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Article |
SERCA pump activity is physiologically regulated by presenilin and regulates amyloid β production
Correspondence to Frank M. LaFerla: laferla{at}uci.edu
In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca2+ is maintained at low levels by pumping Ca2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca2+-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca2+, whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca2+ clearance and exaggerating inositol 1,4,5-trisphosphate–mediated Ca2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid β production. Our results point to a physiological role for the presenilins in Ca2+ signaling via regulation of the SERCA pump.
Abbreviations used in this paper: Aβ, amyloid β; AD, Alzheimer's disease; AICD, APP intracellular domain; APP, amyloid precursor protein; CCE, capacitative Ca2+ entrance; FAD, familial AD; IP3, inositol 1,4,5-trisphosphate; MEF, mouse embryonic fibroblast; nAChR, nicotinic acetylcholine receptor; pcDNA, pseudo-cDNA; PSDKO, presenilin double knockout; SERCA, sarco ER Ca2+-ATPase.
© 2008 Green et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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-secretase–mediated cleavage of the amyloid precursor protein (APP) to form amyloid β (Aβ) peptides (Duff et al., 1996) and disruption of intracellular Ca2+ homeostasis (LaFerla, 2002; Demuro et al., 2005). Ca2+ signaling disruptions manifest as enhanced filling of ER Ca2+ stores (Leissring et al., 1999b), attenuation of capacitive Ca2+ entry stores (Leissring et al., 2000; Yoo et al., 2000; Smith et al., 2002; Herms et al., 2003), and by exaggerated liberation of Ca2+ from the ER by the second messenger inositol 1,4,5-trisphosphate (IP3; Leissring et al., 1999b; Yoo et al., 2000; Smith et al., 2002; Stutzmann et al., 2004). Given that mutations in presenilin disrupt intracellular Ca2+ signaling, we set out to determine whether presenilins may serve a physiological role in intracellular Ca2+ homeostasis. In support of a role in Ca2+ homeostasis, overexpression of wild-type PS1 or PS2 in Xenopus laevis oocytes causes enhanced IP3-mediated Ca2+ release, an effect that is exacerbated by mutations in both genes (Leissring et al., 1999b). However, it remains unclear whether the exaggerated IP3-evoked responses result from modulation of the IP3 signaling pathway, such as sensitization of IP3 receptors by presenilins, or as a consequence of overfilling of ER stores. Recently, the presenilins have been reported to be able to form ER leak channels, and it has been reported that mutations in the presenilins disrupt this function (Tu et al., 2006). However, it is unclear how leak channel formation could account for the numerous reports of wild-type presenilin overexpression increasing IP3-mediated calcium release.
Ca2+ pumps, along with Ca2+ release channels, are the key components of Ca2+ regulatory systems in neuronal and nonneuronal cells (Berridge et al., 2000). The sarco ER Ca2+-ATPase (SERCA) pumps have the highest affinity for Ca2+ removal from the cytosol and, together with plasma membrane Ca2+-ATPases and transporters, determine the resting cytosolic Ca2+ concentration. Three differentially expressed genes encode at least five isoforms of the SERCA pump. SERCA1a and -1b are expressed in skeletal muscle, whereas SERCA2a is expressed in cardiac muscle (Aubier and Viires, 1998). SERCA2b, which has a C-terminal extension, is ubiquitously expressed in smooth muscle tissues and nonmuscle tissues including neurons (Baba-Aissa et al., 1998). SERCA3 has limited expression in various nonmuscle tissues (Baba-Aissa et al., 1998).
Given that overfilled ER Ca2+ stores are one consequence of most PS1 mutations, we hypothesized that presenilin may regulate SERCA pump activity. In this paper, we used both gain-of-function and loss-of-function genetic approaches to show that presenilins are required for proper functioning of SERCA activity in both mammalian cell lines and X. laevis oocytes. Notably, we find that presenilins physically associate with SERCA, and modulation of SERCA function via genetic or pharmacological means results in altered Aβ production. Furthermore, SERCA2b knockdown mimics the Ca2+ dynamics seen in presenilin-null cells. Collectively, these results suggest that presenilins regulate and are necessary for normal functioning of the SERCA2b pump, most likely through a direct protein–protein interaction, and that SERCA activity itself impacts Aβ generation.
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PSDKO fibroblasts displayed elevated resting cytosolic Ca2+ levels compared with control cells (Fig. 1, A and B). Application of thapsigargin in the bath perfusion promoted a transient rise of cytosolic [Ca2+] signal as a consequence of constitutively active Ca2+ leakage from the ER, providing a signal proportional to the amount of Ca2+ sequestered in the ER, although it does not take into account differences in Ca2+ efflux across the plasma membrane. PSDKO fibroblasts showed reduced responses to thapsigargin as compared with control fibroblasts (Fig. 1 B). These results are consistent with diminished SERCA activity, as it is this ER Ca2+ pump which helps to maintain low cytosolic resting Ca2+ level by actively pumping Ca2+ from the cytosol into the ER stores, thereby regulating the Ca2+ levels between the two compartments.
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The presenilins are an integral part of the -secretase complex that is responsible for cleavage of the C99 fragment of APP to form Aβ and the APP intracellular domain (AICD). Cells lacking presenilins do not have -secretase activity and consequently produce no Aβ or AICD (Zhang et al., 2000; Hass and Yankner, 2005). Because the absence of the AICD region of APP has been shown to cause deficits in Ca2+ release from ER stores (Leissring et al., 2002), we sought to determine whether the Ca2+ dyshomeostasis in PSDKO cells could be attributed to lack of AICD production rather than to the absence of presenilins themselves. For this purpose, PSDKO fibroblasts were transfected with the AICD (C57) fragment and compared with control pseudo-cDNA (pcDNA)–transfected PSDKO fibroblasts. However, even after 48 h of incubation to allow expression of AICD (Fig. 1 D), control and AICD-expressing PSDKO fibroblasts showed no significant differences in basal or thapsigargin-evoked Ca2+ levels. Given that overexpression of AICD into PSDKO cells did not recover either basal or thapsigargin-evoked Ca2+ levels, we then overexpressed PS1, PS2, or both together in PSDKO cells. Overexpression of either PS1, PS2, or both together rescued both the basal and thapsigargin-evoked Ca2+ levels to the same degree (Fig. 2, A and B).
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PS1 and PS2 mimic SERCA2b-accelerated cytosolic Ca2+ clearance
Based on these data from presenilin-null cell lines, we moved to a more regulatable system to directly establish whether presenilins modulate SERCA pump activity. We used the X. laevis oocyte expression system to monitor the clearance of Ca2+ ions from the cytosol after a transient influx across the plasma membrane. For this purpose, oocytes were induced to express the Ca2+-permeable nicotinic acetylcholine receptor (nAChR) that served as a "Ca2+ switch," allowing precisely controlled cytosolic Ca2+ transients to be evoked by pulsing the membrane potential to strongly negative voltages to increase the electrochemical driving force for Ca2+ entry. Oocytes were loaded with the Ca2+-sensitive dye Oregon Green BAPTA 1 and were voltage-clamped at a holding potential of 0 mV to minimize Ca2+ influx. In the presence of 100–500 nM acetylcholine, a brief (300 ms) hyperpolarizing pulse to –150 mV produced a transient Ca2+ fluorescence signal because of Ca2+ influx through open nicotinic channels. The decay rate of the fluorescence signals after termination of the voltage pulse was then used to quantify the rate of Ca2+ sequestration from the cytosol. For video footage of Ca2+ entry and subsequent clearance from the cytosol in this system please see Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200706171/DC1).
Fig. 3 A shows mean traces of Oregon Green fluorescence, illustrating differences in the decay rate of the Ca2+ signal in control, PS1-, PS2- and SERCA2b-expressing oocytes.
The decay presumably reflects a summation of several factors (e.g., diffusion of Ca2+ ions into the interior of the oocyte, mitochondrial uptake, and extrusion across the plasma membrane) in addition to sequestration into the ER by SERCA pumps. Consistent with this, decay kinetics were best fit by double-exponential processes (Fig. 2, B–D), with time constants of a few hundred milliseconds and a few seconds (Fig. 2 E). Sequestration by SERCA pumps is expected to be reflected primarily in the slower component (
2) and, in agreement with this interpretation, 2 was accelerated almost twofold in SERCA2b-overexpressing oocytes as compared with control cells.
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Pharmacological inhibition of SERCA prevents presenilin-mediated acceleration of cytosolic Ca2+ sequestration
Overexpression of either PS1, 2, or SERCA resulted in an accelerated sequestration of the cytosolic Ca2+ after a controlled Ca2+ influx across the plasma membrane, with PS2 having the most robust effect (Fig. 3). To prove that overexpression of presenilin was accelerating Ca2+ clearance from the cytosol by increasing SERCA activity, we measured Ca2+ clearance in the presence of thapsigargin, a specific inhibitor of SERCA. If our hypothesis was true, then presenilin should no longer accelerate clearance of Ca2+ from the cytosol in the presence of thapsigargin, compared with control oocytes also in the presence of thapsigargin. We incubated control and PS2-expressing oocytes in 30 µM thapsigargin in the bathing solution for 30 min. After a 30-min incubation, we applied a 300-ms hyperpolarization pulse, as before, to allow a controlled influx of Ca2+ across the plasma membrane into the cytosol and then tracked the clearance of this Ca2+ into the intracellular stores. In both control and PS2-expressing oocytes, thapsigargin reduced the speed of the Ca2+ fluorescence decay, with PS2-expressing oocytes showing the strongest effect, which is consistent with impairment of PS2 modulation of SERCA activity (Fig. 4, A and B).
Double-exponential curve fitting revealed that both control and PS2-expressing oocytes had similar 1 and 2 values, despite PS2-overexpressing oocytes having markedly faster 2 values compared with control oocytes in the absence of thapsigargin (Fig. 4 C).
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1 and 2 components with PS1M146V over control (Fig. 5 B). Rates of decay were faster than wild-type PS1 expression alone (1, 0.373 vs. 0.276; 2, 1.919 vs. 1.114), suggesting that this mutation impacts Ca2+ cytosolic sequestration more effectively than wild-type PS1 protein.
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50% (Fig. 6 D), and the decay of IP3-evoked Ca2+ transients was accelerated (Figs. 6, B and C).
These findings show that an increase in SERCA pump activity mimics the phenotype previously seen with presenilin overexpression.
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55 and 25 kD) are the same as of presenilin holoprotein (55 kD) or carboxy fragment (22 kD), and although using different species of polyclonal antibodies revealed bands at the correct weights, we could not absolutely determine whether they were presenilin or cross-reactivity with intraspecies IgG chains.
SERCA activity regulates Aβ production
Our data indicate that presenilins are required for normal SERCA function and physiological maintenance of cellular calcium homeostasis. Moreover, given that presenilins are integral for the production of Aβ, we investigated whether SERCA function influenced Aβ production. To address this issue, we used pharmacological and gain-of-function and loss-of-function genetic approaches. First, we considered the consequences of overexpressing SERCA2b in CHO cells stably expressing APP. After 48 h, we found that higher SERCA2b levels caused a marked increase in Aβ40 production (Fig. 8 A).
Conversely, reducing SERCA2b via siRNA-mediated knockdown caused a significant decrease in both Aβ40 and Aβ42 levels (Fig. 8 B). Pharmacological inhibition of SERCA2b with thapsigargin rapidly reduced Aβ40 and Aβ42 production (Fig. 8, C and D), which is in agreement with SERCA2b knockdown. The sum of these findings indicates that SERCA pump activity impacts Aβ production.
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As both presenilin and SERCA have activities that can be modulated by several proteins as well as by one another, this presents an interesting question: if presenilins regulate SERCA function, then can SERCA also regulate -secretase activity? Several studies have illustrated how modulating Ca2+ influx can modulate production of the Aβ peptide (Querfurth and Selkoe, 1994; Pierrot et al., 2004), as well as increasing intracellular store release (Querfurth et al., 1997). Indeed, any regimen that affects intracellular Ca2+ will invariably alter the activity of SERCA, as it is itself modulated by cytosolic and ER Ca2+ concentration as models have shown (Yano et al., 2004). In this paper, we demonstrate through both pharmacological and genetic means that modulation of SERCA2b function results in altered Aβ production with decreased SERCA function leading to decreased Aβ and increased SERCA function leading to increased Aβ. Therefore, it seems possible that SERCA activity plays a modulatory role in -secretase function or at least in APP processing leading to further generation of the Aβ peptide. Curiously, it has been reported that either stimulation (Pierrot et al., 2004) or inhibition (Yoo et al., 2000) of capacitative Ca2+ entrance (CCE) leads to increased production of Aβ42. Our results show that thapsigargin treatment, which depletes ER stores, diminishes Aβ production, yet depletion of ER stores leads to increased Ca2+ influx via CCE. This suggests that depleting ER stores of Ca2+ has a stronger effect on preventing Aβ production than CCE activation has on inhibiting it.
Numerous proteins, such as calsenilin, ryanodine receptor, and calmyrin (for review see Chen and Schubert, 2002), have been shown to interact with and bind the presenilins as well as the known components of the -secretase complex (nicastrin, PEN2, and Aph1), making the presenilins part of a very large multiprotein complex with multiple functions. Our results suggest that the interaction between SERCA and presenilin is an additional, but very important, interaction that serves to regulate sequestration of calcium into the ER stores, making presenilin a key component of cellular calcium homeostasis. A puzzling aspect is how mutations in the presenilins that modulate -secretase activity could cause the increases in intracellular Ca2+ signaling reported (Leissring et al., 1999a,b). Conversely, inhibitors of -secretase, which bind to the active site of presenilin in the -secretase complex, completely diminish ER Ca2+ release (Leissring et al., 2002; Kasri et al., 2006). Mutations in presenilin have been reported to disrupt the formation of ER leak channels, thereby preventing passive Ca2+ leak, which then leads to store overfilling (Tu et al., 2006) and, hence, exaggerated IP3-mediated release. Whether these same mutations also affect presenilins' modulation of SERCA function remains to be seen, but many of these FAD-linked mutations are associated with an increase, or at least change, in -secretase activity, making the prospect plausible. Indeed, we have shown here that the FAD-linked PS1 mutation M146V does show enhanced clearance of cytosolic Ca2+ compared with wild-type PS1. The requirement of SERCA activity to have presenilins present may provide problems for drugs that inhibit the actions of the -secretase complex to therapeutically reduced Aβ levels in AD. Such drugs have already been shown to abolish ER Ca2+ release in cell cultures (Leissring et al., 2002; Kasri et al., 2006) but their chronic in vivo use has yet to be documented. Certainly, reducing or abolishing ER Ca2+ release in neurons will have massive implications on memory and behavior, which suggests that BACE may be a more prudent drug target to reduce Aβ in AD.
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Materials and methods |
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RNA interference
A 20:1 stock solution of opti-MEM (Invitrogen) to Mirus TransIT-LT1 transfection reagent (Mirus Bio) was incubated at room temperature for 20 min. siRNAs were added and the solution was incubated for an additional 20 min at room temperature. This mixture was then added to cells, already in opti-MEM, to a final siRNA concentration of 25 nM of each partial sequence. The cells were then incubated in 5% CO2 atmosphere at 37°C for 12 h. Subsequently, cells were rescued with DME containing 10% FBS for an additional 12 h in 5% CO2 atmosphere at 37°C. Thereafter, cells were given the same siRNA treatment for an additional 12 h and rescued for the next 12 h with DME containing 10% FBS in 5% CO2 atmosphere at 37°C. Cells were then lysed and the lysates were stored at –80°C for further immunoblotting analysis. SERCA2b siRNA was acquired as a smartpool containing the following partial sequences: GUCAAUGUCGGUUU, CAAAGUUCCUGCUG, GAUCAUGUCUGUCA, and GAUAUAAGGUUAAC.
Oocyte calcium imaging and photolysis of caged IP3
Plasmids containing cDNA clones encoding for human PS1, PS2, SERCA2b, and the mouse muscle 
, β, , and 
nAChR subunits (S.F. Heinemann, Salk Institute, La Jolla, CA) were linearized and transcribed in vitro with SP6 or T3 RNA polymerases as previously described (Leissring et al. 1999b). The RNA transcripts were extracted with phenol-chloroform, precipitated with ethanol, and suspended in RNase-free water at a concentration of 1 µg/µl.
Defolliculated stage-VI oocytes from X. laevis were prepared as previously described (Demuro et al. 2005) and injected the next day with 46 nl of the appropriate cRNA mixtures. 3 d after cRNA injection and 1–4 h before Ca2+ imaging experiments, oocytes were injected with 23 nl of 2 mM of Oregon Green BAPTA-1 (Invitrogen) alone or together with 0.5 mM of caged Ins(1,4,5)P3 (D-myo-inositol 1,4,5-trisphosphate P4(5)-{1-(2-nitrophenyl)ethyl]ester; Invitrogen) for experiments involving IP3-mediated Ca2+ release.
For experiments imaging the clearance of Ca2+ after plasma membrane influx (Fig. 3), oocytes were voltage clamped using a conventional two-microelectrodes technique. The membrane potential was held at 0 mV during superfusion with ACh (100–500 nM) in Ringer's solution and was briefly (300 ms) stepped to –150 mV to strongly increase the electrical driving force for Ca2+ influx. Oocytes were imaged at room temperature by wide-field fluorescence microscopy using an inverted microscope (IX 71; Olympus) equipped with a 60x oil-immersion objective, a 488-nm argon-ion laser for fluorescence excitation, and a charge-coupled device camera (Cascade 128+; Roper Scientific) for imaging fluorescence emission (510–600 nm) at frame rates of up to 500 s–1. Fluorescence signals were monitored from a 40 x 40-µm region within the animal hemisphere of the oocyte and are expressed as a ratio (
F/Fo) of the mean change in fluorescence (F) relative to the resting fluorescence before stimulation (Fo) using MetaMorph software (MDS Analytical Technologies). Mean values of Fo were obtained by averaging over several frame before stimulation.
Experiments measuring Ca2+ liberation in response to photolysis of caged IP3 (Fig. 4) were performed using a linescan confocal microscope (IX-70 inverted microscope with a 40x oil-immersion fluor objective lens, using a 488-nm beam from a 100-mW argon laser [Callamaras and Parker, 1999]) to image fluorescence signals evoked by flashes of UV light (340–400 nm) from a mercury arc lamp, illuminating a disc of 100-µm diameter surrounding the 50-µm scan line in the animal hemisphere of the oocyte.
Protein immunoblotting
Protein extracts were prepared from cells using M-per (Thermo Fisher Scientific) extraction buffer and Complete Mini Protease Inhibitor Tablets (Roche). Protein concentrations were determined by the Bradford method. Equal amounts of protein (10 µg) were separated by SDS/PAGE on a 4–12% Bis/Tris gel (Invitrogen), transferred to PDVF membranes, blocked for 1 h in 5% vol/vol nonfat milk in Tris-buffered saline, pH 7.5, supplemented with 0.2% Tween 20, and incubated overnight at 4°C with primary antibody. Antibodies and dilutions used in this study include -SERCA2b (1:20,000; F. Wuytack, Katholieke Universiteit Leuven, Leuven, Netherlands), CTF20 (1:5,000; EMD), and -Actin (1:10,000; Sigma-Aldrich). Membranes were washed five times and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Quantitative densitometric analyses were performed on digitized images of immunoblots with Scion Image 4.0 (Scion Corporation).
Aβ ELISA
MaxiSorp immunoplates (Thermo Fisher Scientific) were coated with BAN50 at a concentration of 5 µg/ml in 0.1 M NaCO3 buffer, pH 9.6, and blocked with 1% Block Ace (Snow Brand Milk Products, Ltd.). Synthetic Aβ standards, internal controls, and samples were run at least in duplicate. After overnight incubation at 4°C, wells were probed with either HRP-conjugated BA27 (for Aβ1-40) or BC05 (for Aβ1-42) for 2–3 h at 37°C. 3,3',5,5'-tetramethylbenzidine was used as the chromogen, and the reaction was stopped by 6% O-phosphoric acid and read at 450 nm on a plate reader (Molecular Dynamics). Data are reported as mean per live cell + SEM, and statistical significance was evaluated using Student's t test.
Immunoprecipitation
50 µg MEF cell lysate was incubated with 40 µl of Protein A Sepharose beads (Sigma-Aldrich) for 1 h and centrifuged, and the supernatant was recovered. A further 40 µl of beads was added along with anti-PS1 (Cell Signaling Technology), PS2 (G. Thinakaran, University of Chicago, Chicago, IL), or p35 (Santa Cruz Biotechnology, Inc.) as a control (1:100), and the volume was made up to 1 ml with water and incubated overnight at 4°C overnight. After pelleting the beads, the supernatant was discarded and the beads were washed with STEN buffer (0.15 M NaCl, 0.05 M Tris HCl, 0.002 M EDTA, and 2% NP-40, pH 7.6) and then STEN containing 0.1% SDS. The beads were then pelleted and 4x loading buffer was added (Invitrogen). The samples were boiled for 10 min and spun down again, and the supernatant was run on a 4–12% Bis/Tris gel. SERCA2b was probed using SERCA2b (1:20,000; F. Wuytack).
Confocal microscopy
Fluorescent immunolabeling followed a standard two-way technique (primary antibody followed by fluorescent secondary antibody). Free-floating sections were rinsed in TBS, pH 7.4, and then blocked (0.25% Triton X-100 and 5% normal goat serum in TBS) for 1 h. Sections were incubated in primary antibody overnight at 4°C, rinsed in PBS, and incubated for 1 h in either fluorescently labeled anti–rabbit or anti–mouse secondary antibodies (Alexa 488, 1:200; Invitrogen). Confocal images were captured on a confocal system (Radiance 2100;Bio-Rad Laboratories). All double-labeled specimens were imaged using the 
-strobing function to prevent nonspecific cross-excitation of fluorophores.
Statistics
Data are presented as mean ± 1 SEM, with n = number of cells examined. An unpaired Student's t test was used to determine statistical significance (P < 0.05).
Online supplemental material
Video 1 shows representative calcium clearance from oocyte cytosol after a 300-ms influx through nAChR. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200706171/DC1.
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Acknowledgments |
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This work was supported by the National Institutes of Health (grants AG17968, AG16573, and GM48071).
Submitted: 26 June 2007
Accepted: 28 May 2008
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