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Address correspondence to Adam D. Linstedt, Dept. of Biological Sciences, Carnegie Mellon University, 4400 5th Ave., Pittsburgh, PA 15213. Tel.: (412) 268-1249. Fax: (412) 268-7129. E-mail: linstedt{at}andrew.cmu.edu
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Abstract |
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Key Words: Golgi structure; tether; docking; mitosis; COPI
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Introduction |
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Our current understanding concludes that kinases active at the G2/M transition, notably cyclin-dependent kinase I and mitogen-activated protein kinase kinase/extracellular signalregulated protein kinase (Achárya et al., 1998; Lowe et al., 1998; Kano et al., 2000), carry out phosphorylation reactions that lead, either directly or indirectly, to the inhibition of Golgi vesicle docking and fusion, which are fundamental processes underlying transport in the Golgi stack. The transport of newly synthesized proteins, termed cargo, through the Golgi stack involves their progressive encounter with modifying enzymes enriched in either the cis-, medial-, or trans-cisternal subcompartments. This is in part due to both the vesicle-mediated transport of cargo between adjacent cisternae (Rothman, 1994) and the ongoing anterograde progression of cargo-containing cisternae through the stack (Bonfanti et al., 1998). In the latter case, cisternal progression occurs as retrieval vesicles carry the modifying enzymes from later subcompartments backward to earlier subcompartments (Glick et al., 1997; Love et al., 1998; Lanoix et al., 1999). Thus, at M-phase, the continued formation of vesicles containing cargo and/or enzymes without their docking and fusion would lead to vesiculation of the organelle (Warren et al., 1995). Because the identified mitotic Golgi vesicles contain Golgi enzymes and other Golgi residents, it follows that these vesicles are probably produced by the same sorting reaction that produces retrieval vesicles during normal interphase trafficking. Alternatively, these vesicles could be produced by a mitotic modification of the cargovesicle sorting reaction allowing the incorporation of Golgi residents (Warren, 1985). In either case, evidence indicates that the mitotic Golgi vesicle formation reaction is largely mediated by the coatomer protein I (COPI)* complex (Misteli and Warren, 1994, 1995), which suggests that COPI vesicle docking and fusion components may be inhibited at the G2/M transition.
Importantly, the proteins implicated in COPI vesicle docking undergo alterations in their phosphorylation state at mitosis. The mitotic phosphorylation of at least one of these, Golgi matrix protein 130 (GM130), reduces its ability to bind its putative docking partner p115 (Nakamura et al., 1997). Collectively, the proteins GM130, Golgi reassembly stacking protein 65 (GRASP65), p115, and giantin are referred to as tethering proteins because they are thought to form a long complex capable of linking COPI vesicles to the Golgi apparatus over considerable distances (Sönnichsen et al., 1998). Giantin is a COOH-terminally anchored Golgi integral membrane protein (Linstedt et al., 1995) at least partially present in COPI vesicles (Sönnichsen et al., 1998). Giantin has a long rod-shaped cytoplasmic domain containing a p115 binding site in its predicted outermost NH2-terminal coiled-coil segment (Lesa et al., 2000; Linstedt et al., 2000). This site binds an acidic domain at the COOH terminus of the peripheral membrane protein p115 (Linstedt et al., 2000). This same acidic stretch in p115 also binds GM130 (Nelson et al., 1998; Linstedt et al., 2000). Giantin and GM130 compete to bind to p115 under certain in vitro conditions (Linstedt et al., 2000), however under different conditions, p115 is able to link giantin to GM130 (Dirac-Svejstrup et al., 2000). The simultaneous binding of p115 to Golgi cisternalocalized GM130 and COPI-localized giantin could link COPI vesicles to the Golgi membrane (Sönnichsen et al., 1998) because GM130 is stably bound to GRASP65, which is attached to the Golgi apparatus via a lipid anchor (Barr et al., 1997). Indeed, in vitro association of COPI vesicles to the Golgi membrane is blocked by antigiantin antibody treatment of the COPI vesicles, anti-GM130 treatment of the Golgi membranes, or depletion of p115 (Sönnichsen et al., 1998). Therefore, inhibition of the p115GM130 interaction by mitotic phosphorylation of GM130 by cyclin-dependent kinase I could account for mitotic Golgi vesiculation (Lowe et al., 1998).
Nevertheless, the precise role of the Golgi-localized tether proteins is complicated by their implication in a diverse set of reactions. The initial characterization of p115 was based on its essential role in an in vitro transport reaction designed to measure cis- to medial-Golgi transfer of vesicular stomatitis virus glycoprotein (Waters et al., 1992). Subsequently, it was also found to mediate fusion of transcytotic vesicles to acceptor membranes (Barroso et al., 1995). More recently, p115, giantin, GM130, and GRASP65 were revealed as a requirement for both cisternal regrowth and cisternal stacking (two subreactions during in vitro Golgi reassembly after mitotic fragmentation) (Shorter and Warren, 1999). GRASP65, as well as the related GRASP55, also play roles in Golgi stacking independent of GM130 (Barr et al., 1998; Shorter et al., 1999). p115 is also required for ER to Golgi transport in semi-intact cells, where it is recruited to COPII-coated vesicles by transient interaction with Rab1 followed by stable interaction with a vesicle SNARE protein complex containing syntaxin5, sly1, membrin, and rbet1 (Allan et al., 2000), and mediates translocation of ERGolgi intermediate compartment (ERGIC) from peripheral sites to the Golgi stack (Alvarez et al., 1999). Interestingly, once transport intermediates reach the Golgi region, evidence suggests that GM130/GRASP65 functions first, perhaps in binding vesicle-localized p115, whereas subsequent steps involve giantin (Alvarez et al., 2000).
The diversity of these reactions suggests that p115 and its binding partners mediate a number of distinct steps involving membranemembrane recognition. As the purpose of docking is to achieve a specific interaction between vesicle and target, it is important to determine whether specificity at each step is achieved by the use of distinct binding partners. Furthermore, the number of distinct in vitro reactions requiring tether proteins certainly raises the question of whether the corresponding in vivo reactions require them as well. For example, the belief that inhibition of p115GM130 interactions causes mitotic Golgi breakdown is not supported by the finding that the microinjection of a peptide that prevents binding of p115 to GM130 leads to no apparent Golgi breakdown (Seemann et al., 2000). On the other hand, the microinjection of anti-p115 antibodies causes Golgi breakdown (Alvarez et al., 1999). This discrepancy leads to the hypothesis that p115 has an essential role in Golgi biogenesis independent of its interaction with GM130. As GM130 and giantin bind the same domain in p115, the injected GM130 NH2-terminal peptide would presumably also inhibit p115giantin complex formation. Therefore, p115's essential role could be independent of both giantin and GM130, the two proteins that it is thought to bridge during COPI vesicle docking.
We sought to test this hypothesis and further characterize the role of p115 in the regulation of Golgi structure in relation to its binding partners giantin and GM130 in vivo. Antibodies were made against the corresponding binding sites of each protein and injected into HeLa cells. Unlike giantin and GM130, the inhibition of p115 alone in interphase cells induced apparent COPI-dependent Golgi vesiculation. This suggests that p115 plays a role in maintenance of Golgi structure that may be relevant to mitotic Golgi breakdown, but this role is independent of its interactions with giantin and GM130.
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Results |
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To test whether the loss of cellular p115 after anti-p115 microinjection was due to targeted degradation, we performed similar microinjection experiments in the presence and absence of a proteasome inhibitor. As expected, control cells examined 3 h after injection exhibited no detectable p115 staining (Fig. 1 E). In contrast, cells treated with the proteasome inhibitor MG132 during the 3 h after injection incubation exhibited readily detectable juxtanuclear p115 staining (Fig. 1 F). The staining pattern was markedly punctate suggesting that the injected antibodies had cross-linked p115 causing it to cluster on the Golgi membrane. In the absence of proteasome activity this failed to cause its degradation. These observations suggest that the binding of the injected antibody somehow targets p115 to the proteasome perhaps by inducing its recognition by a ubiquitin ligase. In any case, this provides an opportunity to study various cell functions in the apparent absence of p115.
Absence of p115 induces apparent COPI-dependent Golgi vesiculation
To test the effect of p115 loss on Golgi structure, we stained the Golgi apparatus in cells that had been incubated for various times after microinjection of anti-p115. The staining was performed using a monoclonal antibody that recognizes Golgi phosphoprotein 130 (GPP130), an integral membrane component of the cis-Golgi apparatus (Linstedt et al., 1997). The Golgi pattern remained normal 10 min after injection (Fig. 2 A), but 30 min after injection, the Golgi apparatus was noticeably fragmented (Fig. 2 B). 2 h after injection, the Golgi apparatus was present as dispersed fragments and there was a noticeable increase in diffuse staining (Fig. 2 C). 4 h after injection, the Golgi apparatus was almost entirely present as diffuse cytoplasmic staining with a few larger structures, or remnants, still visible (Fig. 2 D). A similar pattern of Golgi breakdown was observed when giantin or GM130 was used to mark the Golgi apparatus, and also when cells were allowed to pass through mitosis after injection, as described below (unpublished data). Injection of control anti-GST antibodies had no detectable effect on the Golgi apparatus at similar time points (unpublished data). Thus, loss of p115 led to a dramatic breakdown of at least the early Golgi structure.
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In light of these observations, we tested whether cells lacking detectable giantin retained the capability to undergo Golgi reassembly after either BFA-induced Golgi redistribution to the ER or mitosis-induced Golgi vesiculation. Indeed, the entire cycle of BFA-induced GolgiER fusion and Golgi re-emergence upon BFA washout was indistinguishable in microinjected and adjacent, uninjected cells, even though the experiments were carried out under conditions that reduced giantin levels below detection (unpublished data). Furthermore, cells lacking detectable giantin were able to divide (Fig. 6 E) and exhibited normal Golgi patterns (Fig. 6 F). For these experiments, isolated single cells on each coverslip were injected, incubated for 20 h, and analyzed. At the end of the incubation, each injected cell had doubled indicating passage through mitosis after injection. Normal Golgi reassembly was also evident in microinjected cells that were costained for microtubules (Fig. 6 G) and the Golgi complex (Fig. 6 H), and postmitotic pairs were identified by the short microtubule bundle at the site of cytokinesis. Therefore, cells lacking detectable giantin were viable, exhibited a normal Golgi staining pattern, and underwent cycles of Golgi disassembly/reassembly.
Microinjection of anti-GM130 antibodies that prevent GM130p115 complex formation
Polyclonal antibodies were also generated against the NH2-terminal domain of GM130, which contains its p115 binding site. Microinjection of these antibodies with subsequent incubations up to 16 h had no apparent effect on the staining patterns for giantin (Fig. 7 A), p115 (Fig. 7 B), GPP130 (unpublished data), and KDEL receptor (unpublished data). Not only was the interphase Golgi structure normal in anti-GM130injected cells, but BFA-induced GolgiER fusion and Golgi reemergence upon BFA washout was indistinguishable in microinjected and adjacent, uninjected cells (unpublished data). Also, when isolated single cells on each coverslip were injected and incubated for 20 h, the cells divided normally and the daughter cells reassembled a normal Golgi apparatus as indicated by staining for GPP130 (Fig. 7 C).
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In the first experiment, an immobilized GSTGM130 fusion protein containing its p115 binding site (Linstedt et al., 2000) was incubated with p115-containing cell extracts in the presence of anti-GM130 or control antibodies. Despite the relatively high concentration of GM130 present in the assay, the anti-GM130 antibody showed a concentration-dependent inhibition of p115 binding, whereas control anti-GST antibodies did not (Fig. 8 A). Thus, the anti-GM130 antibody had inhibitory activity against p115GM130 complex formation, yet its microinjection did not displace p115 from the Golgi complex (Fig. 7 B). This observation is actually consistent with previous reports indicating that versions of p115 lacking the GM130 binding domain are still Golgi apparatus targeted (Nelson et al., 1998); that is, p115 appears to have at least one other Golgi-localized receptor. The receptor in question is not giantin because the giantin binding site in p115 maps to the same position as the GM130 binding site (Linstedt et al., 2000). Furthermore, co-injection of both antigiantin and anti-GM130 also did not displace p115 or cause any detectable Golgi abnormality (unpublished data). Therefore, to confirm that anti-GM130 antibodies disrupt p115GM130 interaction without blocking p115 membrane binding, we performed a second binding experiment.
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Discussion |
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Although there was not an essential role for the tether complex, our observations confirmed that p115 is required for Golgi structural maintenance (Alvarez et al., 1999). This indicates that p115 acts in a pathway that is independent, or at least can function independently, of interactions with giantin and GM130. Absence of p115 induced apparent accumulation of Golgi vesicles suggesting that Golgi vesicle docking is in fact a key role of p115 (Waters et al., 1992). Anti-p115induced Golgi vesiculation was prevented by CBM-mediated COPI displacement, pointing to a COPI origin for the accumulated vesicles. This is compatible with the in vitro requirement of p115 for binding of COPI-coated vesicles to isolated Golgi membranes (Sönnichsen et al., 1998).
Presumably, cessation of ER to Golgi transport in anti-p115injected cells also contributed to loss of Golgi structure, as Golgi structure depends on input of membrane and other factors from the ER (Storrie et al., 1998), and p115 is required for ER to Golgi transport in semi-intact cells (Alvarez et al., 1999; Allan et al., 2000). There is also evidence that p115 is required for ER to Golgi transport in intact cells, as p115 is required for Golgi reassembly after BFA washout (Fig. 5 B; Alvarez et al., 1999), a reaction that involves transport of this type. Importantly, in semi-intact cells, inhibition of p115 blocks transport at the step where vesicular tubular clusters of the ERGIC would otherwise translocate from peripheral sites in toward the juxtanuclear Golgi complex (Alvarez et al., 1999). A role for p115 at this step would explain why peripheral ERGIC was maintained in the absence of p115 (Fig. 5 A) despite vesiculation of the Golgi complex. In fact, one possibility is that p115's sole required function is in the docking of Golgi-derived COPI-coated retrieval vesicles with the ERGIC. This reaction may contribute components that are essential to the translocation and maturation of pre-Golgi elements as they form new cis-cisternae. Components required for translocation and maturation might include receptors for cytoplasmic dynein and early Golgi enzymes, respectively. This view explains both COPI vesicle accumulation and arrest of vesicular tubular cluster translocation/maturation in the absence of p115. It is also consistent with recruitment of p115 into a SNARE complex during COPII formation (Allan et al., 2000), as this complex would then be used for the subsequent docking and fusion of COPI retrieval vesicles with ERGIC membranes. Furthermore, this reaction may be analogous to p115-dependent cisternal growth during in vitro Golgi reassembly (Shorter and Warren, 1999).
If p115's essential function for Golgi structure maintenance is to mediate COPI vesicle docking and fusion with the ERGIC, then this reaction apparently involves p115 binding partners other than giantin and GM130. Why then are these proteins required in p115-dependent reactions in vitro? One possibility is that giantin and GM130 do function together with p115 in intact cells, but that they function in transport steps other than COPI vesicle/ERGIC docking. Further, these other functions that involve p115, giantin, and GM130 must play a facilitating role rather than an essential role. This would fit with their description as tethers, because tethering is expected to be the outermost interaction in a series of interactions between vesicle and target membranes, which culminate in targeted membrane fusion. In the absence of tethering, downstream components, such as the interaction of cognate SNARE proteins, may suffice, albeit less efficiently. The situation could be dramatically different for in vitro assays. Because of dilution and disruption of spatial organizing features such as the cytoskeleton and membrane stacking, a requirement for tethering may be revealed. Thus, giantin and/or GM130 may function together with p115 in steps such as docking at the early Golgi complex (Alvarez et al., 1999; Allan et al., 2000), intra-Golgi COPI vesicle transport (Waters et al., 1992), and Golgi stack formation (Shorter and Warren, 1999); however not in a capacity required for Golgi biogenesis in vivo. It is also formally possible that GM130 and giantin are mutually redundant or that their respective functions are redundant with as yet unidentified proteins. However, the fact that co-injection of both antigiantin and anti-GM130 antibodies had no detectable effect on Golgi structure argues against mutual redundancy; and the lack of any identified proteins with meaningful sequence similarity in the human genome database argues against unidentified family members.
The idea that p115 plays both an essential role independent of giantin and GM130 as well as a nonessential role involving giantin and GM130 helps explain the noted lack of Golgi structural perturbation in cells where the GM130 NH2 terminus was used to displace p115 from the Golgi apparatus (Seemann et al., 2000). Displacement of p115 from the Golgi complex, may block the nonessential role but have no effect on the essential role, as the essential role may involve, not Golgi-localized p115, but rather ERGIC-localized p115 initially recruited to membranes during COPII vesicle formation (Allan et al., 2000). This model could also account for the apparent sequential action of p115 relative to giantin and GM130 in semi-intact cell transport from the ER to the medial-Golgi complex (Alvarez et al., 2000), as the putative role of p115 in ERGIC/COPI docking is upstream of its putative role in docking at, or within, the Golgi complex. Also, the model is consistent with the fact that the Saccharomyces cerevisiae homologue of p115, Uso1p, is essential for viability (Nakajima et al., 1991), whereas this yeast does not have homologues for either giantin or GM130. Furthermore, the Uso1p acidic domain, which would be expected to mediate interaction with giantin or GM130 homologues, if they existed, is not required for growth (Seog et al., 1994).
Does tether inhibition at mitosis lead to Golgi vesiculation? Golgi breakdown in interphase cells in response to anti-p115induced p115 degradation mimicked, to a first approximation, Golgi vesiculation in mitotic cells. This suggests that inhibition of p115 may play a major role in mitotic Golgi vesiculation. Indeed, there is evidence suggesting that at least the nonessential role of p115 is inhibited at mitosis, as p115's ability to interact with giantin and GM130 is reduced by dephosphorylation (Dirac-Svejstrup et al., 2000) and p115 becomes dephosphorylated at M-phase (Sohda et al., 1998). On the other hand, p115 inhibition is not likely to be the sole requirement for mitotic breakdown because the time course of the interphase breakdown (t1/2 2 h) was considerably slower than that which occurs from prophase to metaphase (<30 min), even after accounting for the rate of p115 degradation (t1/2
1 h). With regard to giantin and GM130, absence of detectable giantin (Fig. 6) and prevention of GM130p115 interactions (Fig. 7) did not cause Golgi breakdown. This argues that mitotic phosphorylation of GM130 (Lowe et al., 1998), which prevents p115 binding (Nakamura et al., 1997), and mitotic phosphorylation of giantin (unpublished results) are not sufficient to cause Golgi vesiculation, although inhibition of these proteins by phosphorylation may still contribute to the vesiculation reaction. Further, these results suggest that one of the reactions that may be involved, in addition to p115 inhibition at mitosis, would be the inhibition of those transport factors that act immediately after the tethers in the docking and fusion of Golgi-derived vesicles.
Also noteworthy, was the phenomena of antibody-induced antigen degradation. To our knowledge this has not been previously observed, perhaps because many other studies have not stained for the corresponding antigen in antibody-injected cells. Because the loss of staining was time, temperature, and proteasome dependent, and because it followed an apparent clustering of the antigen, we suspect that antibody-induced cross-linking of the antigen somehow triggered binding and ubiquitination of the antigens by a ubiquitin ligase. In the case of giantin and the Golgi-localized fraction of p115, this was probably followed by proteasome recruitment to the Golgi membrane. The roles of cross-linking, ubiquitination, and proteasome localization are important subjects for future study. Giantin and p115 are long-lived proteins suggesting that targeted degradation does not normally play an important role in their regulation. Rather, the antibody-induced degradation of these antigens suggests that if the mechanism can be understood, it may be possible to engineer antibodies that cause degradation of any antigen of interest. This could be significant for further tests of in vivo function by creation of "knock-outs" without requiring targeted gene disruption or antisense RNA. In the cases of anti-p115induced p115 degradation and antigiantin-induced giantin degradation examined here, this phenomena provided strong support for the hypothesis that the essential function of p115 in Golgi structure is independent of its hypothesized role in a giantinp115GM130GRASP65 tether and sets the stage for further work into the roles of these proteins.
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Materials and methods |
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Microinjection and analysis
HeLa cells were cultured on glass coverslips as described (Lee and Linstedt, 1999). For CBM treatments, the coverslips were coated with SuperFibronectin (Sigma-Aldrich). Before microinjection, the culture medium was adjusted to 25 mM Hepes-KOH (pH 7.4) and the thawed antibody aliquots were centrifuged at 100,000 rpm for 1 h in a TLA 100.3 rotor (Beckman Coulter). The cells were maintained at 37°C, injected using an Eppendorf microinjection system, and then were returned to the incubator. Staining and digital image acquisition were as previously described (Linstedt et al., 1997). Using the injected antibody staining pattern and the Trace Contour command in Adobe Photoshop, the injected cells were outlined. In certain cases, the staining of the injected antibody was omitted to exclude any possibility of bleedthrough. For digitonin release, each coverslip was incubated for 6 min at room temperature in 0.5 ml of 0.04 mg/ml digitonin in KHM (100 mM KCl, 25 mM Hepes-KOH 7.4, 2.5 mM magnesium acetate) before fixation. Mean cytoplasmic fluorescence was measured from 10,125 pixels in each cell, manually excluding the juxtanuclear area, using the Histogram function in Adobe Photoshop. Background, taken as the average pixel value outside the cells, was subtracted.
Binding experiments
For measuring binding of p115 to GST-GM130, normal rat kidney (NRK) cells were lysed in HKT (10 mM Hepes-KOH, pH 7.2, 100 mM KCl, 1 mM EDTA, 0.5% Triton X-100) and the lysate cleared by centrifugation at 100,000 rpm in the TLA 100.3 rotor. The cleared lysate (0.1 ml) was then added to a mixture of 5 µl GST-GM130coated glutathione-agarose beads (0.04 mg/ml) and the indicated amounts of the anti-GM130 or anti-GST polyclonal antibodies. After rotation for 60 min at 4°C, the beads were washed four times with 1 ml of HKT and eluted with 0.5 ml of 0.6 M KCl in HKT. Immunoblotting of the eluate was as described (Linstedt et al., 2000). To test for co-immunoprecipitation of p115GM130 complexes from antibody-treated membranes, NRK cells were homogenized in KHM using a 25-gauge needle and centrifuged to obtain a postnuclear supernatant. The postnuclear supernatant was then underlayed with 10 µl of 80% sucrose and centrifuged at 16,000 g for 20 min at 4°C. The membranes were collected and incubated with various amounts of anti-GM130 or antigiantin polyclonal antibodies for 60 min on ice. The membranes were then solubilized with HKT and the lysate was centrifuged at 50,000 rpm in the TLA 100.3 rotor for 30 min. The cleared lysate was rotated at 4°C for 60 min with 20 µl of packed Affi-Gel beads that had been coupled to the anti-p115 polyclonal antibody (Bio-Rad Laboratories). Washing, elution, and detection were then performed as before. To assay membrane-associated p115 after anti-GM130 or antigiantin incubation, membranes were prepared and incubated with antibodies exactly as just described. The antibody-treated membranes were then adjusted to 1 ml KHM, underlayed with 10 µl of 80% sucrose, and centrifuged as before. After four such washes the amount of p115 and GM130 was determined by immunoblotting.
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Footnotes |
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Acknowledgments |
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This work was supported by a National Institutes of Health grant GM-56779-02 to A.D. Linstedt.
Submitted: 1 May 2001
Revised: 12 July 2001
Accepted: 4 September 2001
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References |
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