1 Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia
2 The University of California, Davis, UC Davis Medical Center, Research III, Suite 3300, 4645 2nd Avenue, Sacramento, CA 95817, USA
Correspondence
Barbara S. Coulson
barbarac{at}unimelb.edu.au
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ABSTRACT |
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Present address: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, Ohio State University, Wooster, OH 44691, USA.
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INTRODUCTION |
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Integrins and their natural ligands are essential for epithelial cell adhesion to the extracellular matrix, immune responses and lymphocyte trafficking (Hynes, 2002; Mittelbrunn et al., 2004
). The
4 integrin subunit pairs with either
1 or
7 to form part of a subfamily that also includes
9
1 (Hynes, 2002
). The
4
1 integrin is expressed on mesenchymal cells in the intestinal lamina propria. These cells are important for mucosal inflammation and repair (Choy et al., 1990
; Pender et al., 2000
; Powell et al., 1999
). The
4 integrins are highly expressed on B and T lymphocytes, dendritic cells, monocytes and natural killer cells (Hynes, 2002
; Pender et al., 2000
). Intestinal epithelial cells express
9
1 (Basora et al., 1998
; Yokosaki et al., 1999
). The intestinal homing receptor,
4
7, binds to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on post-capilliary venules. In contrast,
4
1 preferentially engages fibronectin and vascular cell adhesion molecule-1 (VCAM-1) to induce intestinal mesenchymal cell migration (Pender et al., 2000
) and control B- and T-lymphocyte development, proliferation and survival (Mittelbrunn et al., 2004
; Tidswell et al., 1997
).
Protection from rotavirus reinfection is primarily mediated by rotavirus-specific intestinal immunoglobulin A (IgA) in animals (Franco & Greenberg, 1999), and correlates with this IgA in humans (Coulson et al., 1992
). The presence of rotavirus-specific, antibody-secreting cells in the intestine correlates with their detection in blood (Brown et al., 2000
; Yuan et al., 1996
). Human rotavirus-specific, antibody-secreting B cells and CD4+ T cells predominantly express
4
7 (Gonzalez et al., 2003
; Rott et al., 1997
). In mice, rotavirus-specific B cells require
4
7 for protective immunity (Williams et al., 1998
; Youngman et al., 2002
), and memory T cells strongly express
4
7 (Rose et al., 1998
; Rott et al., 1997
). Thus, effective rotavirus immune responses depend on MAdCAM-1 recognition by lymphocytes expressing
4
7.
Potential ligand sequences for 4
1 and
4
7 are found in the outer capsid proteins of most rotaviruses. The sequences IleAspAla (IDA) and LeuAspVal (LDV) in fibronectin that are important for
4
1 and
4
7 recognition (Chan et al., 1992
; Komoriya et al., 1991
; Ruegg et al., 1992
; Sharma et al., 1999
; Wayner & Kovach, 1992
) are present in VP5* and the disintegrin-like domain of VP7, respectively. This VP7 domain also contains the sequence LeuAspIle (LDI) that is closely related to LDV (Coulson et al., 1997
; Hewish et al., 2000
). Simian rotavirus SA11 binds cell surface-expressed
4
1, facilitating infection and replication (Hewish et al., 2000
). Mouse polyomavirus also contains LDV sequence, and uses
4
1 as an entry factor (Caruso et al., 2003
).
Two aims of this study were to determine if human and porcine rotaviruses interact with 4
1 and to detect any rotavirus recognition of
4
7. The presence in rotavirus VP5* of the TyrGlyLeu (YGL) sequence used by osteopontin to bind
4 and
9 integrins (Barry et al., 2000
; Green et al., 2001
; Yokosaki et al., 1999
) is reported here for the first time, leading to the hypothesis that this YGL sequence might mediate rotavirus recognition of
4 integrins. As
4 and
9 integrins share ligand specificity, rotavirus usage of
9
1 was examined. The roles of rotavirus IDA, LDV, LDI and YGL sequences in
4 integrin recognition were studied here for the first time in cells with demonstrable
4 expression.
Integrin subunits combine extracellularly to form a head and two legs. These domains exhibit structural rearrangements between the closed, low affinity conformation and the open, high affinity activated state (Hynes, 2002; Xiao et al., 2004
). The N-terminal
440 aa of integrin
-subunits contain regions important for ligand binding and consist of seven sequence repeats that are predicted to fold into a
-propeller domain in the integrin head (Springer, 1997
). Propeller blades are connected by loop structures. Loops that are critical for
4 adhesion to VCAM-1, fibronectin and MAdCAM-1 are grouped in the upper face of the
-propeller (Higgins et al., 2000
; Irie et al., 1997
; Ruiz-Velasco et al., 2000
). Another hypothesis tested here was that rotaviruses bind
4
1 through the recognition of the same
4 sequences as natural
4 ligands.
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METHODS |
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Cell lines and viruses.
The generation and maintenance of the cell lines utilized in this study and monitoring of their cell surface integrin expression by flow cytometry were carried out as described previously (Eto et al., 2000; Hewish et al., 2000
; Higgins et al., 2000
; Irie et al., 1995
, 1997
; Londrigan et al., 2000
). The cell lines utilized were: human K562, Caco-2 and rhabdomyosarcoma (RD), hamster CHO B2 and CHO K1, and monkey MA104 cells; K562 cells transfected with cDNA encoding human integrin subunits
3 (
3-K562),
4 (
4-K562),
9 (
9-K562) and empty vector (PBJ-K562); CHO B2 cells transfected with cDNA encoding human
4 integrin-subunit (CHO B2
4), mutant
4 in which
4-subunit loops were replaced with the corresponding
5 integrin-subunit regions R1 (residues 4052), R2 (residues 112131), R3a (residues 151164), R3b (residues 181189), R3c (residues 186191), R4 (residues 237247) and R5 (residues 282288) (CHO B2
4 R1, CHO B2
4 R2, CHO B2
4 R3a, CHO B2
4 R3b, CHO B2
4 R3c, CHO B2
4 R4 and CHO B2
4 R5, respectively) and human
4 and
7 (
4
7-CHO B2); CHO K1 cells transfected with cDNA encoding human
4 (CHO K1
4) and
4 with point mutations Y120A/G130A (CHO K1
4 Y120A/G130A), Y187A (CHO K1
4 Y187A) and G190A (CHO K1
4 G190A), and human
4 and
7 (
4
7-CHO K1). The presence of the expected epitopes on recombinant human
4 and
7 expressed on the cell surface was confirmed by flow cytometry using mAbs P4C2 and P4G9 for wild-type and mutant
4, respectively, and mAb Fib27 for
7. The origins of the rotaviruses that were used in this study, and their cultivation in MA104 cells, have been described previously (Graham et al., 2003
; Kirkwood et al., 1993
).
Virus binding, infectivity and growth assays.
The assays used to determine the titre of infectious rotavirus bound to cells, and measure peptide and mAb inhibition of virus binding were carried out as described previously (Coulson et al., 1997; Graham et al., 2003
, 2004
; Hewish et al., 2000
; Londrigan et al., 2003
). Briefly, washed cells were incubated at 37 °C with peptides (0·5 mM) at pH 7·5 for 1 h or mAbs (10 µg ml1) for 2 h. This antibody concentration was determined by flow cytometry to be the lowest saturating concentration. In the presence of peptide or antibody, cells were cooled on ice for 20 min. Trypsin-activated virus (m.o.i. of 3·5 unless otherwise stated) was cooled to 4 °C and allowed to attach to cells on ice for 1 h. Cell-bound virus titres were determined by indirect immunofluorescent staining of infected MA104 cells, and expressed as focus-forming units per ml (f.f.u. ml1) (Hewish et al., 2000
). The effects of peptides at 0·5 mM (unless otherwise stated), and mAbs on virus infectivity (m.o.i. of 0·02) and growth (m.o.i. of 0·35) were measured by a fluorescence focus reduction-assay (Coulson et al., 1997
; Graham et al., 2004
; Hewish et al., 2000
; Londrigan et al., 2003
). For mock infection, cell cultures were inoculated with lysates of confluent, uninfected MA104 cells that had been trypsin-treated and diluted as for the virus infectivity activation. The error bars on the graphs presented represent the standard deviation (SD).
Flow cytometric assay of virus infectivity.
The proportion of PBJ-K562 and 4-K562 cells that were rotavirus-infected was determined by flow cytometry, as these cells showed non-adherent growth. Cells (5x105) that had been infected with trypsin-activated rotaviruses RRV, CRW-8, Wa or RV-5 (m.o.i. of 10) for 16 h were washed once with PBS containing 1 % (v/v) fetal calf serum (PBS/FCS). Cells were permeabilized by methanol fixation for 7 min at 20 °C and washed with PBS/FCS. Cell-associated viral antigen was detected with rabbit antiserum to RRV, using as a negative control normal rabbit serum in which antibodies to rotavirus were present at a titre of <1 : 100 by ELISA (Graham et al., 2003
). Cells were further processed for flow cytometry using a two-step stain. This flow cytometric method was validated using rotavirus-infected MA104 cells, by comparison with visual counting of infected cells in fixed monolayers stained by indirect immunofluorescence with the same antiserum (Hewish et al., 2000
).
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RESULTS |
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The identity of the viral gene(s) involved in 4
1 usage was examined using laboratory-generated reassortants that contained a heterologous VP7 gene segment. Several of these reassortants were used previously to map rotavirus recognition of other integrins (Graham et al., 2003
). In three reassortants, the
4
1-binding phenotype segregated with VP4 (Table 1
). A further three reassortants bound
4
1, consistent with the phenotypes of their parental viruses. Rotavirus usage of
4
1 did not co-segregate with any other viral gene.
Rotavirus binding to 4
1 and
4
7 depended on the ligand-binding domain of
4 and was inhibited by integrin ligand peptides
The ability of rotaviruses to bind recombinant human 4
7 was tested by comparing virus binding to CHO B2,
4-CHO B2 and
4
7-CHO B2 cells. Flow cytometric histograms of
4 expression, determined with mAb P4C2, were identical for
4-CHO B2 and
4
7-CHO B2 cells, so their cell surface levels of
4 were indistinguishable. The relative linear median fluorescence intensity (MFI) of
4 expression was calculated from these histograms, using previously described methodology (Graham et al., 2003
), as 3·5 and 3·2, respectively. As expected, only
4
7-CHO B2 cells expressed the
7-subunit, which was detected with anti-
7 antibody Fib27. The MFI of
7 expression on
4
7-CHO B2 cells was 2·8. CHO B2 cells did not express
4 or
7, as they showed an MFI of 1·0 for these integrin subunits.
4-CHO B2 cells did not express
7 (MFI of 0·8). Similar numbers of
4-K562 cells and K562 cells transfected with
4
7 bind fibronectin CS-1 and VCAM-1 (Guerrero-Esteo et al., 1998
; Munoz et al., 1997
; Ruiz-Velasco et al., 2000
). Thus, alteration of the
4
1 activation state on CHO cells by
7 expression is unlikely.
SA11, RRV and Wa bound to human 4 combined with hamster
1 on
4-CHO B2 cells, but bound to a higher level to
4
7-CHO B2 cells that expressed both human
4 combined with hamster
1, and human
4
7 (Fig. 1
). Thus, these viruses bound to both
4
7 and
4
1 on
4
7-CHO B2 cells. Similar results were obtained using
4
7-CHO K1 and
4-CHO K1 cells (data not shown). SA11 binding to
4-K562 cells was abolished by cellular treatment with mAb P4C2 (Hewish et al., 2000
). This antibody maps to the second and third N-terminal repeats (R2 and R3) of
4 that contain residues critical for ligand binding (Irie et al., 1995
, 1997
; Kamata et al., 1995
). P4C2 also eliminated RRV and Wa binding to
4-K562 cells, as virus titres bound in the presence of this antibody were indistinguishable from those bound to PBJ-K562 and
3-K562 cells (Fig. 2
). P4C2 similarly abrogated rotavirus binding to
4
7. In contrast, the function-blocking mAb Fib27 that maps to aa 176237 of
7 (Tidswell et al., 1997
) had no effect on virus binding to
4
7. Similarly, function-blocking anti-
1 antibody 4B4 that inhibited SA11 binding to MA104 cells (Coulson, 1997
) did not affect RRV binding to
4
1. CRW-8 binding to CHO and K562 cells was independent of
4
1 or
4
7 and unaffected by the anti-
4 or -
7 mAbs.
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Infectivity and growth of some rotaviruses were mediated through 4
1 and
4
7
SA11 binding to 4
1 resulted in increased SA11 yield (Hewish et al., 2000
). The abilities of other rotaviruses to infect and replicate via
4
1 were determined. In the presence of
4
1, the number of K562 cells infected by RRV and CRW-8 (detected by flow cytometry) increased from a mean±SD of 45·3±1·9 to 56·9±2·0 % and from 0·1±0·0 to 24·7±0·4 %, respectively (Fig. 3a
). RRV and CRW-8 yields increased four- to sixfold due to
4 expression (Fig. 3b
). The negligible proportion of
4-K562 cells infected by Wa (0·2±0·1 %) was similar to that in K562 cells (0·1±0·1 %) and mock-infected cells (0·2±0·1 %), and no infectious Wa or RV-5 was produced by PBJ-K562,
3-K562 or
4-K562 cells at 2472 h post-infection (p.i.) (data not shown). RRV and CRW-8 growth in
4-K562 cells was inhibited by SVVYGLR and IDAPS (Fig. 3c
), the same peptides that abolished virus binding to
4 integrins (Fig. 2
). Thus, although SA11, RRV, Wa and RV-5 all bound
4
1, only SA11 and RRV were able to utilize this binding for infection and virus growth in K562 cells. Conversely, CRW-8 interacted with
4
1 to greatly increase its infectivity, but did not use this integrin for initial cell binding.
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CHO B2 cells lack 5 integrin expression, so they lost adhesion during prolonged incubation at 37 °C (Schreiner et al., 1989
). Therefore, rotavirus infectivity mediated by
4
7 was determined using
4
7-CHO K1 cells. The mean±SD of the respective SA11 and RRV titres in f.f.u. ml1 produced at an m.o.i. of 40 in
4
7-CHO K1 cells (5800±750 and 6500±1000) were significantly higher than those in CHO K1 cells (2400±750 and 3300±850) at 16 h p.i. (t-test, P<0·0001). Wa and CRW-8 did not infect either cell line. Thus, binding of SA11 and RRV, but not Wa, to
4
7 resulted in increased infectivity in CHO cells.
Rotaviruses did not utilize 9
1 for cell attachment or entry
SA11, RRV and CRW-8 did not use recombinant 9
1 for K562 cell infection (Fig. 3b
) or replication (Fig. 4a
, lower panel). SA11 (Fig. 4a
, upper panel) and Wa (data not shown) did not bind
9
1. Caco-2 cell expression of
9 was detected by flow cytometry with anti-
9 mAb Y9A2 (data not shown). This antibody did not affect SA11, RRV and Wa infection of Caco-2 cells, and peptide SVVYGLR did not affect SA11 infection of Caco-2 cells (Fig. 4b
). Thus,
9
1 was not a rotavirus receptor or entry co-factor.
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Domains and sequence of the 4-subunit required for rotavirus binding
The 4 domains crucial for rotavirus binding were located using swapping mutagenesis. Predicted protruding loops at or near
4
1 sites necessary for natural ligand binding were replaced with corresponding
5 regions (Higgins et al., 2000
; Irie et al., 1997
). Rotaviruses do not bind
5
1. Therefore, if the swapped
4 region is necessary for virus binding, swapping to the
5 region will prevent virus binding to
4. The region swapping will affect virus binding only if the exchanged regions are sufficiently distinct in sequence. The R3b and R3c domains are conservative in sequence between
4 and
5. The only point mutations in the putative ligand-binding region of
4 (aa 108268) of 61 tested that abolish natural
4 ligand binding are non-conservative and located in R2 (Y120/G130), R3b (Y187) and R3c (G190) (Higgins et al., 2000
; Irie et al., 1995
, 1997
). As shown in Fig. 6
,
4 was detected at similar levels on all CHO cell lines transfected to express these swapping and point mutations in
4, using anti-
4 antibody P4G9 that maps to an epitope outside the swapped domains (Irie et al., 1997
; Kamata et al., 1995
). Binding of RRV, SA11 and Wa to
4
1 on CHO B2 cells was abolished by swapping R2 or R4, but was unaffected by swapping R1, R3a, R3b, R3c or R5 (Fig. 7
). The point mutations eliminated Wa, RRV and SA11 binding to
4
1. CRW-8 did not bind
4
1 on any cell line tested, showing the specificity of the Wa, SA11 and RRV binding (Fig. 7
). CS-1 peptide, VCAM-1 and MAdCAM-1 also required
4 R2 and R4, but not R1, R3a, R3b, R3c or R5 for binding (Higgins et al., 2000
; Irie et al., 1997
). Thus, the same
4 regions and amino acids are critical for
4 binding by both rotaviruses and these natural
4 ligands.
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DISCUSSION |
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A further novel finding is the likely involvement of the conserved VP5* YGL sequence in 4 recognition by rotaviruses. The peptide SVVYGLR inhibited
4
1 and
4
7 binding by rotaviruses and virus infectivity, and
4
1 binding by rotaviruses segregated with VP4 in reassortants. It is proposed that VP4 binds
4
1 using YGL. In the X-ray crystallographic structure of RRV VP5* containing aa 252523 (VP5CT), most of the Tyr residue in the YGL sequence is exposed on the surface (Dormitzer et al., 2004
). The shorter sequence SVVYG is sufficient to mediate adhesion to
4 integrins (Gao et al., 2004
). Possibly, exposure of the Tyr and Gly residues in YGL may be sufficient for binding. The YGL sequence might become accessible through opening of a possible diglycine hinge in overlying
-sheets (Dormitzer et al., 2004
).
The VP5* IDA sequence was not necessary for 4 integrin binding by rotaviruses, consistent with the IDA location in a predicted heptad repeat region that may be inaccessible (Bremont et al., 1992
; Lopez et al., 1991
). Peptide IDAPS elimination of rotavirus binding to
4 integrins probably occurred via non-IDA viral sequence, similarly to IDAPS inhibition of fibronectin binding to
4 through EILDVP mimicry (Sharma et al., 1999
). SVVYGLR and EILDVP peptides compete for
4
1 binding, and have similar affinities for
4
1 (Barry et al., 2000
). As SVVYGLR, IDAPS and EILDVP peptides inhibit ligand binding through related
4 regions, the differential effects of these peptides on rotavirus binding and infection probably relate to sequence differences in parts of the peptides with portions of the
4-binding regions in rotavirus. The VP7 LDV sequence was not involved in
4 integrin binding, as the presence of LDVT, LDVT mutation and EILDVP blockade did not affect
4 interactions with rotaviruses. This lack of inhibition by EILDVP also suggests that the VP7 LDI is not necessary for
4 recognition by rotaviruses.
Anti-VP5* mAb 2G4, and anti-VP7 antibodies RV-4 : 2 and F45 : 2, inhibited rotavirus4
1 binding to a titre similar to their neutralization titres. Acute- and convalescent-phase sera from children with gastroenteritis also specifically inhibited Wa binding to
4
1 (K. L. Graham, F. E. Fleming, P. Halasz, Y. Takada and B. S. Coulson, unpublished results). These findings support the proposed roles for VP4 and VP7 in
4
1 recognition by rotaviruses.
Rotavirus usage of 4 integrins but not
9
1 was unexpected, as SVVYGLR is a common ligand for these integrins. Mutation of the SVVYGLR peptide showed that Leu and Tyr are important for
4 binding (Green et al., 2001
), whereas the Tyr appears to be critical for
9
1 binding (Yokosaki et al., 1999
). The first Val has a role in
4 binding, and mutation of the Arg increased activity against
4
1 but reduced activity against
4
7 (Green et al., 2001
). Non-conservative amino acid changes for the Val and Arg adjacent to YGL in VP5* could prevent
9
1 binding by rotaviruses.
It is at present unclear how rotavirus interactions with 4 fit with current models of rotavirus cell entry. SA11 and RRV binding to
4
1 and
4
7 may facilitate virus cell entry by endocytosis or direct membrane penetration (Lopez & Arias, 2004
), as proposed previously for rotavirus usage of other integrins (Graham et al., 2003
). Human rotavirus binding to
4 might affect cellular functions that relate to antirotaviral responses, without facilitating infection. CRW-8 cell binding is independent of cellular protein synthesis (Jolly et al., 2001
), and OSU, another porcine rotavirus of the same P serotype as CRW-8, binds ganglioside GM3 (Rolsma et al., 1998
). Therefore, CRW-8 may bind GM3, facilitating usage of
4 integrins in a co-receptor role that results in cell entry. The identification of several possible modes of rotavirus interactions with
4 in this study will allow further dissection of the mechanisms by which different rotaviruses utilize these integrins.
The adjacent R2, R3b, R3c and R4 loops of 4 in the upper face of the predicted
-propeller structure of the integrin head were critical for rotavirus
4 recognition. However, loops R1 and R5 that are located distal from R2, R3b R3c and R4 on the upper or lower face, respectively (Higgins et al., 2000
; Irie et al., 1997
; Springer, 1997
), were not involved in rotavirus
4 binding. The
4 loops required for binding by rotaviruses, MAdCAM-1, fibronectin and VCAM-1 are, therefore, identical (Higgins et al., 2000
; Irie et al., 1995
, 1997
). Judging from their predicted positions, these loops are highly exposed on the head of activated
4 and ideally placed to mediate binding to rotaviruses and natural ligands. The increased rotavirus infectivity after
4
1 activation supports this conclusion.
This binding of rotaviruses to 4 integrins through the same
4 domains as natural ligands strongly suggests that virus
4 binding is likely to affect the functions of these competing ligands. The blockade of rotavirus
4 binding by SVVYGLR reinforces this conclusion, as SVVYGLR completely inhibits
4
7 binding to MAdCAM-1 and
4
1 binding to fibronectin CS-1, and partially inhibits
4
1 binding to VCAM-1 (Green et al., 2001
). Rotavirus
4
7 binding might affect immune responses dependent on
4
7 recognition of MAdCAM-1 for intestinal homing, and modulate the protection afforded against reinfection by rotavirus-specific
4
7hi B and T cells (Gonzalez et al., 2003
; Rose et al., 1998
; Rott et al., 1997
; Williams et al., 1998
; Youngman et al., 2002
). Potential immune modulation by rotavirusintegrin interactions is a topic for further study.
Lamina propria mesenchymal cells are the only resident gut cell known to express 4 (Choy et al., 1990
; Pender et al., 2000
). Possibly,
4
1 on these cells may bind rotaviruses. Rotavirus disease may be associated with viraemia (Blutt et al., 2003
), so virus in the circulation could contact immune cells expressing
4. Rotavirus recognition of activated
4
1 might facilitate virus carriage and infection. Lymphoid cells mediate rotavirus escape from the gut (Mossel & Ramig, 2003
), which might involve virus
4 integrin binding. Rotavirus infection has been associated with encephalitis (Iturriza-Gomara et al., 2002
) and pancreatic islet autoantibody responses (Honeyman et al., 2000
). Rotavirus
4
7 binding might disseminate virus within the intestine and to the pancreas and brain where MAdCAM-1 is also expressed. These issues are important subjects for continuing research.
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ACKNOWLEDGEMENTS |
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Received 12 April 2005;
accepted 12 September 2005.