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Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.

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This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

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The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca2+ stores via inositol 1,4,5-trisphosphate-gated Ca2+ channels is required for nuclear assembly in Xenopus egg extracts. Other work suggested that lumenal Ca2+ stores are required for nuclear protein import in mammalian cells in vivo, but not in vitro. Here, we rigorously tested the role of lumenal Ca2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca2+ stores were depleted by pretreating the extracts with Ca2+ ionophores (ionomycin, A23187) or inhibitors of Ca(2+)-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca(2+)-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca2+ stores confirms a previous unpublished observation by Greber and Gerace, and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca2+. Finally, we concluded that, contrary to our expectations, lumenal Ca2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.

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In higher eukaryotes, the entire nucleus disassembles during prometaphase of the cell cycle and later reassembles around daughter chromosomes. Remarkably, the complex events that occur to create a functional nucleus in vivo can be duplicated in vitro by using cell-free extracts. Current experiments are aimed at understanding the molecular mechanisms of assembly and disassembly of the nuclear pore complexes and nuclear membranes, and the functional roles of four identified inner membrane proteins, two of which bind to both chromatin and the nuclear lamina.

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1. In calf pulmonary artery endothelial (CPAE) cells loaded with fura-2, the effects of ATP on Ca2+ entry were mediated entirely by the ability of P2U purinoceptors to stimulate InsP3 formation, empty intracellular Ca2+ stores and thereby activate capacitative Ca2+ entry. 2. Restoration of extracellular Ca2+ to cells with empty intracellular stores evoked transient increases in cytosolic [Ca2+] ([Ca2+]i) which then declined to an elevated plateau. These overshoots in [Ca2+]i were not a consequence of store refilling nor of desensitization of the capacitative pathway. Similar responses were recorded from cells in which Ca2+ uptake into mitochondria had been inhibited by microinjection of Ruthenium Red. The amplitudes of the capacitative Ca2+ signals decreased at lower extracellular [Ca2+], but [Ca2+]i invariably overshot before slowly declining to an elevated plateau. Even modest increases in [Ca2+]i therefore caused a delayed attenuation of the Ca2+ signal evoked by capacitative Ca2+ entry. 3. Modest pre-elevation of [Ca2+]i inhibited the ability of subsequent capacitative Ca2+ entry to further increase [Ca2+]i. The onset of the inhibition was slow (half-time (t1/2), approximately 100 s) and more tightly correlated with the preceding peak [Ca2+]i than with the [Ca2+]i immediately preceding Ca2+ entry. Recovery was also slow and complete only after [Ca2+]i had returned to its basal level for 320 +/- 3 s. 4. In thapsigargin-treated cells loaded with mag-fura-2, the peak [Ca2+]i that followed restoration of extracellular Ca2+ was accompanied by an abrupt approximately 2.5-fold decrease in the rate of Mn2+ entry, which then continued indefinitely at the reduced rate, demonstrating a rapid partial inactivation of the capacitative pathway. 5. The half-time for Ca2+ removal from the cytosol was significantly slower during the rising (t 1/2 = 22 +/- 2.5 s) than during the falling (t 1/2 = 7.1 +/- 0.7 s) phase of the Ca2+ overshoot evoked by addition of extracellular Ca2+ to thapsigargin-treated cells. 6. We conclude that an increase in [Ca2+]i rapidly inhibits the capacitative pathway and more slowly activates mechanisms that remove Ca2+ from the cytosol. Reversal of either or both of these regulatory mechanisms can occur only a considerable time after [Ca2+]i has been completely restored to its resting level. These mechanisms are likely to protect cells from excessive increases in [Ca2+]i and contribute to oscillatory changes in [Ca2+]i.

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Cytosolic Ca2+ biphasically regulates Ins(1,4,5)P3-stimulated Ca2+ mobilization in liver [Marshall and Taylor (1993) J. Biol. Chem. 268, 13214-13220]. We have investigated the mechanisms underlying this biphasic control of Ca2+ mobilization in permeabilized hepatocytes by comparing the effects of Sr2+, Ba2+ and Ca2+ on the liver Ins(1,4,5)P3 receptor. Both Ca2+ and Sr2+ increased the binding of [3H]Ins(1,4,5)P3 to liver membranes by converting receptors from a low-affinity (KD approximately 35 nM) to a high-affinity (KD approximately 5 nM) state. Ba2+ (< or = 20 microM) did not affect [3H]Ins(1,4,5)P3 binding. At concentrations similar to those that caused an enhancement of [3H]Ins(1,4,5)P3 binding, Sr2+ (EC50 = 570 nM) and Ca2+ (EC50 = 200 nM) increased the sensitivity of the intracellular Ca2+ stores to Ins(1,4,5)P3. Further modest elevations in [Ca2+] (EC50 = 1.5 microM) inhibited Ins(1,4,5)P3-stimulated Ca2+ mobilization, whereas Sr2+ caused inhibition only when its concentration was very substantially increased (EC50 approximately 900 microM). Sr2+ is therefore only 3-fold less potent than Ca2+ in causing sensitization of Ins(1,4,5)P3-stimulated Ca2+ release, but 600-fold less potent in causing inhibition. Ba2+ neither sensitized ([Ba2+] < or = 20 microM) nor inhibited ([Ba2+] < or = 1 mM) Ins(1,4,5)P3-stimulated Ca2+ release, and did not inhibit either the sensitization of Ca2+ release evoked by Sr2+ or the inhibition of Ca2+ release evoked by Ca2+. Our results suggest that two distinct Ca(2+)-binding sites, which differ in their selectivities for bivalent cations, mediate the interconversion of Ins(1,4,5)P3 receptors between at least three different conformational states. These two Ca(2+)-binding sites, which may reside either on the Ins(1,4,5)P3 receptor itself or on distinct regulatory proteins, can be distinguished by their different selectivities for bivalent cations.

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Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a soluble second messenger responsible for the generation of highly organized Ca2+ signals in a variety of cell types. These Ca2+ signals control many cellular responses, including cell growth, fertilization, smooth muscle contraction and secretion. Ins(1,4,5)P3 is produced at the plasma membrane following receptor activation, but rapidly diffuses into the cytosol, where it binds to specific receptors through which it mobilizes intracellular Ca2+ stores. The actions of Ins(1,4,5)P3 within cells are tightly controlled: enzymes control the rapid generation and metabolism of Ins(1,4,5)P3 following receptor activation; multiple Ins(1,4,5)P3 receptor subtypes and splice variants exist, some of which are differentially expressed between cell types and at different stages of development; and Ins(1,4,5)P3 receptors are the targets for a number of allosteric regulators, including protein kinases, ATP and divalent cations. Understanding how cells control the Ca(2+)-mobilizing activity of Ins(1,4,5)P3 will be important if we are to unravel the mechanisms that underlie the complex arrangements of Ca2+ signals.

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The increase in cytosolic free Ca2+ concentration that follows mobilization of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate Ins(1,4,5)P3 has been reported to modulate the sensitivity of Ins(1,4,5)P3 receptors. We have examined the effects of cytosolic Ca2+ on Ins(1,4,5)P3-stimulated Ca2+ mobilization in permeabilized hepatocytes. Increasing the free Ca2+ concentration in the medium ([Ca2+]m) caused a concentration-dependent increase in the sensitivity of the stores to Ins(1,4,5)P3; the concentration of Ins(1,4,5)P3 that caused half-maximal Ca2+ mobilization (EC50) decreased from 261 +/- 11 nM (n = 3) to 50 +/- 4 nM (n = 8) as [Ca2+]m was increased from approximately 7 nM to 1.6 microM. The EC50 for this effect of Ca2+ was approximately 250 nM. In addition, higher [Ca2+]m (> 600 nM) reduced the extent of Ca2+ release induced by a maximal concentration of Ins(1,4,5)P3; elevating [Ca2+]m to 2.6 microM reduced the proportion of Ca2+ releasable by Ins(1,4,5)P3 by 94 +/- 8% (n = 3). Both effects of Ca2+ were independent of Ca2(+)-stimulated Ins(1,4,5)P3 formation. When elevated [Ca2+]m was returned to control levels, the sensitization of Ins(1,4,5)P3-mediated Ca2+ mobilization reversed completely, whereas the reduction in the size of the Ins(1,4,5)P3-sensitive Ca2+ pool was reversed by only 59 +/- 12% (n = 5) after 20 s and was not further reversed after 100 s. The two distinct effects of Ca2+ on Ins(1,4,5)P3-mediated Ca2+ release combined to control the amount of Ca2+ released by a submaximal concentration of Ins(1,4,5)P3 in a biphasic manner.

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Increases in intracellular free Ca2+ concentration ([Ca2+]i), whether initiated by changes in plasma membrane potential or receptor-stimulated polyphosphoinositide hydrolysis, can be astonishingly complex, often occurring as repetitive Ca2+ spikes and regenerative Ca2+ waves that propagate through the cell and sometimes into neighbouring cells. The key to understanding these complex Ca2+ signals lies in understanding the interactions between the different pools from which Ca2+ can rapidly enter the cytosol and the activities of the various Ca(2+)-transporting systems that reverse the process.

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