiking was attenuated by tetrodotoxin (TTX) and low calcium solutions, and abolished by prior exhaustion of intracellular calcium stores. Further, DA receptor antagonists reduced these transients, whereas DA induced rises with similar spiking kinetics. Amphetamine, which also results in elevated levels...

Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

Science.gov (United States)

Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

2015-12-01

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

Calmyonemin: a 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate).

Science.gov (United States)

David, C; Viguès, B

1994-01-01

Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calcium-binding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein cross-reacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myoneme-mediated retraction of the ciliature.

Analysis of Spontaneous and Nerve-Evoked Calcium Transients in Intact Extraocular Muscles in Vitro

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Feng, Cheng-Yuan; Hennig, Grant W.; Corrigan, Robert D.; Smith, Terence K.; von Bartheld, Christopher S.

2012-01-01

Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2max duration of 2–12 s, velocity of 25–50 μm/s) and two fast “flash-like” types (Type 1, 30–90 ms; Type 2, 90–150 ms 1/2max duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs. PMID:22579493

Calcium signal communication in the central nervous system.

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Braet, Katleen; Cabooter, Liesbet; Paemeleire, Koen; Leybaert, Luc

2004-02-01

The communication of calcium signals between cells is known to be operative between neurons where these signals integrate intimately with electrical and chemical signal communication at synapses. Recently, it has become clear that glial cells also exchange calcium signals between each other in cultures and in brain slices. This communication pathway has received utmost attention since it is known that astrocytic calcium signals can be induced by neuronal stimulation and can be communicated back to the neurons to modulate synaptic transmission. In addition to this, cells that are generally not considered as brain cells become progressively incorporated in the picture, as astrocytic calcium signals are reported to be communicated to endothelial cells of the vessel wall and can affect smooth muscle cell tone to influence the vessel diameter and thus blood flow. We review the available evidence for calcium signal communication in the central nervous system, taking into account a basic functional unit -the brain cell tripartite- consisting of neurons, glial cells and vascular cells and with emphasis on glial-vascular calcium signaling aspects.

Aggregate complexes of HIV-1 induced by multimeric antibodies.

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Stieh, Daniel J; King, Deborah F; Klein, Katja; Liu, Pinghuang; Shen, Xiaoying; Hwang, Kwan Ki; Ferrari, Guido; Montefiori, David C; Haynes, Barton; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Rerks-Ngarm, Supachai; Michael, Nelson L; Robb, Merlin L; Kim, Jerome H; Denny, Thomas N; Tomaras, Georgia D; Shattock, Robin J

2014-10-02

Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry. The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA. These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

Nuclear calcium signaling induces expression of the synaptic organizers Lrrtm1 and Lrrtm2.

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Hayer, Stefanie N; Bading, Hilmar

2015-02-27

Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2-4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca(2+)/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Nuclear Calcium Signaling Induces Expression of the Synaptic Organizers Lrrtm1 and Lrrtm2*

Science.gov (United States)

Hayer, Stefanie N.; Bading, Hilmar

2015-01-01

Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2–4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca2+/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2. PMID:25527504

Mammary-Specific Ablation of the Calcium-Sensing Receptor During Lactation Alters Maternal Calcium Metabolism, Milk Calcium Transport, and Neonatal Calcium Accrual

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Mamillapalli, Ramanaiah; VanHouten, Joshua; Dann, Pamela; Bikle, Daniel; Chang, Wenhan; Brown, Edward

2013-01-01

To meet the demands for milk calcium, the lactating mother adjusts systemic calcium and bone metabolism by increasing dietary calcium intake, increasing bone resorption, and reducing renal calcium excretion. As part of this adaptation, the lactating mammary gland secretes PTHrP into the maternal circulation to increase bone turnover and mobilize skeletal calcium stores. Previous data have suggested that, during lactation, the breast relies on the calcium-sensing receptor (CaSR) to coordinate PTHrP secretion and milk calcium transport with calcium availability. To test this idea genetically, we bred BLG-Cre mice with CaSR-floxed mice to ablate the CaSR specifically from mammary epithelial cells only at the onset of lactation (CaSR-cKO mice). Loss of the CaSR in the lactating mammary gland did not disrupt alveolar differentiation or milk production. However, it did increase the secretion of PTHrP into milk and decreased the transport of calcium from the circulation into milk. CaSR-cKO mice did not show accelerated bone resorption, but they did have a decrease in bone formation. Loss of the mammary gland CaSR resulted in hypercalcemia, decreased PTH secretion, and increased renal calcium excretion in lactating mothers. Finally, loss of the mammary gland CaSR resulted in decreased calcium accrual by suckling neonates, likely due to the combination of increased milk PTHrP and decreased milk calcium. These results demonstrate that the mammary gland CaSR coordinates maternal bone and calcium metabolism, calcium transport into milk, and neonatal calcium accrual during lactation. PMID:23782944

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration

Directory of Open Access Journals (Sweden)

Rubén Aquino-Martínez

2017-11-01

Full Text Available Abstract Background Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4 on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved. Methods A circular calvarial bone defect (5 mm diameter was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR. Results CaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration. Conclusions Specific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host’s undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced

Radioimmunoscintigraphy with monoclonal antibody Technetium-99m-Anti-EGF-Receptor (R3-MAB) for the detection of head and neck tumours, metastasis and recurrence. Final report for the period 15 April 1995 - 15 April 1997

International Nuclear Information System (INIS)

Oliva Gonzalez, J.P.

1998-03-01

A clinical study was carried out to determine the sensitivity of radioimmunoscintigraphy (RIS) using indigenously produced mouse monoclonal antibody (MAB) against epidermal growth factor receptor in the detection of primary, recurrent and metastatic malignant epithelial tumours of the head and neck region in 13 patients. The MAB was labelled with 99m Tc and imaging was carried out using gamma camera and SPECT. The results were correlated with histopathological findings. RIS gave a sensitivity of 76.9%. This study showed that the indigenously produced MAB can be used for the detection of malignant epithelial tumours in the head and neck region but the MAB will be further characterized to improve its sensitivity in the detection of the neoplasia. (author)

The effect of radiopacifiers agents on pH, calcium release, radiopacity, and antimicrobial properties of different calcium hydroxide dressings.

Science.gov (United States)

Ordinola-Zapata, Ronald; Bramante, Clovis Monteiro; García-Godoy, Franklin; Moldauer, Bertram Ivan; Gagliardi Minotti, Paloma; Tercília Grizzo, Larissa; Duarte, Marco Antonio Hungaro

2015-07-01

The aim of this study was to evaluate the antimicrobial activity, pH level, calcium ion release, and radiopacity of calcium hydroxide pastes associated with three radiopacifying agents (iodoform, zinc oxide, and barium sulfate). For the pH and calcium release tests, 45 acrylic teeth were utilized and immersed in ultrapure water. After 24 h, 72 h, and 7 days the solution was analyzed by using a pH meter and an atomic absorption spectrophotometer. Polyethylene tubes filled with the pastes were used to perform the radiopacity test. For the antimicrobial test, 25 dentin specimens were infected intraorally in order to induce the biofilm colonization and treated with the pastes for 7 days. The Live/Dead technique and a confocal microscope were used to obtain the ratio of live cells. Parametric and nonparametric statistical tests were performed to show differences among the groups (P calcium release test on the 7th day (P > 0.05). The calcium hydroxide/iodoform samples had the highest radiopacity and antimicrobial activity against the biofilm-infected dentin in comparison to the other pastes (P Calcium hydroxide mixed with 17% iodoform and 35% propylene glycol into a paste had the highest pH, calcium ion release, radiopacity, and the greatest antimicrobial action versus similar samples mixed with BaSO4 or ZnO. © 2015 Wiley Periodicals, Inc.

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine

Directory of Open Access Journals (Sweden)

Sabine Buhner

2017-11-01

Full Text Available Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations.Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%.Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39 peaking 90 s (64/144 after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9. Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15 in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1–3.Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare.

Enzymatic pH control for biomimetic depostion of calcium phosphate coatings

NARCIS (Netherlands)

Nijhuis, A.W.G.; Nejadnik, M.R.; Nudelman, F.; Walboomers, X.F.; Riet, te J.; Habibovic, P.; Birgani, Z.T.; Li, Y.B.; Bomans, P.H.H.; Jansen, J.A.; Sommerdijk, N.A.J.M.; Leeuwenburgh, S.C.G.

2014-01-01

The current study examines the enzymatic decomposition of urea into carbon dioxide and ammonia as a means to increase the pH during biomimetic deposition of calcium phospate (CaP) onto implant surfaces. The kinetics of the enzymatically induced pH increase were studied by monitoring pH, calcium

Enzymatic pH control for biomimetic deposition of calcium phosphate coatings

NARCIS (Netherlands)

Nijhuis, A.W.G.; Nejadnik, M.R.; Nudelman, F.; Walboomers, X.F.; Riet, J. te; Habibovic, P.; Tahmasebi Birgani, Z.; Li, Y.; Bomans, P.H.; Jansen, J.A.; Sommerdijk, N.A.; Leeuwenburgh, S.C.G.

2014-01-01

The current study examines the enzymatic decomposition of urea into carbon dioxide and ammonia as a means to increase the pH during biomimetic deposition of calcium phosphate (CaP) onto implant surfaces. The kinetics of the enzymatically induced pH increase were studied by monitoring pH, calcium

An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

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Rast, Georg; Weber, Jürgen; Disch, Christoph; Schuck, Elmar; Ittrich, Carina; Guth, Brian D

2015-01-01

Human induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose. We introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control. Simultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical "chronotropic" compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient. Despite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

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Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Lucchi, Chiara; Correia-de-Sá, Paulo; Videira, Arnaldo

2015-10-01

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics. Copyright © 2015 Elsevier B.V. All rights reserved.

Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists

International Nuclear Information System (INIS)

Eltze, M.; Boer, R.; Sanders, K.H.; Boss, H.; Ulrich, W.R.; Flockerzi, D.

1990-01-01

The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[ 3 H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity

Calcium-induced conformational changes of Thrombospondin-1 signature domain: implications for vascular disease.

Science.gov (United States)

Gupta, Akanksha; Agarwal, Rahul; Singh, Ashutosh; Bhatnagar, Sonika

2017-06-01

Thrombospondin1 (TSP1) participates in numerous signaling pathways critical for vascular physiology and disease. The conserved signature domain of thrombospondin 1 (TSP1-Sig1) comprises three epidermal growth factor (EGF), 13 calcium-binding type 3 thrombospondin (T3) repeats, and one lectin-like module arranged in a stalk-wire-globe topology. TSP1 is known to be present in both calcium-replete (Holo-) and calcium-depleted (Apo-) state, each with distinct downstream signaling effects. To prepare a homology model of TSP1-Sig1 and investigate the effect of calcium on its dynamic structure and interactions. A homology model of Holo-TSP1-Sig1 was prepared with TSP2 as template in Swissmodel workspace. The Apo-form of the model was obtained by omitting the bound calcium ions from the homology model. Molecular dynamics (MD) simulation studies (100 ns) were performed on the Holo- and Apo- forms of TSP1 using Gromacs4.6.5. After simulation, Holo-TSP1-Sig1 showed significant reorientation at the interface of the EGF1-2 and EGF2-3 modules. The T3 wire is predicted to show the maximum mobility and deviation from the initial model. In Apo-TSP1-Sig1 model, the T3 repeats unfolded and formed coils with predicted increase in flexibility. Apo-TSP1-Sig1model also predicted the exposure of the binding sites for neutrophil elastase, integrin and fibroblast growth factor 2. We present a structural model and hypothesis for the role of TSP1-Sig1 interactions in the development of vascular disorders. The simulated model of the fully calcium-loaded and calcium-depleted TSP1-Sig1 may enable the development of its interactions as a novel therapeutic target for the treatment of vascular diseases.

Involvement of the cervical sympathetic nervous system in the changes of calcium homeostasis during turpentine oil-induced stress in rats.

Science.gov (United States)

Stern, J E; Ladizesky, M G; Keller Sarmiento, M I; Cardinali, D P

1993-03-01

Hypocalcemia is a common finding during stress. The objective of this study was to examine: (a) the changes in circulating calcium, parathyroid hormone (PTH) and calcitonin (CT) concentration in rats stressed by being given a subcutaneous injection of turpentine oil, and (b) the involvement of the sympathetic cervical pathway in stress-induced changes of calcium homeostasis. Four hours after receiving turpentine oil or vehicle, rats were subjected either to hypocalcemia, by being given EDTA intraperitoneally, or to hypercalcemia, by being injected CaCl2 intraperitoneally. Significant changes in serum calcium (10% decrease), serum PTH (28% increase) and CT levels (40% decrease) were observed in stressed rats. EDTA administration brought about a significantly greater hypocalcemia, and a higher PTH secretory response in turpentine oil-stressed rats. During stress, the increase of serum calcium after CaCl2 was significantly smaller, and the rise of CT was greater than in controls. In the case of CT the changes were still observed in rats subjected to superior cervical ganglionectomy (SCGx) 14 days earlier. In the case of PTH, the increase found in stressed rats, but not the augmented response after EDTA, was blunted by SCGx. The potentiation of hypocalcemia brought about by turpentine oil was no longer observed in SCGx rats. In vehicle-treated controls, SCGx delayed PTH response to hypocalcemia, but did not affect the increased response of CT to CaCl2 challenge. The results indicate that a number of changes in calcium homeostasis arise during turpentine oil stress in rats. SCGx was effective to modify the set point for PTH release, but played a minor role in affecting the augmentation of CT release during stress.

Inotropic effect, binding properties, and calcium flux effects of the calcium channel agonist CGP 28392 in intact cultured embryonic chick ventricular cells

International Nuclear Information System (INIS)

Laurent, S.; Kim, D.; Smith, T.W.; Marsh, J.D.

1985-01-01

CGP 28392 is a recently described dihydropyridine derivative with positive inotropic properties. To study the mechanism of action of this putative calcium channel agonist, we have related the effects of CGP 28392 on contraction (measured with an optical video system) and radioactive calcium uptake to ligand-binding studies in cultured, spontaneously beating chick embryo ventricular cells. CGP 28392 produced a concentration-dependent increase in amplitude and velocity of contraction (EC 50 = 2 x 10(-7) M; maximum contractile effect = 85% of the calcium 3.6 mM response). Nifedipine produced a shift to the right of the concentration-effect curve for CGP 28392 without decreasing the maximum contractile response, suggesting competitive antagonism (pA2 = 8.3). Computer analysis of displacement of [ 3 H]nitrendipine binding to intact heart cells by unlabeled CGP 28392 indicated a K /sub D/ = 2.2 +/- 0.95 x 10(-7) M, in good agreement with the EC 50 for the inotropic effect. CGP 28392 increased the rate of radioactive calcium influx (+39% at 10 seconds) without altering beating rate, while nifedipine decreased radioactive calcium influx and antagonized the CGP 28392-induced increase in calcium influx. Our results indicate that, in intact cultured myocytes, CGP 28392 acts as a calcium channel agonist and competes for the dihydropyridine-binding site of the slow calcium channel. In contrast to calcium channel blockers, CGP 28392 increases calcium influx and enhances the contractile state

[Calcium suppletion for patients who use gastric acid inhibitors: calcium citrate or calcium carbonate?].

NARCIS (Netherlands)

Jonge, H.J. de; Gans, R.O.; Huls, G.A.

2012-01-01

Various calcium supplements are available for patients who have an indication for calcium suppletion. American guidelines and UpToDate recommend prescribing calcium citrate to patients who use antacids The rationale for this advice is that water-insoluble calcium carbonate needs acid for adequate

Enzymatic pH Control for Biomimetic Deposition of Calcium Phosphate Coatings

NARCIS (Netherlands)

Nijhuis, A.W.; Reza Nejadnik, M.; Nudelman, F.; Walboomers, X.F.; te Riet, J.; Habibovic, Pamela; Tahmasebi Birgani, Zeinab; Yubao, L.; Bomans, P.H.H.; Jansen, J.A.; Sommerdijk, N.A.J.M.; Leeuwenburgh, S.C.G.

2014-01-01

The current study has focused on enzymatic decomposition of urea into carbon dioxide and ammonia as a means to increase the pH during biomimetic deposition of Calcium Phospate (CaP) onto implant surfaces. The kinetics of the enzymatically induced pH increase were studied by monitoring pH, calcium

Effects of potassium concentration on firing patterns of low-calcium epileptiform activity in anesthetized rat hippocampus: inducing of persistent spike activity.

Science.gov (United States)

Feng, Zhouyan; Durand, Dominique M

2006-04-01

It has been shown that a low-calcium high-potassium solution can generate ictal-like epileptiform activity in vitro and in vivo. Moreover, during status epileptiform activity, the concentration of [K+]o increases, and the concentration of [Ca2+]o decreases in brain tissue. Therefore we tested the hypothesis that long-lasting persistent spike activity, similar to one of the patterns of status epilepticus, could be generated by a high-potassium, low-calcium solution in the hippocampus in vivo. Artificial cerebrospinal fluid was perfused over the surface of the exposed left dorsal hippocampus of anesthetized rats. A stimulating electrode and a recording probe were placed in the CA1 region. By elevating K+ concentration from 6 to 12 mM in the perfusate solution, the typical firing pattern of low-calcium ictal bursts was transformed into persistent spike activity in the CA1 region with synaptic transmission being suppressed by calcium chelator EGTA. The activity was characterized by double spikes repeated at a frequency approximately 4 Hz that could last for >1 h. The analysis of multiple unit activity showed that both elevating [K+]o and lowering [Ca2+]o decreased the inhibition period after the response of paired-pulse stimulation, indicating a suppression of the after-hyperpolarization (AHP) activity. These results suggest that persistent status epilepticus-like spike activity can be induced by nonsynaptic mechanisms when synaptic transmission is blocked. The unique double-spike pattern of this activity is presumably caused by higher K+ concentration augmenting the frequency of typical low-calcium nonsynaptic burst activity.

Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in High Glucose-Induced Apoptosis in Rat Cardiac Myocytes and Murine Pancreatic β-Cells.

Science.gov (United States)

Yao, Takako; Fujimura, Tsutomu; Murayama, Kimie; Okumura, Ko; Seko, Yoshinori

2017-10-18

We previously identified a novel apoptosis-inducing humoral factor in the conditioned medium of hypoxic/reoxygenated-cardiac myocytes. We named this novel post-translationally-modified secreted-form of eukaryotic translation initiation factor 5A Oxidative stress-Responsive Apoptosis-Inducing Protein (ORAIP). We confirmed that myocardial ischemia/reperfusion markedly increased plasma ORAIP levels and rat myocardial ischemia/reperfusion injury was clearly suppressed by neutralizing anti-ORAIP monoclonal antibodies (mAbs) in vivo. In this study, to investigate the mechanism of cell injury of cardiac myocytes and pancreatic β-cells involved in diabetes mellitus (DM), we analyzed plasma ORAIP levels in DM model rats and the role of ORAIP in high glucose-induced apoptosis of cardiac myocytes in vitro. We also examined whether recombinant-ORAIP induces apoptosis in pancreatic β-cells. Plasma ORAIP levels in DM rats during diabetic phase were about 18 times elevated as compared with non-diabetic phase. High glucose induced massive apoptosis in cardiac myocytes (66.2 ± 2.2%), which was 78% suppressed by neutralizing anti-ORAIP mAb in vitro. Furthermore, recombinant-ORAIP clearly induced apoptosis in pancreatic β-cells in vitro. These findings strongly suggested that ORAIP plays a pivotal role in hyperglycemia-induced myocardial injury and pancreatic β-cell injury in DM. ORAIP will be a biomarker and a critical therapeutic target for cardiac injury and progression of DM itself.

Calcium status in premenopausal and post menopausal women

International Nuclear Information System (INIS)

Qureshi, H.J.; Hussain, G.; Bashir, M.U.; Latif, N.; Riaz, Z.

2010-01-01

Background: In postmenopausal women, the two major causes of bone loss are oestrogen deficiency after menopause and age related processes. Bone turnover increases to high levels and oestrogen deficiency may induce calcium loss by indirect effects on extra skeletal calcium homeostasis. Objective of this study was to evaluate calcium status in pre-menopausal and postmenopausal women. Methods: This cross sectional study was carried out in 34 premenopausal women and 33 postmenopausal women, in Department of Physiology, Services Institute of Medical Sciences, Lahore. Height and weight of each woman were taken to find out the body mass index (BMI). Serum calcium, parathyroid hormone and calcitonin levels of each subject were determined. Results: Premenopausal women were obese (BMI>30 Kg/m/sup 2/) while postmenopausal women were overweight (BMI>25 Kg/m/sup 2/). Serum calcium levels were significantly lower in postmenopausal women than in pre-menopausal women, while serum parathyroid hormone levels were significantly higher in postmenopausal woman. Serum calcitonin level was not significantly different in the two groups. Conclusion: Postmenopausal women are calcium deficient and have increased bone turnover as indicated by increased serum parathyroid hormone levels. (author)

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase.

Science.gov (United States)

Duan, Wenjuan; Zhou, Juefei; Li, Wei; Zhou, Teng; Chen, Qianqian; Yang, Fuyu; Wei, Taotao

2013-04-01

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

Protection of Dentate Hilar Cells from Prolonged Stimulation by Intracellular Calcium Chelation

Science.gov (United States)

Scharfman, Helen E.; Schwartzkroin, Philip A.

1989-10-01

Prolonged afferent stimulation of the rat dentate gyrus in vivo leads to degeneration only of those cells that lack immunoreactivity for the calcium binding proteins parvalbumin and calbindin. In order to test the hypothesis that calcium binding proteins protect against the effects of prolonged stimulation, intracellular recordings were made in hippocampal slices from cells that lack immunoreactivity for calcium binding proteins. Calcium binding protein--negative cells showed electrophysiological signs of deterioration during prolonged stimulation; cells containing calcium binding protein did not. When neurons without calcium binding proteins were impaled with microelectrodes containing the calcium chelator BAPTA, and BAPTA was allowed to diffuse into the cells, these cells showed no deterioration. These results indicate that, in a complex tissue of the central nervous system, an activity-induced increase in intracellular calcium can trigger processes leading to cell deterioration, and that increasing the calcium binding capacity of a cell decreases its vulnerability to damage.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

Science.gov (United States)

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Myeloablative radioimmunotherapy with {sup 188}Re-CD66mAb before stem cell transplantation. No increase of proinflammatory cytokine levels of TNF-{alpha}; Myeloablative Radioimmuntherapie mit {sup 188}Re-CD66mAb vor Stammzelltransplantation. Kein Anstieg proinflammatorischer Zytokinspiegel von TNF-{alpha}

Energy Technology Data Exchange (ETDEWEB)

Mutschler, J.; Reske, S.N. [Universitaetsklinik Ulm (Germany). Klinik fuer Nuklearmedizin; Steinbach, G. [Universitaetsklinik Ulm (Germany). Abt. Klinische Chemie; Bunjes, D. [Universitaetsklinik Ulm (Germany). Medizinische Klinik III; Buchmann, I. [Universitaetsklinik Heidelberg (Germany). Abt. fuer Nuklearmedizin

2009-07-01

Tumour necrosis factor-{alpha} (TNF-{alpha}) serum levels may increase due to intensive conditioning regimes with high-dose chemotherapy and total body irradiation (TBI) before stem cell transplantation. This increases the risk for developing acute graft versus host disease (aGvHD) after stem cell transplantation. In this prospective study we investigated the influence of radioimmunotherapy with {sup 188}Re-CD-66-mAb on changes on TNF-{alpha} serum levels. Patients, methods: In 18 patients we measured TNF-{alpha} before and up to 96 hours after radioimmunotherapy, in 2 patients in addition following TBI, in 9 patients also following chemotherapy. For measuring TNF-{alpha} we used an automated immunochemiluminescence assay (Immulite 1000 DPC Biermann, Bad Nauheim). The mean follow up period to record incidence of aGVHD was 100 days after stem cell transplantation. Compared to the basal levels before, the levels of TNF-{alpha} after conditioning with {sup 188}Re-CD-66-mAb did not increase significantly and remained in the physiological range. In contrast, these initial physiological cytokine levels increased and became pathological following 48 h after total body irradiation (13.2 {+-} 6.6 pg/ml) and chemotherapy (10.8 {+-} 15.7 pg/ml). In our study we found a low incidence of aGvHD (22.2%, n = 4/18). Conclusion: These results demonstrate that additional conditioning therapy with {sup 188}Re-CD-66-mAb does not increase proinflammatory cytokine levels of TNF-{alpha}. This finding may indicate that additive radioimmunotherapy may not be a significant factor for increasing the rate of conditioning- associated aGvHD. (orig.)

The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate

Science.gov (United States)

Song, Guodong; Habibovic, Pamela; Bao, Chongyun; Hu, Jing; van Blitterswijk, Clemens A.; Yuan, Huipin; Chen, Wenchuan; Xu, Hockin H.K.

2013-01-01

Osteoinductive biomaterials are promising for bone repair. There is no direct proof that bone marrow mesenchymal stem cells (BMSCs) home to non-osseous sites and participate in ectopic bone formation induced by osteoinductive bioceramics. The objective of this study was to use a sex-mismatched beagle dog model to investigate BMSC homing via blood circulation to participate in ectopic bone formation via osteoinductive biomaterial. BMSCs of male dogs were injected into female femoral marrow cavity. The survival and stable chimerism of donor BMSCs in recipients were confirmed with polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Biphasic calcium phosphate (BCP) granules were implanted in dorsal muscles of female dogs. Y chromosomes were detected in samples harvested from female dogs which had received male BMSCs. At 4 weeks, cells with Y-chromosomes were distributed in the new bone matrix throughout the BCP granule implant. At 6 weeks, cells with Y chromosomes were present in newly mineralized woven bone. TRAP positive osteoclast-like cells were observed in 4-week implants, and the number of such cells decreased from 4 to 6 weeks. These results show that osteoprogenitors were recruited from bone marrow and homed to ectopic site to serve as a cell source for calcium phosphate-induced bone formation. In conclusion, BMSCs were demonstrated to migrate from bone marrow through blood circulation to non-osseous bioceramic implant site to contribute to ectopic bone formation in a canine model. BCP induced new bone in muscles without growth factor delivery, showing excellent osteoinductivity that could be useful for bone tissue engineering. PMID:23298780

Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility.

Directory of Open Access Journals (Sweden)

Jian Sun

2016-05-01

Full Text Available A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling.

Influence of whole-body irradiation on calcium and phosphate homeostasis in the rat

International Nuclear Information System (INIS)

Pento, J.T.; Kenny, A.D.

1975-01-01

Previous irradiation studies have revealed marked alterations in calcium metabolism. Moreover, the maintenance of calcium homeostasis with parathyroid hormone or calcium salts has been reported to reduce radiation lethality. Therefore, the present study was designed to evaluate the influence of irradiation on calcium homeostasis in the rat. Nine hundred rad of whole-body irradiation produced a significant depression of both plasma calcium and phosphate at 4 days postirradiation. This effect of irradiation was observed to be dose-dependent over a range of 600 to 1200 rad, and possibly related to irradiation-induced anorexia. The physiological significance of these observations is discussed

Enhancement of CD147 on M1 macrophages induces differentiation of Th17 cells in the lung interstitial fibrosis.

Science.gov (United States)

Geng, Jie-jie; Zhang, Kui; Chen, Li-na; Miao, Jin-lin; Yao, Meng; Ren, Ying; Fu, Zhi-guang; Chen, Zhi-nan; Zhu, Ping

2014-09-01

Lung interstitial fibrosis is a chronic lung disease, and few effective therapies are available to halt or reverse the progression of the disease. In murine and human lung fibrosis, the expression of CD147 is increased. However, the role of CD147 in lung fibrosis has not been identified, and it remains to be determined whether lung fibrosis would be improved by decreasing the expression of CD147. A murine bleomycin-induced lung interstitial fibrosis model was used in the experiments, and HAb18 mAbs and CsA were administered during the induction of lung fibrosis. In our study, we found that the HAb18 mAbs markedly reduced the collagen score and down-regulated M1 macrophages and Th17 cells. In vitro, flow cytometry analysis showed that M1 macrophages induced higher Th17 differentiation than M2 macrophages. After treatment with HAb18 mAbs or after reducing the expression of CD147 by lentivirus interference in M1 macrophages, the level of Th17 cells were significantly inhibited. In conclusion, HAb18 mAbs or CsA treatment ameliorates lung interstitial fibrosis. CD147 promoted M1 macrophage and induced the differentiation of Th17 cells in lung interstitial fibrosis, perhaps by regulating some cytokines such as IL-6, IL-1β, IL-12 and IL-23. These results indicated that CD147 may play an important role in the development of lung interstitial fibrosis. Copyright © 2014 Elsevier B.V. All rights reserved.

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

DEFF Research Database (Denmark)

Jorgensen, N R; Geist, S T; Civitelli, R

1997-01-01

mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein......Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium...... stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...

The interplay between HIF-1 and calcium signalling in cancer.

Science.gov (United States)

Azimi, Iman

2018-04-01

The interplay between hypoxia-inducible factor-1 (HIF-1) and calcium in cancer has begun to be unravelled with recent findings demonstrating the relationships between the two in different cancer types. This is an area of significance considering the crucial roles of both HIF-1 and calcium signalling in cancer progression and metastasis. This review summarises the experimental evidence of the crosstalk between HIF-1 and specific calcium channels, pumps and regulators in the context of cancer. HIF-1 as a master regulator of hypoxic transcriptional responses, mediates transcription of several calcium modulators. On the other hand, specific calcium channels and pumps regulate HIF-1 activity through controlling its transcription, translation, stabilisation, or nuclear translocation. Identifying the interplay between HIF-1 and components of the calcium signal will give new insights into mechanisms underlying cellular responses to physiological and pathophysiological cues, and may provide novel and more efficient therapeutic strategies for the control of cancer progression. Copyright © 2018 Elsevier Ltd. All rights reserved.

Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

International Nuclear Information System (INIS)

Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

2011-01-01

We had earlier shown that exposure to arsenic (0.50 μM) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca 2+ ) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca 2+ homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca 2+ levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: → Altered Ca 2+ homeostasis leads to arsenic-induced HKM apoptosis. → Calpain-2 plays a critical role in the process. → ERK is pro-apoptotic in arsenic-induced HKM apoptosis. → Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

Effect of ethionine on hepatic mitochondrial and microsomal calcium uptake

International Nuclear Information System (INIS)

Agarwal, A.K.; Zinermon, W.D.; Latoni, L.

1988-01-01

Ethionine, an ethyl analog of methionine, produces a variety of physiological and pathological effects in animals. These range from acute effects in the liver, kidney, pancreas, and other organs to liver carcinogenesis. Female rats when injected with ethionine exhibit a rapid decrease in hepatic adenosine triphosphate levels followed by a marked inhibition of RNA and protein synthesis and accumulation of triglycerides. Since calcium transport in mitochondria and microsomes is ATP dependent, it becomes interesting to find out if ethionine administration has any effect on subcellular calcium transport. Calcium has recently gained an increased controversy regarding its role in chemical induced lethal cell damage. Certain groups believe that influx of extracellular calcium across the damaged plasma membrane might actually mediate the irreversible damage to the cell, whereas according to other, entry of calcium into the cell is secondary to the damage. The present study was carried out to investigate the calcium [ 45 Ca] transport in mitochondria and microsomes following ethionine administration. The effect of carbon tetrachloride on calcium uptake in ethionine treated rats was also studied

Development of a degradable cement of calcium phosphate and calcium sulfate composite for bone reconstruction

International Nuclear Information System (INIS)

Guo, H; Wei, J; Liu, C S

2006-01-01

A new type of composite bone cement was prepared and investigated by adding calcium sulfate (CS) to calcium phosphate cement (CPC). This composite cement can be handled as a paste and easily shaped into any contour, which can set within 5-20 min, the setting time largely depending on the liquid-solid (L/S) ratio; adding CS to CPC had little effect on the setting time of the composite cements. No obvious temperature increase and pH change were observed during setting and immersion in simulated body fluid (SBF). The compressive strength of the cement decreased with an increase in the content of CS. The degradation rate of the composite cements increased with time when the CS content was more than 20 wt%. Calcium deficient apatite could form on the surface of the composite cement because the release of calcium into SBF from the dissolution of CS and the apatite of the cement induced the new apatite formation; increasing the content of CS in the composite could improve the bioactivity of the composite cements. The results suggested that composite cement has a reasonable setting time, excellent degradability and suitable mechanical strength and bioactivity, which shows promising prospects for development as a clinical cement

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

Science.gov (United States)

Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

2016-06-01

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium pantothenate, calcium chloride double salt... FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.330 Calcium pantothenate, calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may...

47-mG2a: A Mouse IgG2a-Type of PcMab-47 Useful for Detecting Podocalyxin in Esophageal Cancers by Immunohistochemistry.

Science.gov (United States)

Kaneko, Mika K; Itai, Shunsuke; Yamada, Shinji; Kato, Yukinari

2018-04-09

Esophageal cancer is one of the highly malignant cancers. It comprises two of the most common histological tumor types: squamous cell carcinoma (SCC) and adenocarcinoma. SCC accounts for about 90% of esophageal cancers. Despite developments in treatment strategies, the prognosis and survival rate remain poor. Podocalyxin (PODXL) is a highly glycosylated type-I transmembrane protein. It is expressed in normal tissues such as kidney, heart, breast, and pancreas. Upregulation of PODXL correlates with tumor progression, invasion, and metastasis. Therefore, this glycoprotein could be a potential biomarker for predicting the prognosis of some cancers, for instance, brain, colorectal, oral, lung, bladder, prostate, and ovarian cancers. We previously developed a specific and sensitive anti-PODXL monoclonal antibody (mAb), PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ). We showed their utility in immunohistochemical analysis of oral cancers. Herein, we demonstrate that PcMab-47 and 47-mG 2a can also be used to detect esophageal squamous cell carcinoma (ESCC) with this technique. These two antibodies, respectively, stained 123/130 (94.6%) and 127/130 (97.7%) ESCC cases, indicating that they can detect PODXL with high sensitivity in this carcinoma. Of more than 3+ cases, 47-mG 2a was more effective than PcMab-47, respectively, staining 56/127 (44.1%) and 41/123 (33.3%). Therefore, 47-mG 2a can be used for the detection of PODXL in ESCC using immunohistochemical analysis.

ORIGINAL ARTICLES Calcium supplementation to prevent pre ...

African Journals Online (AJOL)

During early pregnancy BP normally falls, climbing slowly ... Resource Centre for Randomised Trials, Institute of Health Sciences, Old Road. Headington, Oxford, UK ... Subgroup analyses were used to test whether these effects ..... Prevention of pregnancy-induced hypertension by calcium supplementation in angiotensin.

The therapeutic CD38 monoclonal antibody daratumumab induces programmed cell death via fcg receptor-mediated cross-linking

DEFF Research Database (Denmark)

Overdijk, Marije B.; Jansen, J. H. Marco; Nederend, Maaike

2016-01-01

RIIb as well as activating FcgRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcgRmediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA......Emerging evidence suggests that FcgR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce...... programmed cell death (PCD) of CD38+ multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcgR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRg-chain knockout or NOTAM mice carrying a signaling-inactive FcRg-chain, we found that the inhibitory Fcg...

Aeroallergen analyses and their clinical relevance. I. Immunochemical quantification of allergens by RAST-inhibition, Mab-ELISA, basophil histamine release, and counter current immuno electrophoresis

DEFF Research Database (Denmark)

Johnsen, C R; Abrahamsen, L; Stahl Skov, P

1991-01-01

The aim was to compare IgE and IgG4 RAST-inhibition assay (RI), monoclonal antibody ELISA (Mab-ELISA), counter current immuno electrophoresis (CCIE) and histamine release from basophil leukocytes (HR) for allergen quantification with special reference to aeroallergen detection. As components......-U/ml) than IgE-RI (2*10(3) SQ-U/ml). The ranges of allergen detection limits for the Mab-ELISA were equal for cat and Derm. pter. (10-10(2) SQ-U/ml). The range of allergen detection limits for CCIE, assaying dog were 10(4)-10(5) SQ-U/ml. The ranges of allergen detection limits for HR were equal for cat...

Aberrant Splicing Induced by Dysregulated Rbfox2 Produces Enhanced Function of CaV1.2 Calcium Channel and Vascular Myogenic Tone in Hypertension.

Science.gov (United States)

Zhou, Yingying; Fan, Jia; Zhu, Huayuan; Ji, Li; Fan, Wenyong; Kapoor, Isha; Wang, Yue; Wang, Yuan; Zhu, Guoqing; Wang, Juejin

2017-12-01

Calcium influx from activated voltage-gated calcium channel Ca V 1.2 in vascular smooth muscle cells is indispensable for maintaining myogenic tone and blood pressure. The function of Ca V 1.2 channel can be optimized by alternative splicing, one of post-transcriptional modification mechanisms. The splicing factor Rbfox2 is known to regulate the Ca V 1.2 pre-mRNA alternative splicing events during neuronal development. However, Rbfox2's roles in modulating the key function of vascular Ca V 1.2 channel and in the pathogenesis of hypertension remain elusive. Here, we report that the proportion of Ca V 1.2 channels with alternative exon 9* is increased by 10.3%, whereas that with alternative exon 33 is decreased by 10.5% in hypertensive arteries. Surprisingly, the expression level of Rbfox2 is increased ≈3-folds, presumably because of the upregulation of a dominant-negative isoform of Rbfox2. In vascular smooth muscle cells, we find that knockdown of Rbfox2 dynamically increases alternative exon 9*, whereas decreases exon 33 inclusion of Ca V 1.2 channels. By patch-clamp studies, we show that diminished Rbfox2-induced alternative splicing shifts the steady-state activation and inactivation curves of vascular Ca V 1.2 calcium channel to hyperpolarization, which makes the window current potential to more negative. Moreover, siRNA-mediated knockdown of Rbfox2 increases the pressure-induced vascular myogenic tone of rat mesenteric artery. Taken together, our data indicate that Rbfox2 modulates the functions of vascular Ca V 1.2 calcium channel by dynamically regulating the expressions of alternative exons 9* and 33, which in turn affects the vascular myogenic tone. Therefore, our work suggests a key role for Rbfox2 in hypertension, which provides a rational basis for designing antihypertensive therapies. © 2017 American Heart Association, Inc.

Determination of percent calcium carbonate in calcium chromate

International Nuclear Information System (INIS)

Middleton, H.W.

1979-01-01

The precision, accuracy and reliability of the macro-combustion method is superior to the Knorr alkalimetric method, and it is faster. It also significantly reduces the calcium chromate waste accrual problem. The macro-combustion method has been adopted as the official method for determination of percent calcium carbonate in thermal battery grade anhydrous calcium chromate and percent calcium carbonate in quicklime used in the production of calcium chromate. The apparatus and procedure can be used to measure the percent carbonate in inorganic materials other than calcium chromate. With simple modifications in the basic apparatus and procedure, the percent carbon and hydrogen can be measured in many organic material, including polymers and polymeric formulations. 5 figures, 5 tables

Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium-dependent protein kinases in Ulva compressa exposed to copper excess.

Science.gov (United States)

González, Alberto; Cabrera, M de Los Ángeles; Henríquez, M Josefa; Contreras, Rodrigo A; Morales, Bernardo; Moenne, Alejandra

2012-03-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H(2)O(2), ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H(2)O(2) increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H(2)O(2) accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H(2)O(2). In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H(2)O(2), and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein

The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L.

Science.gov (United States)

Wang, F; Zhu, W; Liu, T; Sun, Z; Ju, S; Ju, S; Yu, G; Xie, W; Deng, Z; Lu, B; Zhang, X

2007-01-01

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

2016-01-29

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

International Nuclear Information System (INIS)

Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue; Tsuchihashi, Kenji; Ueda, Shiho; Masuko, Takashi; Saya, Hideyuki; Nagano, Osamu

2016-01-01

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

Science.gov (United States)

Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

2017-10-01

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Polyamines mediate abnormal Ca2+ transport and Ca2+-induced cardiac cell injury in the calcium paradox

International Nuclear Information System (INIS)

Trout, J.J.; Koenig, H.; Goldstone, A.D.; Lu, C.Y.; Fan, C.C.

1986-01-01

Ca 2+ -free perfusion renders heart cells Ca 2+ -sensitive so that readmission of Ca 2+ causes a sudden massive cellular injury attributed to abnormal entry of Ca 2+ into cells (Ca paradox). Hormonal stimulation of Ca 2+ fluxes was earlier shown to be mediated by polyamines (PA). 5 min perfusion of rat heart with Ca 2+ -free medium induce a prompt 40-50% decline in levels of the PA putrescine (PUT), spermidine and spermine and their rate-regulatory synthetic enzyme ornithine decarboxylase (ODC), and readmission of Ca 2+ -containing medium abruptly ( 2+ reperfusion-induced increases in ODC and PA and also prevented increased 45 Ca 2+ uptake and heart injury, manifested by loss of contractility, release of enzymes (CPK, LDH), myoglobin and protein, and E.M. lesions (contracture bands, mitochondrial changes). 1 mM PUT negated DFMO inhibition, repleted heart PA and restored Ca 2+ reperfusion-induced 45 Ca 2+ influx and cell injury. These data indicate that the Ca 2+ -directed depletion-repletion cycle of ODC and PA triggers excessive transsarcolemmal Ca 2+ transport leading to the calcium paradox

Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast

OpenAIRE

Busti, Stefano; Mapelli, Valeria; Tripodi, Farida; Sanvito, Rossella; Magni, Fulvio; Coccetti, Paola; Rocchetti, Marcella; Nielsen, Jens; Alberghina, Lilia; Vanoni, Marco

2016-01-01

Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and met...

Myogenic activation and calcium sensitivity of cannulated rat mesenteric small arteries

NARCIS (Netherlands)

VanBavel, E.; Wesselman, J. P.; Spaan, J. A.

1998-01-01

Pressure-induced activation of vascular smooth muscle may involve electromechanical as well as nonelectromechanical coupling mechanisms. We compared calcium-tone relations of cannulated rat mesenteric small arteries during pressure-induced activation, depolarization (16 to 46 mmol/L K+), and

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

Science.gov (United States)

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I

2015-08-03

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

The thickness of odontoblast-like cell layer after induced by propolis extract and calcium hydroxide

Directory of Open Access Journals (Sweden)

Irfan Dwiandhono

2016-12-01

Full Text Available Background: Propolis is a substance made from resin collected by bees (Apis mellifera from variety of plants, mixed with its saliva and various enzymes to build a nest. Propolis has potential antimicrobial and antiinflammatory agents with some advantages over calcium hydroxide (Ca(OH2. Ca(OH2 has been considered as the “gold standard” of direct pulp-capping materials, but there are still some weakness of its application. First, it can induce pulp inflammation which last up to 3 months. Second, the tissue response to Ca(OH2 is not always predictable. Third, the tunnel defect can probably formed in dentinal bridge with possible bacterial invasion in that gap. Purpose: This study was aimed to determine and compare the thickness of odontoblast-like cells layer after induced by propolis extract and Ca(OH2 in rat’s pulp tissue. Method: Class 1 preparation was done in maxillary first molar tooth of wistar mice until the pulp opened. The Ca(OH2 and propolis extract was applied to induce the formation of odontoblast-like cells, the cavity was filled with RMGIC. The teeth were extracted (after 14 and 28 days of induction. The samples were then processed for histological evaluation. Result: There were significant differences between the thickness of odontoblast-like cells after induced by propolis extract and Ca(OH2. Conclusion: The propolis extract as the direct pulp capping agent produces thicker odontoblast-like cell layer compared to Ca(OH2.

Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin

Science.gov (United States)

Yang, T.; Poovaiah, B. W.

2002-01-01

Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

Parathyroid Hormone Induces Bone Cell Motility and Loss of Mature Osteocyte Phenotype through L-Calcium Channel Dependent and Independent Mechanisms.

Directory of Open Access Journals (Sweden)

Matthew Prideaux

Full Text Available Parathyroid Hormone (PTH can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R, which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1 promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA, exchange proteins activated by cAMP (Epac, protein kinase C (PKC or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these

Mechanistic Exploration of Cancer Stem Cell Marker Voltage-Dependent Calcium Channel α2δ1 Subunit-mediated Chemotherapy Resistance in Small-Cell Lung Cancer.

Science.gov (United States)

Yu, Jiangyong; Wang, Shuhang; Zhao, Wei; Duan, Jianchun; Wang, Zhijie; Chen, Hanxiao; Tian, Yanhua; Wang, Di; Zhao, Jun; An, Tongtong; Bai, Hua; Wu, Meina; Wang, Jie

2018-05-01

Purpose: Chemoresistance in small-cell lung cancer (SCLC) is reportedly attributed to the existence of resistant cancer stem cells (CSC). Studies involving CSC-specific markers and related mechanisms in SCLC remain limited. This study explored the role of the voltage-dependent calcium channel α2δ1 subunit as a CSC marker in chemoresistance of SCLC, and explored the potential mechanisms of α2δ1-mediated chemoresistance and strategies of overcoming the resistance. Experimental Design: α2δ1-positive cells were identified and isolated from SCLC cell lines and patient-derived xenograft (PDX) models, and CSC-like properties were subsequently verified. Transcriptome sequencing and Western blotting were carried out to identify pathways involved in α2δ1-mediated chemoresistance in SCLC. In addition, possible interventions to overcome α2δ1-mediated chemoresistance were examined. Results: Different proportions of α2δ1 + cells were identified in SCLC cell lines and PDX models. α2δ1 + cells exhibited CSC-like properties (self-renewal, tumorigenic, differentiation potential, and high expression of genes related to CSCs and drug resistance). Chemotherapy induced the enrichment of α2δ1 + cells instead of CD133 + cells in PDXs, and an increased proportion of α2δ1 + cells corresponded to increased chemoresistance. Activation and overexpression of ERK in the α2δ1-positive H1048 cell line was identified at the protein level. mAb 1B50-1 was observed to improve the efficacy of chemotherapy and delay relapse as maintenance therapy in PDX models. Conclusions: SCLC cells expressing α2δ1 demonstrated CSC-like properties, and may contribute to chemoresistance. ERK may play a key role in α2δ1-mediated chemoresistance. mAb 1B50-1 may serve as a potential anti-SCLC drug. Clin Cancer Res; 24(9); 2148-58. ©2018 AACR . ©2018 American Association for Cancer Research.

The alpha hemolisina of Escherichia Coli induces increases in the calcium citoplasmico of neutrofilos and monocytes human beings

International Nuclear Information System (INIS)

Garcia, J.

2000-01-01

Escherichia coli alpha hemolysin (AH) and the calcium ionophores ionomycin and 4 Br A23187 caused increases in cell fluorescence, indicative of elevations in cytoplasmic calcium, in fura 2-loaded human polymorphonuclear leukocytes(PMN) and monocytes (MN). The increase in fluorescence caused by AH was dose dependent. Quelation of extracellular calcium with EGTA prevented fluorescence increases in PMN exposed to 2 HU50/ml AH, but did not prevent a small increase in 4 μM, ionomycin-treated PMN, indicating that ionomycin treatment under conditions of calcium quelation can mobilize calcium from internal stores, and that entry of external calcium accounts for most of the increases in cell fluorescence in cells treated with both AH and calcium ionophores. AH, as well as calcium ionophores and the chemotactic peptide FMLP caused rease of myeloperoxidase (MPO) from PMM suggesting that increments in intracellular calcium cause degramulation with release of granule contents (Author) [es

Evolution of the Calcium Paradigm: The Relation between Vitamin D, Serum Calcium and Calcium Absorption

Directory of Open Access Journals (Sweden)

Borje E. Christopher Nordin

2010-09-01

Full Text Available Osteoporosis is the index disease for calcium deficiency, just as rickets/osteomalacia is the index disease for vitamin D deficiency, but there is considerable overlap between them. The common explanation for this overlap is that hypovitaminosis D causes malabsorption of calcium which then causes secondary hyperparathyroidism and is effectively the same thing as calcium deficiency. This paradigm is incorrect. Hypovitaminosis D causes secondary hyperparathyroidism at serum calcidiol levels lower than 60 nmol/L long before it causes malabsorption of calcium because serum calcitriol (which controls calcium absorption is maintained until serum calcidiol falls below 20 nmol/L. This secondary hyperparathyroidism, probably due to loss of a “calcaemic” action of vitamin D on bone first described in 1957, destroys bone and explains why vitamin D insufficiency is a risk factor for osteoporosis. Vitamin D thus plays a central role in the maintenance of the serum (ionised calcium, which is more important to the organism than the preservation of the skeleton. Bone is sacrificed when absorbed dietary calcium does not match excretion through the skin, kidneys and bowel which is why calcium deficiency causes osteoporosis in experimental animals and, by implication, in humans.

Calcium absorption

International Nuclear Information System (INIS)

Carlmark, B.; Reizenstein, P.; Dudley, R.A.

1976-01-01

The methods most commonly used to measure the absorption and retention of orally administered calcium are reviewed. Nearly all make use of calcium radioisotopes. The magnitude of calcium absorption and retention depends upon the chemical form and amount of calcium administered, and the clinical and nutritional status of the subject; these influences are briefly surveyed. (author)

Calcium channel alpha-2-delta-1 protein upregulation in dorsal spinal cord mediates spinal cord injury-induced neuropathic pain states.

Science.gov (United States)

Boroujerdi, Amin; Zeng, Jun; Sharp, Kelli; Kim, Donghyun; Steward, Oswald; Luo, Z David

2011-03-01

Spinal cord injury (SCI) commonly results in the development of neuropathic pain, which can dramatically impair the quality of life for SCI patients. SCI-induced neuropathic pain can be manifested as both tactile allodynia (a painful sensation to a non-noxious stimulus) and hyperalgesia (an enhanced sensation to a painful stimulus). The mechanisms underlying these pain states are poorly understood. Clinical studies have shown that gabapentin, a drug that binds to the voltage-gated calcium channel alpha-2-delta-1 subunit (Ca(v)α2δ-1) proteins is effective in the management of SCI-induced neuropathic pain. Accordingly, we hypothesized that tactile allodynia post SCI is mediated by an upregulation of Ca(v)α2δ-1 in dorsal spinal cord. To test this hypothesis, we examined whether SCI-induced dysregulation of spinal Ca(v)α2δ-1 plays a contributory role in below-level allodynia development in a rat spinal T9 contusion injury model. We found that Ca(v)α2δ-1 expression levels were significantly increased in L4-6 dorsal, but not ventral, spinal cord of SCI rats that correlated with tactile allodynia development in the hind paw plantar surface. Furthermore, both intrathecal gabapentin treatment and blocking SCI-induced Ca(v)α2δ-1 protein upregulation by intrathecal Ca(v)α2δ-1 antisense oligodeoxynucleotides could reverse tactile allodynia in SCI rats. These findings support that SCI-induced Ca(v)α2δ-1 upregulation in spinal dorsal horn is a key component in mediating below-level neuropathic pain states, and selectively targeting this pathway may provide effective pain relief for SCI patients. Spinal cord contusion injury caused increased calcium channel Ca(v)α2δ-1 subunit expression in dorsal spinal cord that contributes to neuropathic pain states. Copyright © 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

KAUST Repository

Schmöckel, Sandra M.

2015-02-27

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

Different NaCl-Induced Calcium Signatures in the Arabidopsis thaliana Ecotypes Col-0 and C24

KAUST Repository

Schmö ckel, Sandra M.; Garcia, Alexandre F.; Berger, Bettina; Tester, Mark A.; Webb, Alex A. R.; Roy, Stuart J.

2015-01-01

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

Phylogenetic diversity of Pasteurellaceae and horizontal gene transfer of leukotoxin in wild and domestic sheep.

Science.gov (United States)

Kelley, Scott T; Cassirer, E Frances; Weiser, Glen C; Safaee, Shirin

2007-01-01

Wild and domestic animal populations are known to be sources and reservoirs of emerging diseases. There is also a growing recognition that horizontal genetic transfer (HGT) plays an important role in bacterial pathogenesis. We used molecular phylogenetic methods to assess diversity and cross-transmission rates of Pasteurellaceae bacteria in populations of bighorn sheep, Dall's sheep, domestic sheep and domestic goats. Members of the Pasteurellaceae cause an array of deadly illnesses including bacterial pneumonia known as "pasteurellosis", a particularly devastating disease for bighorn sheep. A phylogenetic analysis of a combined dataset of two RNA genes (16S ribosomal RNA and RNAse P RNA) revealed remarkable evolutionary diversity among Pasteurella trehalosi and Mannheimia (Pasteurella) haemolytica bacteria isolated from sheep and goats. Several phylotypes appeared to associate with particular host species, though we found numerous instances of apparent cross-transmission among species and populations. Statistical analyses revealed that host species, geographic locale and biovariant classification, but not virulence, correlated strongly with Pasteurellaceae phylogeny. Sheep host species correlated with P. trehalosi isolates phylogeny (PTP test; P=0.002), but not with the phylogeny of M. haemolytica isolates, suggesting that P. trehalosi bacteria may be more host specific. With regards to populations within species, we also discovered a strong correlation between geographic locale and isolate phylogeny in the Rocky Mountain bighorn sheep (PTP test; P=0.001). We also investigated the potential for HGT of the leukotoxin A (lktA) gene, which produces a toxin that plays an integral role in causing disease. Comparative analysis of the combined RNA gene phylogeny and the lktA phylogenies revealed considerable incongruence between the phylogenies, suggestive of HGT. Furthermore, we found identical lktA alleles in unrelated bacterial species, some of which had been isolated

Radioisotope 45Ca labeling four calcium chemical compounds and tracing calcium bioavailability

International Nuclear Information System (INIS)

Zheng Hui; Zhen Rong; Niu Huisheng; Li Huaifen

2004-01-01

Objective: To build up a new method of the radioisotope 45 Ca labeling four calcium chemical compounds, observe and tracing bioavailability change of calcium labeled with radioisotope 45 Ca. Methods: The calcium gluconate (Ca-Glu), calcium citrate (Ca-Cit), calcium carbonate (Ca-Car) and calcium L-threonate (Ca-Thr)were labeled by radioisotope 45 Ca. Four calcium chemical compounds of 45 Ca labeling were used of calcium content 200 mg/kg in the rats and measure the absorption content and bioavailability of calcium in tissue of heart, lever spleen, stomach, kidney, brain, intestine, whole blood, urine, faeces. Results: 1) Radioisotope 45 Ca labeling calcium chemical compound has high radio intensity, more steady standard curve and recover rate. 2) The absorption of organic calcium chemical compounds is higher than the inorganic calcium chemical compound in the study of calcium bioavailability. Conclusion: The method of tracing with radioisotope 45 Ca labeling calcium chemical compounds has the characteristic of the sensitive, objective, accurate and steady in the study of calcium bioavailability

The impact of calcium assay change on a local adjusted calcium equation.

Science.gov (United States)

Davies, Sarah L; Hill, Charlotte; Bailey, Lisa M; Davison, Andrew S; Milan, Anna M

2016-03-01

Deriving and validating local adjusted calcium equations is important for ensuring appropriate calcium status classification. We investigated the impact on our local adjusted calcium equation of a change in calcium method by the manufacturer from cresolphthalein complexone to NM-BAPTA. Calcium and albumin results from general practice requests were extracted from the Laboratory Information Management system for a three-month period. Results for which there was evidence of disturbance in calcium homeostasis were excluded leaving 13,482 sets of results for analysis. The adjusted calcium equation was derived following least squares regression analysis of total calcium on albumin and normalized to the mean calcium concentration of the data-set. The revised equation (NM-BAPTA calcium method) was compared with the previous equation (cresolphthalein complexone calcium method). The switch in calcium assay resulted in a small change in the adjusted calcium equation but was not considered to be clinically significant. The calcium reference interval differed from that proposed by Pathology Harmony in the UK. Local adjusted calcium equations should be re-assessed following changes in the calcium method. A locally derived reference interval may differ from the consensus harmonized reference interval. © The Author(s) 2015.

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

International Nuclear Information System (INIS)

Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

2011-01-01

Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

Development of novel titanium nitride-based decorative coatings by calcium addition

Energy Technology Data Exchange (ETDEWEB)

Hodroj, A. [Institut Jean Lamour, CNRS UMR 7198, Departement CP2S, Ecole des Mines, Parc de Saurupt, CS 14234, 54042 Nancy cedex (France); Pierson, J.F., E-mail: jean-francois.pierson@ijl.nancy-universite.fr [Institut Jean Lamour, CNRS UMR 7198, Departement CP2S, Ecole des Mines, Parc de Saurupt, CS 14234, 54042 Nancy cedex (France)

2011-08-01

Calcium was added into titanium nitride coatings deposited using a hybrid magnetron sputtering-arc evaporation process. The calcium content in the films was adjusted by the variation of the pulsed DC current applied to the Ca sputtering target. X-ray diffraction analyses suggested that the increase of the calcium content induced the partial substitution of titanium atoms by calcium ones in the TiN lattice and a refinement of the grain size. Optical reflectance investigations showed that the absorption band of TiN was shifted towards higher wavelengths and that (Ti,Ca)N coatings may be suitable for decorative applications. Finally, the decrease of the film reflectivity was interpreted as a consequence of a free electron concentration decrease as confirmed from electrical resistivity measurements.

Development of novel titanium nitride-based decorative coatings by calcium addition

International Nuclear Information System (INIS)

Hodroj, A.; Pierson, J.F.

2011-01-01

Calcium was added into titanium nitride coatings deposited using a hybrid magnetron sputtering-arc evaporation process. The calcium content in the films was adjusted by the variation of the pulsed DC current applied to the Ca sputtering target. X-ray diffraction analyses suggested that the increase of the calcium content induced the partial substitution of titanium atoms by calcium ones in the TiN lattice and a refinement of the grain size. Optical reflectance investigations showed that the absorption band of TiN was shifted towards higher wavelengths and that (Ti,Ca)N coatings may be suitable for decorative applications. Finally, the decrease of the film reflectivity was interpreted as a consequence of a free electron concentration decrease as confirmed from electrical resistivity measurements.

Properties of calcium silicate-monobasic calcium phosphate materials for endodontics containing tantalum pentoxide and zirconium oxide.

Science.gov (United States)

Zamparini, Fausto; Siboni, Francesco; Prati, Carlo; Taddei, Paola; Gandolfi, Maria Giovanna

2018-05-08

The aim of the study was to evaluate chemical-physical properties and apatite-forming ability of three premixed calcium silicate materials containing monobasic calcium phosphate (CaH 4 P 2 O 8 ) bioceramic, tantalum pentoxide and zirconium oxide, recently marketed for endodontics (TotalFill BC-Sealer, BC-RRM-Paste, BC-RRM-Putty). Microchemical and micromorphological analyses, radiopacity, initial and final setting times, calcium release and alkalising activity were tested. The nucleation of calcium phosphates (CaPs) and/or apatite after 28 days ageing was evaluated by ESEM-EDX and micro-Raman spectroscopy. BC-Sealer and BC-RRM-Paste showed similar initial (23 h), prolonged final (52 h) setting times and good radiopacity (> 7 mm Al); BC-RRM-Putty showed fast initial (2 h) and final setting times (27 h) and excellent radiopacity (> 9 mm Al). All materials induced a marked alkalisation (pH 11-12) up to 28 days and showed the release of calcium ions throughout the entire test period (cumulative calcium release 641-806 ppm). After 28 days ageing, a well-distributed mineral layer was present on all samples surface; EDX demonstrated relevant calcium and phosphorous peaks. B-type carbonated apatite and calcite deposits were identified by micro-Raman spectroscopy on all the 28-day-aged samples; the deposit thickness was higher on BC-RRM-Paste and BC-RRM-Putty, in agreement with calcium release data. These materials met the required chemical and physical standards and released biologically relevant ions. The CaSi-CaH 4 P 2 O 8 system present in the materials provided Ca and OH ions release with marked abilities to nucleate a layer of B-type carbonated apatite favoured/accelerated by the bioceramic presence. The ability to nucleate apatite may lead many clinical advantages: In orthograde endodontics, it may improve the sealing ability by the deposition of CaPs at the material-root dentine interface, and in endodontic surgery, it could promote bone and

Calcium supplements

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... this page: //medlineplus.gov/ency/article/007477.htm Calcium supplements To use the sharing features on this page, please enable JavaScript. WHO SHOULD TAKE CALCIUM SUPPLEMENTS? Calcium is an important mineral for the ...

Rumen-protected rice bran to induce the adaptation of calcium metabolism in dairy cows

NARCIS (Netherlands)

Martín-Tereso López, J.

2010-01-01

Dairy cows suffer from hypocalcaemia in the days around calving, which may result in a condition generally known as milk fever. Calcium metabolism sharply shifts at the start of lactation, because Ca needs suddenly become much greater than at the end of gestation. Calcium metabolism is able to adapt

Calcium Nutrition and Extracellular Calcium Sensing: Relevance for the Pathogenesis of Osteoporosis, Cancer and Cardiovascular Diseases

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Peterlik, Meinrad; Kállay, Enikoe; Cross, Heide S.

2013-01-01

Through a systematic search in Pubmed for literature, on links between calcium malnutrition and risk of chronic diseases, we found the highest degree of evidence for osteoporosis, colorectal and breast cancer, as well as for hypertension, as the only major cardiovascular risk factor. Low calcium intake apparently has some impact also on cardiovascular events and disease outcome. Calcium malnutrition can causally be related to low activity of the extracellular calcium-sensing receptor (CaSR). This member of the family of 7-TM G-protein coupled receptors allows extracellular Ca2+ to function as a “first messenger” for various intracellular signaling cascades. Evidence demonstrates that Ca2+/CaSR signaling in functional linkage with vitamin D receptor (VDR)-activated pathways (i) promotes osteoblast differentiation and formation of mineralized bone; (ii) targets downstream effectors of the canonical and non-canonical Wnt pathway to inhibit proliferation and induce differentiation of colorectal cancer cells; (iii) evokes Ca2+ influx into breast cancer cells, thereby activating pro-apoptotic intracellular signaling. Furthermore, Ca2+/CaSR signaling opens Ca2+-sensitive K+ conductance channels in vascular endothelial cells, and also participates in IP3-dependent regulation of cytoplasmic Ca2+, the key intermediate of cardiomyocyte functions. Consequently, impairment of Ca2+/CaSR signaling may contribute to inadequate bone formation, tumor progression, hypertension, vascular calcification and, probably, cardiovascular disease. PMID:23340319

Influence of Secondary Hyperparathyroidism Induced by Low Dietary Calcium, Vitamin D Deficiency, and Renal Failure on Circulating Rat PTH Molecular Forms.

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D'Amour, Pierre; Rousseau, Louise; Hornyak, Stephen; Yang, Zan; Cantor, Tom

2011-01-01

Rats(r) with secondary hyperparathyroidism were studied to define the relationship between vitamin D metabolites and rPTH levels measured by 3 different rat ELISAs. Controls and renal failure (RF) rats were on a normal diet, while 2 groups on a low-calcium (-Ca) or a vitamin D-deficient (-D) diet. RF was induced surgically. Mild RF rats had normal calcium and 25(OH)D but reduced 1,25(OH)(2)D levels (P < .001) with a 2.5-fold increased in rPTH (P < .001). Severe RF rats and those on a -Ca or -D diet had reduced calcium (P < .01) and 25(OH)D levels (P < .05), with rPTH increased by 2 (-Ca diet; P < .05), 4 (-D diet; P < .001), and 20-folds (RF; P < .001) while 1,25(OH)(2)D was high (-Ca diet: P < .001) or low (-D diet, RF: P < .001). 25(OH)D and 1,25(OH)(2)D were positively and negatively related on the -Ca and -D diets, respectively. rPTH molecular forms behaved as expected in RF and on -Ca diet, but not on -D diet with more C-rPTH fragments when less were expected. This may be related to the short-time course of this study compared to prior studies.

Neuroprotective effect of gadolinium: a stretch-activated calcium channel blocker in mouse model of ischemia-reperfusion injury.

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Gulati, Puja; Muthuraman, Arunachalam; Jaggi, Amteshwar S; Singh, Nirmal

2013-03-01

The present study was designed to investigate the potential of gadolinium, a stretch-activated calcium channel blocker in ischemic reperfusion (I/R)-induced brain injury in mice. Bilateral carotid artery occlusion of 12 min followed by reperfusion for 24 h was given to induce cerebral injury in male Swiss mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was assessed using Morris water maze test and motor incoordination was evaluated using rota-rod, lateral push, and inclined beam walking tests. In addition, total calcium, thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), and acetylcholinesterase (AChE) activity were also estimated in brain tissue. I/R injury produced a significant increase in cerebral infarct size. A significant loss of memory along with impairment of motor performance was also noted. Furthermore, I/R injury also produced a significant increase in levels of TBARS, total calcium, AChE activity, and a decrease in GSH levels. Pretreatment of gadolinium significantly attenuated I/R-induced infarct size, behavioral and biochemical changes. On the basis of the present findings, we can suggest that opening of stretch-activated calcium channel may play a critical role in ischemic reperfusion-induced brain injury and that gadolinium has neuroprotective potential in I/R-induced injury.

Absorbability of calcium from calcium-bound phosphoryl oligosaccharides in comparison with that from various calcium compounds in the rat ligated jejunum loop.

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To-o, Kenji; Kamasaka, Hiroshi; Nishimura, Takahisa; Kuriki, Takashi; Saeki, Shigeru; Nakabou, Yukihiro

2003-08-01

Calcium-bound phosphoryl oligosaccharides (POs-Ca) were prepared from potato starch. Their solubility and in situ absorbability as a calcium source were investigated by comparing with the soluble calcium compounds, calcium chloride and calcium lactate, or insoluble calcium compounds, calcium carbonate and dibasic calcium phosphate. The solubility of POs-Ca was as high as that of calcium chloride and about 3-fold higher than that of calcium lactate. An in situ experiment showed that the intestinal calcium absorption rate of POs-Ca was almost comparable with that of the soluble calcium compounds, and was significantly higher (pcalcium groups. Moreover, the total absorption rate of a 1:1 mixture of the calcium from POs-Ca and a whey mineral complex (WMC) was significantly higher (psoluble calcium source with relatively high absorption in the intestinal tract.

Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes

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Petrovič, Pavol; Valent, Ivan; Cocherová, Elena; Pavelková, Jana

2015-01-01

The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases. PMID:26009544

Comparative evaluation of different forms of calcium hydroxide in apexification

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Subhankar Ghosh

2014-01-01

Full Text Available Background: One out of every two children sustains a dental injury most often between 8 and 10 years of age. Majority of these teeth subsequently become non-vital and most often with immature apex. Management of these teeth is an enormous challenge for lack of apical stop. Calcium hydroxide in various formulations has maximum literature support in favor of "successful apexification or induced apical closure." Aim: The aim of the following study is to determine the efficacy of calcium hydroxide in a different formulation to induce apexification. Materials and Methods: The present study was undertaken on 51 children of 8-10 years of age (both sexes at Dr. R Ahmed Dental College and Hospital from April 2006 to March 2007. All children had one or two maxillary permanent central incisor (s, non-vital and apices open. In all the cases, apexification was attempted with either calcium hydroxide mixed with sterile distilled water, or calcium hydroxide plus iodoform in methyl cellulose base, or calcium hydroxide plus iodoform in polysilicone oil base. The success of apexification was determined on the basis of clinical and radiographic criteria. Results: In the pre-operative asymptomatic cases (72.55%, failure occurred in only 5.45% cases and pre-operative symptomatic cases failure rate was as high as 35.71%. Success rate was 94.6% in cases with narrow open apices, whereas 64.28% in wide open apices. In cases with pre-existing apical radiolucencies, successful apexification occurred in 63.63% and success rate was 92.5% in the cases without pre-existing apical radiolucencies. Average time consumed for apexification was minimum with calcium hydroxide plus iodoform in polysilicone oil base. Conclusion: The overall success rate observed to be 86.27%, which is in close proximity to the findings of most of the previous studies across the globe.

Comparative evaluation of different forms of calcium hydroxide in apexification.

Science.gov (United States)

Ghosh, Subhankar; Mazumdar, Dibyendu; Ray, Pradip Kumar; Bhattacharya, Bhaswar

2014-01-01

One out of every two children sustains a dental injury most often between 8 and 10 years of age. Majority of these teeth subsequently become non-vital and most often with immature apex. Management of these teeth is an enormous challenge for lack of apical stop. Calcium hydroxide in various formulations has maximum literature support in favor of successful apexification or induced apical closure. The aim of the following study is to determine the efficacy of calcium hydroxide in a different formulation to induce apexification. The present study was undertaken on 51 children of 8-10 years of age (both sexes) at Dr. R Ahmed Dental College and Hospital from April 2006 to March 2007. All children had one or two maxillary permanent central incisor (s), non-vital and apices open. In all the cases, apexification was attempted with either calcium hydroxide mixed with sterile distilled water, or calcium hydroxide plus iodoform in methyl cellulose base, or calcium hydroxide plus iodoform in polysilicone oil base. The success of apexification was determined on the basis of clinical and radiographic criteria. In the pre-operative asymptomatic cases (72.55%), failure occurred in only 5.45% cases and pre-operative symptomatic cases failure rate was as high as 35.71%. Success rate was 94.6% in cases with narrow open apices, whereas 64.28% in wide open apices. In cases with pre-existing apical radiolucencies, successful apexification occurred in 63.63% and success rate was 92.5% in the cases without pre-existing apical radiolucencies. Average time consumed for apexification was minimum with calcium hydroxide plus iodoform in polysilicone oil base. The overall success rate observed to be 86.27%, which is in close proximity to the findings of most of the previous studies across the globe.

Effect of tocotrienol from Bixa orellana (annatto on bone microstructure, calcium content, and biomechanical strength in a model of male osteoporosis induced by buserelin

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Mohamad NV

2018-03-01

Full Text Available Nur-Vaizura Mohamad, Soelaiman Ima-Nirwana, Kok-Yong Chin Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Background: Patients receiving androgen deprivation therapy experience secondary hypogonadism, associated bone loss, and increased fracture risk. It has been shown that tocotrienol from Bixa orellana (annatto prevents skeletal microstructural changes in rats experiencing primary hypogonadism. However, its potential in preventing bone loss due to androgen deprivation therapy has not been tested. This study aimed to evaluate the skeletal protective effects of annatto tocotrienol using a buserelin-induced osteoporotic rat model. Methods: Forty-six male Sprague Dawley rats aged 3 months were randomized into six groups. The baseline control (n=6 was sacrificed at the onset of the study. The normal control (n=8 received corn oil (the vehicle of tocotrienol orally daily and normal saline (the vehicle of buserelin subcutaneously daily. The buserelin control (n=8 received corn oil orally daily and subcutaneous buserelin injection (75 µg/kg daily. The calcium control (n=8 was supplemented with 1% calcium in drinking water and daily subcutaneous buserelin injection (75 µg/kg. The remaining rats were given daily oral annatto tocotrienol at 60 mg/kg (n=8 or 100 mg/kg (n=8 plus daily subcutaneous buserelin injection (75 µg/kg (n=8. At the end of the experiment, the rats were euthanized and their blood, tibia, and femur were harvested. Structural changes of the tibial trabecular and cortical bone were examined using X-ray micro-computed tomography. Femoral bone calcium content and biomechanical strength were also evaluated. Results: Annatto tocotrienol at 60 and 100 mg/kg significantly prevented the deterioration of trabecular bone and cortical thickness in buserelin-treated rats (P<0.05. Both doses of annatto tocotrienol also improved femoral biomechanical strength and bone calcium content

Hydrogen peroxide-induced DNA damage is independent of nuclear calcium but dependent on redox-active ions.

Science.gov (United States)

Jornot, L; Petersen, H; Junod, A F

1998-01-01

In cells undergoing oxidative stress, DNA damage may result from attack by .OH radicals produced by the Fenton reaction, and/or by nucleases activated by nuclear calcium. In the present study, the participation of these two mechanisms was investigated in HeLa cells. Nuclear-targeted aequorin was used for selectively monitoring Ca2+ concentrations within the nuclei ([Ca2+]n), in conjunction with the cell-permeant calcium chelator bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), the lipid-soluble broad-spectrum metal chelator with low affinity for Ca2+ and Mg2+ N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and the high-affinity iron/copper chelator 1, 10-phenanthroline (PHE). In Ca2+-containing medium, H2O2 induced extensive DNA strand breaks and an increase in [Ca2+]n that was almost identical to that observed in the cytosol ([Ca2+]c). In cells bathed in Ca2+-free/EGTA medium, in which the increases in [Ca2+]n and [Ca2+]c due to H2O2 were significantly reduced, similar levels of DNA fragmentation also occurred. In cells preloaded with BAPTA/AM or TPEN, the small increase of [Ca2+]n normally elicited by H2O2 in Ca2+-free medium was completely buffered, and DNA damage was largely prevented. On the other hand, pretreatment with PHE did not affect the calcium response in the nuclei, but completely prevented DNA strand breakage induced by H2O2. Re-addition of 100 microM CuSO4 and 100 microM FeSO4 to TPEN- and PHE-treated cells prior to H2O2 challenge reversed the effect of TPEN and PHE, whereas 1 mM was necessary to negate the effect of BAPTA/AM. The levels of DNA strand breakage observed, however, did not correlate with the amounts of 8-hydroxy 2'-deoxyguanosine (8-OHdG): H2O2 did not produce 8-OHdG, whereas PHE alone slightly increased 8-OHdG levels. CuSO4 and FeSO4 enhanced the effects of PHE, particularly in the presence of H2O2. Exposure of cells to a mixture of CuSO4/FeSO4 also resulted in a significant increase in

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

Science.gov (United States)

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.

Differential expression of interleukin-8 by polymorphonuclear leukocytes of two closely related species, Ovis canadensis and Ovis aries, in response to Mannheimia haemolytica infection.

Science.gov (United States)

Herndon, Caroline N; Foreyt, William J; Srikumaran, Subramaniam

2010-08-01

The pneumonic lesions and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS; Ovis canadensis) are more severe than those in the related species, domestic sheep (DS; Ovis aries), under both natural and experimental conditions. Leukotoxin (Lkt) and lipopolysaccharide (LPS) are the most important virulence factors of this organism. One hallmark of pathogenesis of pneumonia is the influx of polymorphonuclear leukocytes (PMNs) into the lungs. Lkt-induced cytolysis of PMNs results in the release of cytotoxic compounds capable of damaging lung tissue. Interleukin-8 (IL-8) is a potent PMN chemoattractant. The objective of the present study was to determine if there is differential expression of IL-8 by the macrophages and PMNs of BHS and DS in response to M. haemolytica. Macrophages and PMNs of BHS and DS were stimulated with heat-killed M. haemolytica or LPS. IL-8 expression by the cells was measured by enzyme-linked immunosorbent assays and real-time reverse transcription-PCR (RT-PCR). The PMNs of BHS expressed severalfold higher levels of IL-8 than those of DS upon stimulation. Lesional lung tissue of M. haemolytica-infected BHS contained significantly higher levels of IL-8 than nonlesional tissue. The bronchoalveolar lavage (BAL) fluid of infected BHS also contained higher levels of IL-8 than that of infected DS. Depletion of IL-8 reduced migration of PMNs toward BAL fluid by approximately 50%, indicating that IL-8 is integral to PMN recruitment to the lung during M. haemolytica infection. Excessive production of IL-8, enhanced recruitment of PMNs, and PMN lysis by Lkt are likely responsible for the severity of the lung lesions in M. haemolytica-infected BHS.

Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor

International Nuclear Information System (INIS)

Imai, Y.; Singer, M.S.; Fennie, C.; Lasky, L.A.; Rosen, S.D.

1991-01-01

Lymphocyte attachment to high endothelial venules within lymph nodes is mediated by the peripheral lymph node homing receptor (pnHR), originally defined on mouse lymphocytes by the MEL-14 mAb. The pnHR is a calcium-dependent lectin-like receptor, a member of the LEC-CAM family of adhesion proteins. Here, using a soluble recombinant form of the homing receptor, we have identified an endothelial ligand for the pnHR as an ∼ 50-kD sulfated, fucosylated, and sialylated glycoprotein, which we designate Sgp50 (sulfated glycoprotein of 50 kD). Recombinant receptor binding to this lymph node-specific glycoprotein requires calcium and is inhibitable by specific carbohydrates and by MEL-14 mAb. Sialylation of the component is required for binding. Additionally, the glycoprotein is precipitated by MECA-79, an adhesion-blocking mAb reactive with lymph node HEV. A related glycoprotein of ∼ 90 kD (designated as Sgp90) is also identified

PeakCaller: an automated graphical interface for the quantification of intracellular calcium obtained by high-content screening.

Science.gov (United States)

Artimovich, Elena; Jackson, Russell K; Kilander, Michaela B C; Lin, Yu-Chih; Nestor, Michael W

2017-10-16

Intracellular calcium is an important ion involved in the regulation and modulation of many neuronal functions. From regulating cell cycle and proliferation to initiating signaling cascades and regulating presynaptic neurotransmitter release, the concentration and timing of calcium activity governs the function and fate of neurons. Changes in calcium transients can be used in high-throughput screening applications as a basic measure of neuronal maturity, especially in developing or immature neuronal cultures derived from stem cells. Using human induced pluripotent stem cell derived neurons and dissociated mouse cortical neurons combined with the calcium indicator Fluo-4, we demonstrate that PeakCaller reduces type I and type II error in automated peak calling when compared to the oft-used PeakFinder algorithm under both basal and pharmacologically induced conditions. Here we describe PeakCaller, a novel MATLAB script and graphical user interface for the quantification of intracellular calcium transients in neuronal cultures. PeakCaller allows the user to set peak parameters and smoothing algorithms to best fit their data set. This new analysis script will allow for automation of calcium measurements and is a powerful software tool for researchers interested in high-throughput measurements of intracellular calcium.

Get Enough Calcium

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... Calcium Print This Topic En español Get Enough Calcium Browse Sections The Basics Overview Foods and Vitamins ... women, don't get enough calcium. How much calcium do I need every day? Women: If you ...

Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton.

Science.gov (United States)

Pritchard, Scott; Erickson, Geoffrey R; Guilak, Farshid

2002-11-01

Loading of the spine alters the osmotic environment in the intervertebral disk (IVD) as interstitial water is expressed from the tissue. Cells from the three zones of the IVD, the anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP), respond to osmotic stress with altered biosynthesis through a pathway that may involve calcium (Ca(2+)) as a second messenger. We examined the hypothesis that IVD cells respond to hyperosmotic stress by increasing the concentration of intracellular calcium ([Ca(2+)](i)) through a mechanism involving F-actin. In response to hyperosmotic stress, control cells from all zones decreased in volume and cells from the AF and TZ exhibited [Ca(2+)](i) transients, while cells from the NP did not. Extracellular Ca(2+) was necessary to initiate [Ca(2+)](i) transients. Stabilization of F-actin with phalloidin prevented the Ca(2+) response in AF and TZ cells and decreased the rate of volume change in cells from all zones, coupled with an increase in the elastic moduli and apparent viscosity. Conversely, actin breakdown with cytochalasin D facilitated Ca(2+) signaling while decreasing the elastic moduli and apparent viscosity for NP cells. These results suggest that hyperosmotic stress induces volume change in IVD cells and may initiate [Ca(2+)](i) transients through an actin-dependent mechanism.

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress.

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Mauricio Torres

2010-12-01

Full Text Available Prion-related disorders (PrDs are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES. Altered endoplasmic reticulum (ER homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES. Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.

The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation

International Nuclear Information System (INIS)

Garcia-Rates, Sara; Camarasa, Jordi; Sanchez-Garcia, Ana I.; Gandia, Luis; Escubedo, Elena; Pubill, David

2010-01-01

Previous work by our group demonstrated that homomeric α7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca 2+ increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC 50 values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca 2+ release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca 2+ levels and induced an increase in α-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca 2+ -dependent enzymes such as

The Effects of Dietary Calcium and/or Iron Deficiency upon Murine Intestinal Calcium Binding Protein Activity and Calcium Absorption

OpenAIRE

McDonald, Catherine M.

1980-01-01

Iron deficiency has been shown to impair calcium absorption, leading to decreased bone mass. Vitamin D3-dependent calcium binding protein (CaBP) has been demonstrated to be necessary for the active transport of calcium in the intestine of numerous species. Iron deficiency might affect the activity of the calcium binding protein. Four experimental diets were formulated as follows: Diet 1, iron adequate, calcium adequate; Diet 2, iron deficient, calcium adequate; Diet 3, iron adequate, calci...

Effect of lowering dietary calcium intake on fractional whole body calcium retention

International Nuclear Information System (INIS)

Dawson-Hughes, B.; Stern, D.T.; Shipp, C.C.; Rasmussen, H.M.

1988-01-01

Although fractional calcium absorption is known to vary inversely with calcium intake, the extent and timing of individual hormonal and calcium absorption responses to altered calcium intake have not been defined. We measured fractional whole body retention of orally ingested 47 Ca, an index of calcium absorption, in nine normal women after they had eaten a 2000-mg calcium diet for 8 weeks and a 300-mg calcium diet for 1, 2, 4, and 8 weeks. After the diet change, serum intact PTH (32.2% increase; P = 0.005), serum 1,25-dihydroxyvitamin D [1,25-(OH)2D; 43.8% increase; P = 0.003], and fractional whole body calcium retention (42.8% increase; P = 0.004) increased within 1 week. Although the PTH and calcium retention responses remained fairly constant throughout the low calcium intake period, serum 1,25-(OH)2D concentrations declined toward baseline after week 1. Thus, the late increase in calcium retention may have resulted from calcium absorption that was independent of 1,25-(OH)2D stimulation

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

Energy Technology Data Exchange (ETDEWEB)

Ginkel, Paul R. van; Yan, Michael B. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Bhattacharya, Saswati [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Pediatrics, University of Wisconsin, Madison, WI 53792 (United States); Polans, Arthur S., E-mail: aspolans@wisc.edu [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Kenealey, Jason D. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Nutrition, Dietetics and Food Science, Brigham Young University, Provo, UT 84602 (United States)

2015-11-01

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 cells 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.

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

International Nuclear Information System (INIS)

Ginkel, Paul R. van; Yan, Michael B.; Bhattacharya, Saswati; Polans, Arthur S.; Kenealey, Jason D.

2015-01-01

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 cells 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 3 pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca 2+ -dependent pro-apoptotic pathways inhibit cancer cell growth.

Myeloablative radioimmunotherapy with 188Re-CD66mAb before stem cell transplantation. No increase of proinflammatory cytokine levels of TNF-α

International Nuclear Information System (INIS)

Mutschler, J.; Reske, S.N.; Steinbach, G.; Bunjes, D.; Buchmann, I.

2009-01-01

Tumour necrosis factor-α (TNF-α) serum levels may increase due to intensive conditioning regimes with high-dose chemotherapy and total body irradiation (TBI) before stem cell transplantation. This increases the risk for developing acute graft versus host disease (aGvHD) after stem cell transplantation. In this prospective study we investigated the influence of radioimmunotherapy with 188 Re-CD-66-mAb on changes on TNF-α serum levels. Patients, methods: In 18 patients we measured TNF-α before and up to 96 hours after radioimmunotherapy, in 2 patients in addition following TBI, in 9 patients also following chemotherapy. For measuring TNF-α we used an automated immunochemiluminescence assay (Immulite 1000 DPC Biermann, Bad Nauheim). The mean follow up period to record incidence of aGVHD was 100 days after stem cell transplantation. Compared to the basal levels before, the levels of TNF-α after conditioning with 188 Re-CD-66-mAb did not increase significantly and remained in the physiological range. In contrast, these initial physiological cytokine levels increased and became pathological following 48 h after total body irradiation (13.2 ± 6.6 pg/ml) and chemotherapy (10.8 ± 15.7 pg/ml). In our study we found a low incidence of aGvHD (22.2%, n = 4/18). Conclusion: These results demonstrate that additional conditioning therapy with 188 Re-CD-66-mAb does not increase proinflammatory cytokine levels of TNF-α. This finding may indicate that additive radioimmunotherapy may not be a significant factor for increasing the rate of conditioning- associated aGvHD. (orig.)

CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling.

Science.gov (United States)

Manara, Maria Cristina; Terracciano, Mario; Mancarella, Caterina; Sciandra, Marika; Guerzoni, Clara; Pasello, Michela; Grilli, Andrea; Zini, Nicoletta; Picci, Piero; Colombo, Mario P; Morrione, Andrea; Scotlandi, Katia

2016-11-29

CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.

Comparative evaluation of different forms of calcium hydroxide in apexification

OpenAIRE

Subhankar Ghosh; Dibyendu Mazumdar; Pradip Kumar Ray; Bhaswar Bhattacharya

2014-01-01

Background: One out of every two children sustains a dental injury most often between 8 and 10 years of age. Majority of these teeth subsequently become non-vital and most often with immature apex. Management of these teeth is an enormous challenge for lack of apical stop. Calcium hydroxide in various formulations has maximum literature support in favor of "successful apexification or induced apical closure." Aim: The aim of the following study is to determine the efficacy of calcium hydroxid...

Calcium hydroxide isotope effect in calcium isotope enrichment by ion exchange

International Nuclear Information System (INIS)

Jepson, B.E.; Shockey, G.C.

1984-01-01

The enrichment of calcium isotopes has been observed in ion-exchange chromatography with an aqueous phase of calcium hydroxide and a solid phase of sulfonic acid resin. The band front was exceedingly sharp as a result of the acid-base reaction occuring at the front of the band. Single-stage separation coefficients were found to be epsilon( 44 Ca/ 40 Ca) = 11 x 10 -4 and epsilon( 48 Ca/ 40 Ca) = 18 x 10 -4 . The maximum column separation factors achieved were 1.05 for calcium-44 and 1.09 for calcium-48 with the heavy isotopes enriching in the fluid phase. The calcium isotope effect between fully hydrated aqueous calcium ions and undissociated aqueous calcium hydroxide was estimated. For the calcium-44/40 isotope pair the separation coefficient was 13 x 10 -4 . 20 references, 2 figures

Interactions of Mitochondria/Metabolism and Calcium Regulation in Alzheimer's Disease: A Calcinist Point of View.

Science.gov (United States)

Gibson, Gary E; Thakkar, Ankita

2017-06-01

Decades of research suggest that alterations in calcium are central to the pathophysiology of Alzheimer's Disease (AD). Highly reproducible changes in calcium dynamics occur in cells from patients with both genetic and non-genetic forms of AD relative to controls. The most robust change is an exaggerated release of calcium from internal stores. Detailed analysis of these changes in animal and cell models of the AD-causing presenilin mutations reveal robust changes in ryanodine receptors, inositol tris-phosphate receptors, calcium leak channels and store activated calcium entry. Similar anomalies in calcium result when AD-like changes in mitochondrial enzymes or oxidative stress are induced experimentally. The calcium abnormalities can be directly linked to the altered tau phosphorylation, amyloid precursor protein processing and synaptic dysfunction that are defining features of AD. A better understanding of these changes is required before using calcium abnormalities as therapeutic targets.

Evaluation of cellular influences caused by calcium carbonate nanoparticles.

Science.gov (United States)

Horie, Masanori; Nishio, Keiko; Kato, Haruhisa; Endoh, Shigehisa; Fujita, Katsuhide; Nakamura, Ayako; Kinugasa, Shinichi; Hagihara, Yoshihisa; Yoshida, Yasukazu; Iwahashi, Hitoshi

2014-03-05

The cellular effects of calcium carbonate (CaCO₃) nanoparticles were evaluated. Three kinds of CaCO₃ nanoparticles were employed in our examinations. One of the types of CaCO₃ nanoparticles was highly soluble. And solubility of another type of CaCO₃ nanoparticle was lower. A stable CaCO₃ nanoparticle medium dispersion was prepared and applied to human lung carcinoma A549 cells and human keratinocyte HaCaT cells. Then, mitochondrial activity, cell membrane damage, colony formation ability, DNA injury, induction of oxidative stress, and apoptosis were evaluated. Although the influences of CaCO₃ nanoparticles on mitochondrial activity and cell membrane damage were small, "soluble" CaCO₃ nanoparticles exerted some cellular influences. Soluble CaCO₃ nanoparticles also induced a cell morphological change. Colony formation was inhibited by CaCO₃ nanoparticle exposure. In particular, soluble CaCO₃ nanoparticles completely inhibited colony formation. The influence on intracellular the reactive oxygen species (ROS) level was small. Soluble CaCO₃ nanoparticles caused an increase in C/EBP-homologous protein (CHOP) expression and the activation of caspase-3. Moreover, CaCO₃ exposure increased intracellular the Ca²⁺ level and activated calpain. These results suggest that cellular the influences of CaCO₃ nanoparticles are mainly caused by intracellular calcium release and subsequently disrupt the effect of calcium signaling. In conclusion, there is possibility that soluble CaCO₃ nanoparticles induce cellular influences such as a cell morphological change. Cellular influence of CaCO₃ nanoparticles is caused by intracellular calcium release. If inhaled CaCO₃ nanoparticles have the potential to influence cellular events. However, the effect might be not severe because calcium is omnipresent element in cell. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Intracellular calcium mobilization in human lymphocytes in the presence of synthetic IgG Fc peptides

International Nuclear Information System (INIS)

Plummer, J.M.; Panahi, Y.P.; McClurg, M.R.; Hahn, G.S.; Naemura, J.R.

1986-01-01

Certain synthetic peptides derived from the Fc region of human IgG can suppress the mixed lymphocyte response. These peptides were tested for the ability to induce intracellular calcium mobilization in human lymphocytes using fura-2/calcium fluorescence. T cells were isolated by rosetting and were > 90% OKT3 positive. Lymphocytes were incubated with the acetoxymethyl ester of fura-2 (10 μM) for 60 minutes at 37 0 C. Fluorescence intensity changes at 505 nm were monitored at an excitation lambda of 340 nm. Fura-2 was not cytotoxic compared to quin-2 since fura-2 loaded mononuclear cells incorporated 3 H-thymidine when stimulated by PHA, succinyl Con A, PWM or LPS-STM whereas quin-2 loaded cells showed a dose dependent inhibition of proliferation. Those synthetic peptides (5 to 400 μg/ml) that suppressed the MLR induced a dose dependent increase in intracellular calcium in mononuclear cells, lymphocytes, non-T cells and T cells. The fura-2 calcium fluorescence time course response was similar for peptide, PHA and succinyl Con A. These results suggest that these immunoregulatory peptides suppress 3 H-thymidine incorporation at a point after intracellular calcium mobilization and that fura-2 has advantages over quin-2 in measuring intracellular calcium levels in lymphocytes

Viral aggregating and opsonizing activity in collectin trimers

DEFF Research Database (Denmark)

Hartshorn, Kevan L; White, Mitchell R; Tecle, Tesfaldet

2010-01-01

of bovine serum collectin CL-46 induces aggregation of influenza A virus (IAV) and potently increases IAV uptake by neutrophils. CL-46-NCRD showed calcium-dependent and sugar-sensitive binding to both neutrophils and IAV. Replacement of specific residues of the CRD of human SP-D with those found in bovine...... were further increased by cross-linking of hSP-D-NCRD/R343V trimers with mAbs directed against areas of the hSP-D-NCRD not involved in viral binding. Unlike the wild-type human SP-D hSP-D-NCRD, hSP-D-NCRD/R343V also induced viral aggregation. These results indicate that collectins can act as opsonins...

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8+ T cell responses in mice

Science.gov (United States)

Zhou, Weibin; Moguche, Albanus; Chiu, David; Murali-Krishna, Kaja; Baneyx, François

2014-01-01

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration “cold chain”. Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8+ T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8+ T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. PMID:24275478

Calcium

Science.gov (United States)

... You can get decent amounts of calcium from baked beans, navy beans, white beans, and others. Canned fish. You're in luck if you like sardines and canned salmon with bones. Almond milk. Working Calcium Into Your ...

Influence of dietary calcium on bone calcium utilization

International Nuclear Information System (INIS)

Farmer, M.; Roland, D.A. Sr.; Clark, A.J.

1986-01-01

In Experiment 1, 10 microCi 45 Ca/day were administered to 125 hens for 10 days. Hens were then allocated to five treatments with calcium levels ranging from .08 to 3.75% of the diet. In Experiment 2, hens with morning oviposition times were randomly allocated to 11 treatments that were periods of time postoviposition ranging from 6 hr to 24 hr, in 2-hr increments (Experiment 2). At the end of each 2-hr period, eggs from 25 hens were removed from the uterus. The 18-, 20-, and 22-hr treatments were replicated three times. In Experiment 3, hens were fed either ad libitum or feed was withheld the last 5 or 6 hr before oviposition. In Experiment 4, hens were fed 10 microCi of 45 Ca for 15 days to label skeletal calcium. Hens were divided into two groups and fed a .08 or 3.75% calcium diet for 2 days. On the second day, 25 hens fed the 3.75% calcium diet were intubated with 7 g of the same diet containing .5 g calcium at 1700, 2100, 0100, 0500, and 0700 hr. The measurements used were egg weight, shell weight, and 45 Ca content of the egg shell. Results indicated a significant linear or quadratic regression of dietary calcium levels on 45 Ca accumulation in eggshells and eggshell weight (Experiment 1). As the calcium level of the diet increased, eggshell weight increased and 45 Ca recovery decreased. Utilization of skeletal calcium for shell formation ranged from 28 to 96%. In Experiment 2, the rate of shell calcification was not constant throughout the calcification process but varied significantly

Calcium D-saccharate

DEFF Research Database (Denmark)

Garcia, André Castilho; Hedegaard, Martina Vavrusova; Skibsted, Leif Horsfelt

2016-01-01

Molar conductivity of saturated aqueous solutions of calcium d-saccharate, used as a stabilizer of beverages fortified with calcium d-gluconate, increases strongly upon dilution, indicating complex formation between calcium and d-saccharate ions, for which, at 25 °C, Kassoc = 1032 ± 80, ΔHassoc......° = -34 ± 6 kJ mol-1, and ΔSassoc° = -55 ± 9 J mol-1 K-1, were determined electrochemically. Calcium d-saccharate is sparingly soluble, with a solubility product, Ksp, of (6.17 ± 0.32) × 10-7 at 25 °C, only moderately increasing with the temperature: ΔHsol° = 48 ± 2 kJ mol-1, and ΔSassoc° = 42 ± 7 J mol-1...... K-1. Equilibria in supersaturated solutions of calcium d-saccharate seem only to adjust slowly, as seen from calcium activity measurements in calcium d-saccharate solutions made supersaturated by cooling. Solutions formed by isothermal dissolution of calcium d-gluconate in aqueous potassium d...

Acute Treatment with T-Type Calcium Channel Enhancer SAK3 Reduces Cognitive Impairments Caused by Methimazole-Induced Hypothyroidism Via Activation of Cholinergic Signaling.

Science.gov (United States)

Husain, Noreen; Yabuki, Yasushi; Shinoda, Yasuharu; Fukunaga, Kohji

2018-01-01

Hypothyroidism is a common disorder that is associated with psychological disturbances such as dementia, depression, and psychomotor disorders. We recently found that chronic treatment with the T-type calcium channel enhancer SAK3 prevents the cholinergic neurodegeneration induced by a single intraperitoneal (i.p.) injection of methimazole (MMI; 75 mg/kg), thereby improving cognition. Here, we evaluated the acute effect of SAK3 on cognitive impairments and its mechanism of action following the induction of hypothyroidism. Hypothyroidism was induced by 2 injections of MMI (75 mg/kg, i.p.) administered once per week. Four weeks after the final MMI treatment, MMI-treated mice showed reduced serum thyroxine (T4) levels and cognitive impairments without depression-like behaviors. Although acute SAK3 (1.0 mg/kg, p.o.) administration failed to ameliorate the decreased T4 levels and histochemical destruction of the glomerular structure, acute SAK3 (1.0 mg/kg, p.o.) administration significantly reduced cognitive impairments in MMI-treated mice. Importantly, the α7 nicotinic acetylcholine receptor (nAChR)-selective inhibitor methyllycaconitine (MLA; 12 mg/kg, i.p.) and T-type calcium channel-specific blocker NNC 55-0396 (25 mg/kg, i.p.) antagonized the acute effect of SAK3 on memory deficits in MMI-treated mice. We also confirmed that acute SAK3 administration does not rescue reduced olfactory marker protein or choline acetyltransferase immunoreactivity levels in the olfactory bulb or medial septum. Taken together, these results suggest that SAK3 has the ability to improve the cognitive decline caused by hypothyroidism directly through activation of nAChR signaling and T-type calcium channels. © 2018 S. Karger AG, Basel.

Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

Science.gov (United States)

Ludwar, Bjoern Ch; Evans, Colin G; Cropper, Elizabeth C

2012-07-15

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation). These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3). Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record

Modulation of intracellular calcium homeostasis by trimethyltin chloride in human tumour cells: Neuroblastoma SY5Y and cervix adenocarcinoma HeLa S3

International Nuclear Information System (INIS)

Florea, Ana-Maria; Splettstoesser, Frank; Dopp, Elke; Rettenmeier, Albert W.; Buesselberg, Dietrich

2005-01-01

Physiological modifications of intracellular Ca 2+ ([Ca 2+ ] i ) levels trigger and/or regulate a diversity of cellular activities (e.g. neurotransmitter release, synaptic plasticity, muscular contraction, cell proliferation), while calcium overloads could result in cytotoxicity. Previously, we have shown that trimethyltin chloride (Me 3 SnCl; TMT) modulates calcium homeostasis in cervix adenocarcinoma (HeLa S3) cells [Florea, A.-M., Dopp, E., Buesselberg, D., 2005. TMT induces elevated calcium transients in HeLa cells: types and levels of response. Cell Calcium 37, 252-258]. Here we compare [Ca 2+ ] i -changes induced by trimethyltin chloride in neuroblastoma SY5Y and HeLa S3 cells using calcium-sensitive dyes (fluo-4/AM (fluo-4) and rhod-2/AM (rhod-2)) and laser scanning microscopy (LSM). TMT-induced calcium elevations in neuroblastoma SY5Y as well as in HeLa S3 cells. [Ca 2+ ] i rose to a sustained plateau or to transient spikes. Overall, the detected averaged increase of the maximum calcium elevation were: 0.5 μM ∼125.6%; 5 μM ∼130.1%; 500 μM ∼145% in HeLa S3 cells and 0.5 μM ∼133.3%; 5 μM ∼136.1%; 500 μM ∼147.1% in neuroblastoma SY5Y cells. The calcium rise derived from internal stores did not significantly depend on the presence of calcium in the external solution: ∼109% (no calcium added) versus ∼117% (2 mM calcium; 5 μM TMT) in HeLa cells. This difference was similar in neuroblastoma SY5Y cells, were ∼127% versus ∼136% increase (5 μM TMT) were measured. Staining of calcium stores with rhod-2 showed a TMT-induced [Ca 2+ ] i -decrease in the stores followed by an increase of the calcium concentration in the nuclei of the two cell lines tested. Our results suggest that toxic effects in human tumour cells after exposure to trimethyltin compounds might be due to an elevation of [Ca 2+ ] i

Phosphocitrate inhibits mitochondrial and cytosolic accumulation of calcium in kidney cells in vivo.

Science.gov (United States)

Tew, W P; Malis, C D; Howard, J E; Lehninger, A L

1981-01-01

Synthetic 3-phosphocitrate, an extremely potent inhibitor of calcium phosphate crystallization as determined in a nonbiological physical-chemical assay, has many similarities to a mitochondrial factor that inhibits crystallization of nondiffracting amorphous calcium phosphate. In order to determine whether phosphocitrate can prevent uptake and crystallization of calcium phosphate in mitochondria in vivo, it was administered intraperitoneally to animals given large daily doses of calcium gluconate or parathyroid hormone, a regimen that causes massive accumulation and crystallization of calcium phosphate in the mitochondria and cytosol of renal tubule cells in vivo. Administration of phosphocitrate greatly reduced the net uptake of Ca2+ by the kidneys and prevented the appearance of apatite-like crystalline structures within the mitochondrial matrix and cytosol of renal tubule cells. Phosphocitrate, which is a poor chelator of Ca2+, did not reduce the hypercalcemia induced by either agent. These in vivo observations therefore indicate that phosphocitrate acts primarily at the cellular level to prevent the extensive accumulation of calcium phosphate in kidney cells by inhibiting the mitochondrial accumulation or crystallization of calcium phosphate. Images PMID:6946490

Total body neutron activation analysis of calcium: calibration and normalisation

Energy Technology Data Exchange (ETDEWEB)

Kennedy, N S.J.; Eastell, R; Ferrington, C M; Simpson, J D; Strong, J A [Western General Hospital, Edinburgh (UK); Smith, M A; Tothill, P [Royal Infirmary, Edinburgh (UK)

1982-05-01

An irradiation system has been designed, using a neutron beam from a cyclotron, which optimises the uniformity of activation of calcium. Induced activity is measured in a scanning, shadow-shield whole-body counter. Calibration has been effected and reproducibility assessed with three different types of phantom. Corrections were derived for variations in body height, depth and fat thickness. The coefficient of variation for repeated measurements of an anthropomorphic phantom was 1.8% for an absorbed dose equivalent of 13 mSv (1.3 rem). Measurements of total body calcium in 40 normal adults were used to derive normalisation factors which predict the normal calcium in a subject of given size and age. The coefficient of variation of normalised calcium was 6.2% in men and 6.6% in women, with the demonstration of an annual loss of 1.5% after the menopause. The narrow range should make single measurements useful for diagnostic purposes.

Research of calcium oxide hydration in calcium nitrate solutions

Directory of Open Access Journals (Sweden)

M.A. Oliynyk

2016-09-01

Full Text Available Mineral fertilizers are one of the important factors of agriculture intensification and increasing of food products quantity. The volume of fertilizers production and its domestic consumption in Ukraine indicate that nitrogen fertilizer using only comes nearer to the required number of science-based. One of the most widespread artificial fertilizers is the calcium nitrate. Aim: The aim is to study and theoretically substantiate the processes occurring in the preparation of suspensions of calcium hydroxide Са(ОН2 in solution of calcium nitrate Ca(NО32. Materials and Methods: The technical calcium oxide (quicklime DSTU BV.2.7-90-99, solutions of calcium nitrate of 15, 20, 25, 30, 35 and 40% Ca(NО32 concentrations were used in the work. The content of lime in the preparation of a suspension in the solution changed (in terms of calcium oxide CaO from 150 g/dm3 to the maximum possible. Each of these solutions saturated at 40°С in lime to maximum concentration. Suitable for use in these experiments and in the technology of calcium nitrate obtaining are considered the solutions (suspensions that within 12 hours did not lose their mobility (transportability. Results: The experimental results show that increasing of the concentration of calcium nitrate in solution within the range 15...40%, the amount of lime that you can put into the solution without loss of transportability decreases. Further increasing of lime quantity in solutions concentrations causes to its solidifying, loss of mobility (transportability. Calculations showed that in the presence of calcium nitrate the solubility of Са(ОН2 is reduced nearly by order that can lead to the formation of calcium oxide CaO the solid phase Са(ОН2 on the surface, which also can form hydrogen bonds with the components of the solution. As the probability of formation of hydrogen bonds in solutions is high, there is a possibility of formation of clusters.

Production of precipitated calcium carbonate from calcium silicates and carbon dioxide

International Nuclear Information System (INIS)

Teir, Sebastian; Eloneva, Sanni; Zevenhoven, Ron

2005-01-01

The possibilities for reducing carbon dioxide emissions from the pulp and paper industry by calcium carbonation are presented. The current precipitated calcium carbonate (PCC) production uses mined, crushed calcium carbonate as raw materials. If calcium silicates were used instead, carbon dioxide emissions from the calcination of carbonates would be eliminated. In Finland, there could, thus, be a potential for eliminating 200 kt of carbon dioxide emissions per year, considering only the PCC used in the pulp and paper industry. A preliminary investigation of the feasibility to produce PCC from calcium silicates and the potential to replace calcium carbonate as the raw material was made. Calcium carbonate can be manufactured from calcium silicates by various methods, but only a few have been experimentally verified. The possibility and feasibility of these methods as a replacement for the current PCC production process was studied by thermodynamic equilibrium calculations using HSC software and process modelling using Aspen Plus[reg]. The results from the process modelling showed that a process that uses acetic acid for extraction of the calcium ions is a high potential option for sequestering carbon dioxide by mineral carbonation. The main obstacle seems to be the limited availability and relatively high price of wollastonite, which is a mineral with high calcium silicate content. An alternative is to use the more common, but also more complex, basalt rock instead

Calcium electroporation in three cell lines; a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

2013-01-01

offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium...

A kinetic model of dopamine- and calcium-dependent striatal synaptic plasticity.

Directory of Open Access Journals (Sweden)

Takashi Nakano

2010-02-01

Full Text Available Corticostriatal synapse plasticity of medium spiny neurons is regulated by glutamate input from the cortex and dopamine input from the substantia nigra. While cortical stimulation alone results in long-term depression (LTD, the combination with dopamine switches LTD to long-term potentiation (LTP, which is known as dopamine-dependent plasticity. LTP is also induced by cortical stimulation in magnesium-free solution, which leads to massive calcium influx through NMDA-type receptors and is regarded as calcium-dependent plasticity. Signaling cascades in the corticostriatal spines are currently under investigation. However, because of the existence of multiple excitatory and inhibitory pathways with loops, the mechanisms regulating the two types of plasticity remain poorly understood. A signaling pathway model of spines that express D1-type dopamine receptors was constructed to analyze the dynamic mechanisms of dopamine- and calcium-dependent plasticity. The model incorporated all major signaling molecules, including dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32 kDa (DARPP32, as well as AMPA receptor trafficking in the post-synaptic membrane. Simulations with dopamine and calcium inputs reproduced dopamine- and calcium-dependent plasticity. Further in silico experiments revealed that the positive feedback loop consisted of protein kinase A (PKA, protein phosphatase 2A (PP2A, and the phosphorylation site at threonine 75 of DARPP-32 (Thr75 served as the major switch for inducing LTD and LTP. Calcium input modulated this loop through the PP2B (phosphatase 2B-CK1 (casein kinase 1-Cdk5 (cyclin-dependent kinase 5-Thr75 pathway and PP2A, whereas calcium and dopamine input activated the loop via PKA activation by cyclic AMP (cAMP. The positive feedback loop displayed robust bi-stable responses following changes in the reaction parameters. Increased basal dopamine levels disrupted this dopamine-dependent plasticity. The

Transcellular transport of calcium