Ionomycin

Similar 5-exo cyclization procedures have been widely utilized by Evans in his total syntheses of complex natural compounds, such as the synthesis of ionomycin [12a] and polyether antibiotic X-206 [12b]. A 5-exo cyclization of a y-epoxy alcohol has also been observed under basic conditions [12c]. 

As expected, ionomycin, a calcium ionophore, causes a sustained rise in the free intracellular concentration to approximately 450 nM (Figure 4). In this system, pardaxin induced an increase in intracellular [Ca ] only in the presence of extracellular Ca (Figure 4). These results indicate that pardaxin mediated a Ca influx but did not release Ca from intracellular stores. This influx is most probably mediated directly by pardaxin channels and possibly also indirectly by activation of the Ca channels of the chromaffin cells by the depolarization produced by the pardaxin channels (data not shown). These observations further substantiate our hypothesis 10) that transmembrane fluxes of Na and Ca are involved in the pathological action of pardaxin. 

BK Toeplitz, AI Cohen, PT Funke, WL Parker, JZ Gougoutas. (1979). Structure of ionomycin—A novel diacidic polyether antibiotic having high affinity for calcium ions. J Am Chem Soc 101 3344-3353. 

Sensor any single-polypeptide construct (containing the fluorophores to be used in the experiment) that can be induced to change FRET significantly and homogeneously e.g., Yellow Cameleon in combination with ionomycin. 

In Fig. 9.4, the results are shown of an experiment using a double (annexinA4-EYFP+ annexinA4-mCherry) transfected cell immediately after addition of the calcium ionophore ionomycin (top) and 5 min after application of ionomycin (bottom). Clearly visible is the diffuse localization of annexin A4 before relocation and the more structured localization (into membrane ruffles and filopodia) after relocation. More importantly, the EYFP fluorescence lifetime is quenched from 2.9 ns before translocation to... 

Stability constants for a series of M2+ cations, including Ca2+, with A23187 and with ionomycin have been determined in methanol-water mixtures (Taylor, R. W. Pfeiffer, D. R. Chapman, C. J. Craig, M. E. Thomas, T. P. Pure Appl. Chem. 1993, 65, 579-584) the estimation of stability constants for complexes of the anti-fungal antibiotic pradimicin (BMY-28864) with alkaline earth cations is complicated by the tendency of this antibiotic to aggregate (Hu, M. Ishizuka, Y. Igarashi, Y. Oki, T. Nakanishi, H. Spectrochim. Acta A 1999, 56, 181-191). 

Lautens also used this nickel-catalyzed hydroalumination methodology in the total synthesis of ionomycin 145. The starting compound was a [3.2.1]oxabicyclic alkene 143.144 Their rigid bicyclic structures can be used to introduce functional groups in a highly stereoselective manner. The synthesis of the key intermediate 144 involves the slow addition of DIBAL to the oxabicyclic alkene and the Ni(COD)2/(6T)-BINAP in toluene to afford 144 in 95% yield and 93-95% ee. (Scheme 18). 

Somlyo You showed that there are other mechanisms that come into play, such as Ca2+ sensitization. I would expect an agonist such as a purinergic agonist to activate the Rho/Rho kinase system, which ionomycin does not. 

Blaustein With regard to the stores releasing Ca2+, here you are talking about indirect functional observations when you study the releasable pool. If we actually look at the store itself with furaptra and use high concentrations of an agonist or maximal doses of caffeine, it releases some Ca2+, but the Ca2+ doesn t go down much below 50/tM. You can release more if you use the Ca2+-free solution or ionomycin. The SR is not really empty. Our feeling is that there are multiple stores. There may be some stores that can be depleted relatively easily, whereas other stores are more difficult to deplete. I have some evidence that the junctional stores may be the ones that you are talking about release of Ca2+ is probably not complete . 

Taylor Perhaps I should add that if we empty the stores with vasopressin, which will completely empty them, as opposed to thapsigargin and ionomycin, we can get... 

However, intracellular Ca2+ increases in themselves are insufficient to activate the oxidase because levels of this cation can be artificially increased in the absence of oxidase activity (e.g. by the addition of Ca2+ iono-phores such as ionomycin). Furthermore, activation of the oxidase by agonists such as PMA occurs in the absence of intracellular Ca2+ increases. Thus, if DAG is generated at sufficiently high concentrations to activate protein kinase C, then increases in intracellular Ca2+ are not required for oxidase activation. Furthermore, some agonists (e.g. LTB4 and PAF) generate substantial increases in intracellular Ca2+ but are poor activators of the respiratory burst. Also, there is a marked discrepancy between the concentrations of some agonists (e.g. fMet-Leu-Phe) required to activate either Ca2+ increases or oxidase activity. For example, maximal elevations in Ca2+ can occur at concentrations of fMet-Leu-Phe that are too low to activate the respiratory burst. It therefore appears that intracellular Ca2+ increases are not required to activate the respiratory burst per se, but are involved in the... 

NK cells express receptors for numerous monokines constitutively, and produce IFN-y and other NK-derived cytokines rapidly in response to stimulation by monokines [18, 19]. Freshly isolated CD56 "s human NK cells are the primary source of NK cell-derived immuno-regulatory cytokines, including IFN-y, TNF-(3 (lymphotoxin), IL-10, IL-13 and GM-CSF, whereas the CDSb NK-cell subset produces consistently negligible amounts of these cytokines following stimulation with recombinant monokines in vitro [20]. The production of cytokines by NK cell subsets was investigated following activation with phorbol esters (e.g. phorbol 12-myristate 13-acetate (PMA)) and ionomycin. 

Selected entries from Methods in Enzymology [vol, page(s)] Chelation, 238, 74, 76, 297 buffers [for analysis of exocytosis, 221, 132 preparation, 219, 186 modulation of cytosolic buffering capacity with quin2, 221, 159] fluorescence assay, 240, 724-725, 740-742 fluorescence imaging, 225, 531 238, 303-304, 322-325, 334-335 free intracellular levels after bacterial invasion, 236, 482-489 free calcium in solutions for membrane fusion analysis, calculation and control, 221, 149 homeostasis mechanisms, 238, 80 hormonal elevation, 238, 79 inositol phosphate effect on release, 238, 207 determination of cytosolic levels [computer methods, 238, 73-75 with fura-2, 238, 73, 146 with indo-1, 238, 298, 316-317 with quin-2, 238, 297] hormone effects, 238, 79 ionomycin effects, 238, 79 membrane depolarization effects,... 

The microenvironment may also influence apoptosis. Exposure of isolated thymocytes to TPA plus ionomycin for 24 hours enhanced apoptosis. On the other hand, when ffiymocytes were cultured in intact lobes, a 24 hour TPA plus ionomycin exposure only marginally induced apoptosis. Therefore, it appears that removing thymocytes from their thymic microenvironment makes the cells more susceptible to certain stimuli, possibly by altering their physiological status. (Moore et al., 1992). Viral infection may also alter apoptosis. Epstein-Barr virus infected human Burkitt s lymphoma cells were particularly sensitive to treatment with PdBu (42% apoptosis at 72 hours), whereas its virus free counterpart displayed only 12% apoptosis (Ishii and Gobe, 1993). 

SCHEME 72. Iterative one-pot homologation of reduced polypropionates viaZACA-Pd-catalyzed vinylation and its application to highly efficient catalytic and asymmetric synthesis of key intermediates of ionomycin and borrelidin... 

Fig. 4. In situ end-labeling. Forward light scatter (FSC) vs FITC fluorescence (FL1) of (A) viable and (B) apoptotic Burkitt lymphoma cells. Cells were induced into apoptosis by incubation with the calcium ionophore, ionomycin. Region I, viable cells Region 2, apoptotic cells.

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