|
| ||
|
|
||
Calcium Ionophore (calcium + ionophore)
Selected AbstractsTHE ROLE OF CALCIUM IN FLOW-STIMULATED BIOLUMINESCENCE OF THE RED TIDE DINOFLAGELLATE LINGULODINIUM POLYEDRUMJOURNAL OF PHYCOLOGY, Issue 2000P. Von Dassow Many marine planktonic dinoflagellates emit flashes of light in response to either laminar or turbulent flows as well as direct mechanical stimulation. The production of a flash of light is known to be mediated by a proton-mediated action potential across the vacuolar membrane; the mechanotransduction process initiating this action potential is unknown. Here we report on an investigation into the role of Ca+2 in the mechanotransduction process regulating bioluminescence in the red tide dinoflagellate Lingulodinium polyedrum. Calcium ionophores and low concentrations of the membrane-disrupting agent digitonin stimulated bioluminescence only when calcium was present in the media or added with the agent, indicating that the flash-triggering vacuolar action potential is specifically stimulated by a calcium influx. A variety of known calcium channel blockers or antagonists inhibited mechanically stimulated bioluminescence but did not affect cellular bioluminescent capacity. In many cases the inhibitory affect occurred after only a brief exposure. In addition, gadolinium (Gd+3), a blocker of many stretch-activated ion channels, caused potent inhibition of mechanically stimulated bioluminescence. The order of potency of the transition metals tested was La+3 > Gd+3 > Co+2 > Mn+2 > Ni+2, similar to their potency as blockers of known calcium channels. Experiments with a quantified shear flow demonstrated that flow-stimulated bioluminescence depended on the level of extracellular calcium. Future work will elucidate the signaling pathway involving calcium-mediated flow-stimulated mechanotransduction. Our goal is to use bioluminescence as a proxy for the initial cellular mechanotransduction events triggered by fluid flow. [source] Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeletonCYTOSKELETON, Issue 4 2004Katherine Serrano Abstract The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being ,95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets. Cell Motil. Cytoskeleton 58:242,252, 2004. © 2004 Wiley-Liss, Inc. [source] Parabolic flight primes cytotoxic capabilities of polymorphonuclear leucocytes in humansEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 8 2009I. Kaufmann Abstract Background, Previously performed in vitro studies suggested that gravitational stress may alter functions of immune cells. This study investigated the in vivo effects of parabolic flight manoeuvres as a short-term model of micro- and hypergravity on the cytotoxic and microbicidal polymorphonuclear leucocyte (PMN) functions as the key element of innate immunity. Material and methods, Twenty-one healthy male volunteers underwent 30 subsequent parabolic flight manoeuvres. Each manoeuvre produced 22-s periods of nearly weightlessness close to «0g», with each parabola starting with a pull-up and ending with a pull-out (hypergravity) at 1·8 g for about 20 s each. Blood samples were drawn 24 h prior to take off (T0), after 25,30 parabolas (T1), and 24 h (T2) and 48 h (T3) after flight for determination of (i) leucocyte number and subpopulations, (ii) PMNs' capabilities to produce hydrogen peroxide (H2O2) and to adhere and phagocytose particles and (iii) plasma cytokines known to prime PMN functions [interleukin-8 (IL-8), tumour necrosis factor-, (TNF-,), granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)]. Results, Parabolic flight induced an increase in leucocyte number with a significant elevation of the PMN fraction. The spontaneous H2O2 production by PMNs did not change; however, the capability of PMNs to produce H2O2 in response to soluble stimuli [N -formyl-methionyl-leucyl-phenylalanine (fMLP), fMLP and TNF-,, calcium ionophore (A23187), phorbol myristate acetate (PMA)] was increased. Adhesive and phagocytic properties of PMNs were not altered. Regarding priming cytokines, IL-8 and G-CSF were significantly elevated. Conclusions, Our data indicate that parabolic flight induces priming of the cytotoxic capabilities of PMNs without affecting microbicidal functions. [source] Effects of tumour necrosis factor alpha and interleukin-6 on progesterone and calcium ionophore-induced acrosome reactionINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2009F. Lampiao Summary For human spermatozoa to successfully fertilize the oocyte, they need to undergo a timely acrosome reaction (AR). Factors which disturb the AR may lead to fertilization failure. The objective of this study was to investigate the effects of two cytokines namely tumour necrosis factor alpha (TNF-,) and interleukin-6 (IL-6) on the spontaneous, calcium ionophore-induced and progesterone-induced human sperm AR. Twenty-two normal semen samples were treated with increasing concentrations of TNF-, and IL-6 after spermatozoa were isolated by a double wash swim-up method. The AR was induced by calcium ionophore A23187 and progesterone. The AR was determined by using fluorescein isothiacyanate Pisum sativum agglutinin and observed under fluorescence microscope. Both TNF-, and IL-6 could decrease the spontaneous, ionophore and progesterone-induced AR (p < 0.05) in a dose-dependent manner. TNF-, showed a more potent inhibiting effect than IL-6 by inhibiting the AR at lower concentrations. This study has demonstrated that TNF-, and IL-6 play a role in inhibiting both the non-physiological as well as physiologically elicited AR by calcium ionophore and progesterone respectively. [source] Caspase cleavage of exon 9 deleted presenilin-1 is an early event in apoptosis induced by calcium ionophore A 23187 in SH-SY5Y neuroblastoma cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2001Bogdan O. Popescu Abstract Presenilins (PSs) are mutated in a majority of familial Alzheimer disease (FAD) cases. Mutated PSs may cause FAD by a number of pro-apoptotic mechanisms, or by regulating ,-secretase activity, a protease involved in ,-amyloid precursor protein processing to the neurotoxic ,-amyloid peptide. Besides their normal endoproteolytic processing, PSs are substrates for caspases, being cleaved to alternative N-terminal and C-terminal fragments. So far little is known about the role of PSs cleavage in the apoptotic machinery. Here, we used SH-SY5Y neuroblastoma cells stably transfected with wild-type or exon 9 deleted presenilin 1 (PS1) in a time-course study after the exposure to the calcium ionophore A23187. During and after exposure to A 23187, intracellular calcium levels were higher in exon 9 deleted PS1 cells as compared with non-transfected and wild-type PS1 transfected cells. Cell death and the enrichment of apoptotic cells after A23187 exposure were increased by overexpression of exon 9 deleted PS1 as compared with the control cell lines. Wild-type PS1 cells were compared with exon 9 deleted PS1 cells and the temporal relationship between PS1 and other caspase substrates cleavages was analyzed. Exon 9 deleted PS1 cells exhibited a higher caspase-3 activation and a greater cleavage of PS1 and poly(ADP-ribose) polymerase (PARP) compared with wild-type PS1 cells. Exon 9 deleted PS1 cleavage occurred earlier than other caspase substrate cleavages (i.e., PARP and gelsolin), simultaneous with minimum detectable caspase-3 activation. Therefore, alternative cleavage of PS1 may play an important role for the regulation of the proteolytic cascade activated during apoptosis. J. Neurosci. Res. 66:122,134, 2001. © 2001 Wiley-Liss, Inc. [source] Flavonoid inhibition of platelet procoagulant activity and phosphoinositide synthesisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2003R. Bucki Summary., Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection. Phosphatidylinositol 4,5-bisphosphate (PIP2) was previously reported to play a direct role in phosphatidylserine (PS) exposure, as a Ca2+ target. Thrombin formation and platelet procoagulant activity are dependent on PS exposure. As flavonoids can inhibit phosphoinositide (PPI) kinases, we examined whether changes in PPI metabolism in flavonoid-treated platelets could be involved in their antiplatelet effects. Treatment with the flavonoids quercetin or catechin reduced PS exposure, thrombin formation, PIP2 level and resynthesis after platelet activation with collagen, thrombin or calcium ionophore. Flavonoids also prevented [Ca2+]i increase induced by collagen, but not by the ionophore. The ability of flavonoids to decrease PS exposure induced by ionophore treatment could result from the diminution of PIP2 levels, whereas PS exposure induced by collagen could also be diminished by flavonoids' effects on calcium signaling dependent on PIP2 hydrolysis. These data favor a role for PIP2 in the antiplatelet effects of flavonoids. [source] Ecabet sodium attenuates reactive oxygen species produced by neutrophils after priming with bacterial lipopolysaccharidesLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 6 2003Wataru Munakata Abstract The pathogenic roles of reactive oxygen species (ROS) have been implicated in ulcerative colitis (UC). The aim of this study was to examine the effects of ecabet sodium on ROS produced by human neutrophils, particularly after being primed by bacterial lipopolysaccharides (LPS). Neutrophils were isolated from six healthy volunteers. Each well of a 96-well microplate received neutrophil suspension (1.0 × 105 cells) and the plates were incubated at 37°C for 30 min with or without E. coli LPS (f.c. 0.001 ng/µL). Ecabet sodium (f.c. 0,5.0 mg/mL) was added before starting or after finishing the incubation. Neutrophils were stimulated by opsonized zymosan (OZ; 1.0 mg/mL) or calcium ionophore (A21837; 0.3 µmol/L) and luminol-dependent chemiluminescence response was measured using a Lumi Box H-1000. Ecabet sodium attenuated ROS production at a concentration of 5.0 mg/mL (p < 0.05) in LPS-primed neutrophils. However, attenuating effects were not significantly different when ecabet sodium was added before or after the incubation with E. coli LPS. Ecabet sodium may have some attenuating effects on ROS produced by human neutrophils even after neutrophils are primed by bacterial LPS. These results may explain, in part, the therapeutic effects of ecabet sodium for UC. Copyright © 2003 John Wiley & Sons, Ltd. [source] Ultraviolet B Light-induced Nitric Oxide/Peroxynitrite Imbalance in Keratinocytes,Implications for Apoptosis and NecrosisPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2010Shiyong Wu Elevation of nitric oxide (NO,) can either promote or inhibit ultraviolet B light (UVB)-induced apoptosis. In this study, we determined real-time concentration of NO, and peroxynitrite (ONOO,) and their role in regulation of membrane integrity and apoptosis. Nanosensors (diameter 300,500 nm) were used for direct in situ simultaneous measurements of NO, and ONOO, generated by UVB in cultured keratinocytes and mice epidermis. An exposure of keratinocytes to UVB immediately generated ONOO, at maximal concentration of 190 nm followed by NO, release with a maximal concentration of 91 nm. The kinetics of UVB-induced NO,/ONOO, was in contrast to cNOS agonist stimulated NO,/ONOO, from keratinocytes. After stimulating cNOS by calcium ionophore (CaI), NO, release from keratinocytes was followed by ONOO, production. The [NO,] to [ONOO,] ratio generated by UVB decreased below 0.5 indicating a serious imbalance between cytoprotective NO, and cytotoxic ONOO,,a main component of nitroxidative stress. The NO,/ONOO, imbalance increased membrane damage and cell apoptosis was partially reversed in the presence of free radical scavenger. The results suggest that UVB-induced and cNOS-produced NO, is rapidly scavenged by photolytically and enzymatically generated superoxide (O2,,) to produce high levels of ONOO,, which enhances oxidative injury and apoptosis of the irradiated cells. [source] Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti,citrullinated protein antibodies in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 3 2006Federico Pratesi Objective To test the hypothesis that deimination of viral sequences containing Arg,Gly repeats could generate epitopes recognized by anti,citrullinated protein antibodies (ACPAs) that are present in rheumatoid arthritis (RA) sera. Methods Multiple antigen peptides derived from Epstein-Barr virus (EBV),encoded Epstein-Barr nuclear antigen 1 (EBNA-1) were synthesized, substituting the arginines with citrulline, and were used to screen RA sera. Anti,cyclic citrullinated peptide antibodies were purified by affinity chromatography and tested on a panel of in vitro deiminated proteins. Their ability to bind in vivo deiminated proteins was evaluated by immunoprecipitation, using EBV-infected cell lines. Results Antibodies specific for a peptide corresponding to the EBNA-135,58 sequence containing citrulline in place of arginine (viral citrullinated peptide [VCP]) were detected in 50% of RA sera and in <5% of normal and disease control sera. In addition, affinity-purified anti-VCP antibodies from RA sera reacted with filaggrin-derived citrullinated peptides, with deiminated fibrinogen, and with deiminated recombinant EBNA-1. Moreover, anti-VCP antibodies immunoprecipitated, from the lysate of calcium ionophore,stimulated lymphoblastoid cell lines, an 80-kd band that was reactive with a monoclonal anti,EBNA-1 antibody and with anti,modified citrulline antibodies. Conclusion These data indicate that ACPAs react with a viral deiminated protein and suggest that EBV infection may play a role in the induction of these RA-specific antibodies. [source] NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytesCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2005Qing Liu Abstract Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival. Copyright © 2004 John Wiley & Sons, Ltd. [source] Differences in regulatory pathways identify subgroups of T cell-derived Th2 cytokinesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2000K. Rafiq We analysed regulatory mechanisms involved in the production of Th2 cytokines by freshly isolated human T cells. We used an in vitro culture system in which the primary signal was provided by a cross-linking anti-CD3 MoAb presented on the Fc receptors of P815 cells. Both CD80 and CD86, expressed on transfected P815 cells, were able to provide efficient costimulation for the production of IL-4, IL-5 and IL-13. IL-2 was also highly important for induction of all three Th2 cytokines. However, differences between IL-4 on the one hand and IL-5 and IL-13 on the other hand were observed when sensitivity to cyclosporin A (CsA) was studied. CsA (an inhibitor of calcineurin phosphatase activity) strongly inhibited IL-4 production, but it did either not affect or even increased IL-5 and IL-13 production. In accordance with this, CD80 and phorbol myristate acetate (PMA) (without anti-CD3 or calcium ionophore) were sufficient to induce production of IL-5 and IL-13, but not of IL-4. The subgrouping of Th2 cytokines was further confirmed at another level on the basis of differences in cell sources: IL-4 was predominantly produced by CD4+ T cells, while IL-5 and IL-13 were produced by both CD4+ and CD8+ T cells. Thus, differences in cell sources and in the requirement of the calcium/calcineurin-signalling pathway allowed us to identify two subgroups (IL-4 and IL-5/IL-13) among human Th2-type T cell cytokines. [source] | |||||||||||||