1 Department of Biological Sciences, Imperial College, London SW7 2AZ, UK
2 Dipartimento di Biologia, Difesa e Biotecnologie, Agro-Forestali-Università della Basilicata-Macchia Romana, 85100 Potenza, Italy
3 National Centre for Scientific Research Demokritos, 153 10 Aghia Paraskevi, Athens, Greece
4 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy (Québec), Canada G1V 4C7
5 Instituto di Genetica e Biofisica, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy
Correspondence
Julie A. Olszewski
jaolsz{at}ship.edu
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ABSTRACT |
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Present address: Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy (Québec), Canada G1V 4C7.
Present address: Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain.
Present address: Syngenta, Jealotts Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK.
||Present address: Biology Department, Shippensburg University of Pennsylvania, 1871 Old Main Drive, Shippensburg, PA 17257, USA.
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INTRODUCTION |
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Although PDVs can infect most larval tissues, the greatest infection occurs in haemocytes (Stoltz & Vinson, 1979; Wyder et al., 2003
). Infected haemocytes lose their ability to adhere to a substrate, often show cytoskeleton disruption and may become apoptotic (Schmidt et al., 2001
; Lavine & Strand, 2002
). All of these events ultimately interfere with encapsulation of the parasitoid egg and larvae. In vitro infection of some insect host-cell lines has also been shown to lead to the form of programmed cell death (PCD) known as apoptosis in a subset of the cells, but transformed cell lines recover from this phenotype (Béliveau et al., 2003
; Kim et al., 1996
).
Toxoneuron nigriceps, a parasitoid of Heliothis virescens larvae, is associated with the PDV Toxoneuron nigriceps bracovirus (TnBV) (Stoltz et al., 1976; Stoltz & Vinson, 1979
). Parasitization of H. virescens larvae by T. nigriceps leads to suppression of the immune response and developmental arrest of the host larvae, caused by the combined action of TnBV and teratocyte cells from the parasitoid's embryonic serosa (Pennacchio et al., 2001
). To identify PDV gene products that contribute to these processes, the expression pattern of TnBV genes from parasitized H. virescens larvae was analysed and cDNAs of viral transcripts were obtained (Malva et al., 2004
; Falabella et al., 2003
). The first TnBV gene identified (formerly named CnBV1, but here renamed TnBV1 to accommodate the ICTV reclassification of TnBV) is 467 nt long, contains one intron and encodes a putative protein of 124 aa with a predicted molecular mass of
15 kDa (Varricchio et al., 1999
; GenBank accession no. Y19010). The deduced amino acid sequence of TnBV1 does not show a significant level of identity to other proteins. The early and sustained expression of TnBV1 in fat body and haemocytes of last-instar H. virescens larvae (Varricchio et al., 1999
; P. Falabella & F. Pennachio, unpublished data) suggests that it may have a role in immune suppression. Also, detection of TnBV1 mRNA in the host's prothoracic glands also indicated a possible influence on endocrine and developmental disruption. In order to characterize the function of TnBV1 in the parasitization of H. virescens larvae, we expressed the cDNA in lepidopteran cell lines by using various systems and assessed its effects on cell morphology and viability.
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METHODS |
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Construction and identification of recombinant viruses.
Transfer-vector plasmids were constructed to generate AcMNPV recombinants expressing a TnBV1 cDNA that was selected from a cDNA library of T. nigriceps-parasitized H. virescens larvae (Varricchio et al., 1999). TnBV1 was subcloned from pBluescript (Stratagene) into the XhoI and EcoRI sites of the pSynXIV VI+ X3 vector (O'Reilly et al., 1992
) to yield pSyn-TnBV1. To obtain recombinant virus expressing TnBV1, Sf21 cells, at a density of 2x106 cells per 60 mm dish, were co-transfected with 1 µg vsynVI-gal or vsynVI-egt() viral DNA (O'Reilly et al., 1992
) and 2 µg transfer vector pSyn-TnBV1 plasmid DNA, using DOTAP reagent (Roche). Recombinant viruses were selected by screening for occlusion-positive white plaques and purified between three and seven times (O'Reilly et al., 1992
). The selected viral recombinants were amplified to passage 3 and the presence of the TnBV1 gene was checked by restriction-enzyme digestion, PCR and sequence analysis of viral DNA.
TnBV1 was also cloned into the XhoI and EcoRI sites of the pFastBac1 vector (Invitrogen). In addition, a TnBV1(ATG) mutant, in which the initiator codon ATG was mutated to ATT, was PCR-amplified from the pSyn-TnBV1 template by using the TnBV1nullF and the TnBV1nullR primers (Table 1). The TnBV1(ATG) PCR product was first cloned into pGEM-T Easy (Promega) and the insert was digested with XhoI and EcoRI for cloning in the pFastBac1 vector. pFB-TnBV1 and pFB-TnBV1(ATG) vectors were transformed into DH10Bac Escherichia coli cells (Gibco) and positive clones were selected as described by the manufacturer. To obtain recombinant viruses, high-molecular-mass DNA was purified from the selected clones as recommended by the manufacturer and transfected into Sf21 cells. The Ac-TnBV1 and Ac-TnBV1(ATG) bacmid recombinant viruses were plaque-purified and amplified to passage 3 in Sf21 cells (O'Reilly et al., 1992
). The presence of the TnBV1 gene or its ATG mutant variant was confirmed by PCR and sequence analysis of viral DNA.
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Analysis of insect cells infected with a baculovirus expressing TnBV1.
Phenotypes of the recombinant virus infections were observed by light microscopy (Leica) under a 40x objective lens, analysed by SDS-PAGE and subjected to Western blot analysis, using either High Five or Sf21 cells that were mock-infected or infected with wild-type AcMNPV, parental bacmid or recombinant Ac-TnBV1 or Ac-TnBV1(ATG) virus at an m.o.i. of 10. Cells were harvested 1272 h post-infection (p.i.) and processed for SDS-PAGE and Western blot analysis as described by O'Reilly et al. (1992). Blots were probed with a 1 : 10 000 dilution of anti-p10 DNP 4.17 antibody (a gift from G. W. Blissard, Boyce Thompson Institute, Cornell University, NY, USA) and visualized with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) at a 1 : 10 000 dilution by using an ECL kit (Amersham Biosciences).
Assessment of expression of TnBV1 in transfected High Five cells by RT-PCR.
Transcription of TnBV1 in transfected High Five cells was assessed by RT-PCR. High Five cells were seeded at a density of 4x105 cells per well in a 12-well plate (Corning). Plasmid DNA (2 µg) (pIE1/153A or pIE1/153A.TnBV1) and 0·5 µg of a plasmid carrying the green fluorescent protein marker gene, gfp (pIE1/153A.GFP; Farrell et al., 1998), along with 2·5 µl Lipofectin (Invitrogen) in Express Five medium, were used to transfect cells according to the manufacturer's instructions. Total RNA was extracted at 36 h post-transfection by using Tri-Reagent (Sigma). For first-strand cDNA synthesis, 5 µg total RNA was used in the SuperScript First-Strand Synthesis system for RT-PCR (Gibco). PCRs were carried out by using one-fifth of the RT product as template with the TnBV1for and TnBV1rev primers (Table 1
); PCR products were visualized by gel electrophoresis.
Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) assay.
The effect of TnBV1 expression in Sf21 cells was analysed by fluorescence-activated cell sorting (FACS) coupled with a TUNEL assay. Sf21 cells (1x106) were infected with bacmid or Ac-TnBV1 at an m.o.i. of 10 for 48 h before being fixed, permeabilized (70 % ethanol or methanol) and treated for the TUNEL assay according to the manufacturer's protocol (Mebstain Apoptosis kit II; MBL). For each treatment, 10 000 cells were processed by flow cytometry to measure fluorescein isothiocyanate (FITC) labelling. TUNEL-positive cells were gated electronically at 1 % of the bacmid-infected control. Cells that fluoresced at an intensity higher than the 1 % gate (M1) in the Ac-TnBV1-infected cell population were considered TUNEL-positive. The number of TUNEL-positive cells out of 10 000 was counted in five independent experimental replicates. A generalized linear model with binomial errors, a logit link function and an overdispersion parameter was used to analyse the probability that a TnBV1-infected cell fluoresces at or above the M1 gate for the matched bacmid control. The hypothesis that this probability equals 0·01 was then tested at the =0·05 level with an appropriate t-test on the logit scale (PROC GENMOD; SAS Institute Inc., 1999
).
The effect of TnBV1 transient expression in High Five cells was analysed by microscopy coupled with a TUNEL assay. High Five cells were seeded at a density of 4x105 cells per well in a 12-well plate. Then, 2·0 µg pIE1/153A.TnBV1 plasmid DNA and 0·5 µg of a plasmid carrying the marker gene gfp (pIE1/153A.GFP; Farrell et al., 1998), along with 2·5 µl Lipofectin (Invitrogen) in Express Five medium, were used to transfect cells according to the manufacturer's instructions. Cells transfected with 2·5 µg plasmid pIE1/153A.GFP alone were used as a control. TUNEL labelling was assessed in transfected cells and controls by detecting genomic DNA fragmentation via a DeadEnd Colorimetric TUNEL assay kit (Promega). Slides were observed by transmitted light or phase-contrast (Axiophot; Zeiss) and then fluorescence microscopy at 18 and 36 h post-transfection. TUNEL-positive and -negative cells were counted on five microscopic fields at a magnification of 250x, randomly selected from slides obtained in two independent replicates of the experiment. A generalized linear model with binomial error, a logit link and an overdispersion parameter was fitted to the cell counts (PROC GENMOD; SAS Institute Inc., 1999
). The hypothesis that the proportion of TUNEL-positive cells was higher in the presence of TnBV1 than in the control at 18 and 36 h post-transfection was tested at the
=0·05 level with appropriate contrasts amongst the parameters of the model.
Caspase assay.
The effect of TnBV1 expression in Sf21 cells was analysed enzymically by using a caspase-3 activity assay. Sf21 cells were seeded at a density of 9x105 cells per 35 mm dish and infected with parental bacmid, Ac-TnBV1 or a mutant AcMNPV lacking a functional p35 anti-apoptotic gene (p35 del) (Clem et al., 1991) at an m.o.i. of 5. After 48 h, cells were washed and processed with an EnzChek Caspase-3 assay kit (Molecular Probes), in the presence or absence of a caspase-3 inhibitor, as described in the manufacturer's protocol. The experiment was carried out five times, using two or three dishes per treatment. Two cell counts from each dish were transformed to their natural logarithm to normalize the residuals and stabilize their variance. The variance of the log counts was analysed through a mixed linear model with random effects for the batches of material submitted to a treatment in any replicate and for each dish, and with fixed effects for recombinant virus, inhibitor level and their interaction (PROC MIXED; SAS Institute Inc., 1999
).
Bioassays.
LD50 of recombinant budded virus was determined in fourth-instar H. virescens larvae infected by haemocoelic injection, as described by O'Reilly et al. (1992). Doses of either the bacmid parental or Ac-TnBV1 recombinant virus, ranging from 0·01 to 1000 p.f.u., produced and titrated in Sf21 cells and diluted in TC100 medium (Gibco) supplemented with antibiotics/antimycotics (Sigma), were used for the injection experiments. Control larvae (n=25 or 26) were injected with TC100 medium supplemented with antibiotics/antimycotics (1 µl per insect). Mortality was recorded on a daily basis and total mortality was recorded at day 7 post-injection. Experiments were repeated twice. LD50 was estimated from a mixed non-linear model of the probability of death at each dose, which accounted for mortality by injection with uninfected TC100 medium, for the two replicates and for the random effects of the groups of insects submitted to each dose in each replicate (PROC NLMIXED; SAS Institute Inc., 1999
).
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RESULTS |
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Given that our attempts to select for an occlusion-positive TnBV1 recombinant AcMNPV virus by using homologous recombination in insect cells were unsuccessful, we first employed the Bac-To-Bac system (Invitrogen) to select the recombinant Ac-TnBV1 viral genome in bacteria. This system allows maintenance and manipulation of an AcMNPV-based genome as a bacterial artificial chromosome (bacmid). TnBV1 cDNA or an initiator-methionine mutant (ATG) of TnBV1 was first cloned into the pFastBac transfer vector under the control of the polyhedrin promoter. Once selected for in bacterial cells, the Ac-TnBV1 and Ac-TnBV1(ATG) recombinant viral DNAs were transfected into Sf21 cells and recombinant virus was purified.
The recombinant Ac-TnBV1 and Ac-TnBV1(ATG) viruses replicated in Sf21 cells and produced titres of progeny virus similar to those of the parental or wild-type AcMNPV viruses (data not shown). A time course of virus-infected Sf21 cells was analysed by SDS-PAGE of whole-cell extracts (Fig. 1a). The predicted 14·8 kDa TnBV1-specific band was not detected in Ac-TnBV1-infected cells (Fig. 1a
, lanes 69). However, a 10 kDa band was detected in both AcMNPV- and Ac-TnBV1(ATG)-infected cells at 48 and 72 h p.i. (Fig. 1a
, lanes 4, 5, 12 and 13), but not in Ac-TnBV1-infected cells (Fig. 1a
, lanes 8 and 9). The samples were subjected to Western blot analysis and probed with an antibody specific for the baculovirus protein p10, which is expressed as a very late gene in AcMNPV-infected cells. A 10 kDa specific band corresponding in size to the band seen in the Coomassie blue-stained gel was detected in both AcMNPV- and Ac-TnBV1(ATG)-infected (Fig. 1b
, lanes 3, 4, 5, 12 and 13), but not in Ac-TnBV1-infected, Sf21 cell extracts (Fig. 1b
, lanes 69). The reduction in this very late protein in Ac-TnBV1-infected cells coincided with the time of expected expression of TnBV1 in these cells. We therefore examined whether production of small amounts of TnBV1 hampered progression of baculovirus infection and, consequently, the accumulation of very late viral protein(s).
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Expression of TnBV1 in baculovirus-infected Sf21 cells leads to apoptosis-like PCD
Loss of cell viability can occur following a toxic insult by triggering general necrotic events or from engaging specific PCD pathways. To assess the nature of the phenotypes observed in Ac-TnBV1-infected cells, we tested bacmid- versus Ac-TnBV1-infected Sf21 cells for evidence of apoptosis-like PCD. DNA fragmentation, due to the endonuclease activation associated with apoptosis, results in an increased number of free 3'-hydroxyl groups at DNA ends, which leads to increased labelling in a TUNEL assay (Gavrieli et al., 1992). Sf21 cells were bacmid- or Ac-TnBV1-infected for 48 h before being labelled by the TUNEL assay. Infected cells were processed by flow cytometry and FITC labelling was measured. On average, 27 % of Ac-TnBV1-infected cells fluoresced at a level higher than the 1 % gate determined for the corresponding parental bacmid-infected cells (P=0·0098, Fig. 2
), suggesting that the expression of TnBV1 in AcMNPV-infected cells caused the Sf21 cellular DNA to become specifically fragmented.
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DISCUSSION |
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The role of non-apoptotic or apoptotic-like PCD has, in recent years, begun to be acknowledged in cell-survival and -suicide research (Edinger & Thompson, 2004; Leist & Jäättelä, 2001
). In contrast to apoptosis, the mechanisms underlying other types of cell death, such as necrosis, autophagy and paraptosis, are not yet well-categorized. Generally, these processes are caspase-independent, involve extensive cytoplasmic vacuolation and usually lack specific DNA fragmentation (Syntichaki & Tavernarakis, 2002
; Edinger & Thompson, 2004
; Sperandio et al., 2000
; Wyllie & Golstein, 2001
).
In our initial attempt to overexpress TnBV1, we realized that the expression of this protein in insect cells had a profound effect on their survival. Recombinant AcMNPV virus expressing an intact copy of TnBV1 under the polyhedrin promoter could not be selected for via homologous recombination and screening for occlusion-positive plaques during infection of Sf21 cells. However, when using the Bac-To-Bac system, based on cloning recombinant baculovirus genomes in bacteria, an Ac-TnBV1 recombinant was purified. When cell lysates were screened by SDS-PAGE and Coomassie blue staining, the Ac-TnBV1 recombinant virus did not appear to overexpress a protein of 14·8 kDa, the predicted size of TnBV1 (Fig. 1a). However, marked phenotypic effects were observed in Sf21 cells that were infected with the Ac-TnBV1 virus when compared with cells infected with either the parental bacmid virus or a recombinant virus, Ac-TnBV1(ATG), carrying an initiator-methionine mutation (Fig. 1c
). Sf21 cells infected with Ac-TnBV1 did not attain the swollen appearance typical of infected cells around 24 h p.i. (Fig. 1c
). In fact, Ac-TnBV1-infected Sf21 cells appeared to shrink and had lost viability by 48 h p.i. (Fig. 1c
). This truncated infection resulted in a lack of expression of the very late AcMNPV p10 gene in Ac-TnBV1, but not in Ac-TnBV1(ATG) infections (Fig. 1b
). Thus, the toxic effect of TnBV1 expression within infected Sf21 cells was probably the cause of the inability to obtain an occlusion-positive recombinant virus with an intact copy of TnBV1 by using homologous recombination and selection within insect cells. Occlusion-body formation requires polyhedrin protein production at very late times of infection and, presumably, this was compromised by the premature death of the infected cells. Only viral mutants that had lost the TnBV1 gene gave rise to occlusion-positive plaques.
Expression of TnBV1 in Ac-TnBV1-infected Sf21 cells led to breaks in the host genomic DNA, providing free 3'-hydroxyl ends that became available for TUNEL staining (Fig. 2) and to an increased caspase-3-like protease activity (Table 2
), both indicative of apoptosis. Monitoring of mammalian caspase-3-like activity was chosen following reports that infection of Sf21 cells with the p35 del baculovirus leads to activation of S. frugiperda caspase 1 (Ahmad et al., 1997
), which is similar in activity to mammalian caspase-3 (Seshagiri & Miller, 1997
). Although the phenotype of Ac-TnBV1-infected Sf21 cells did include nuclear and cytoplasmic condensation consistent with apoptosis (Fig. 1c
), we did not observe classical blebbing of apoptotic bodies from the plasma membrane, as is seen with p35 del infections (Clem et al., 1991
). Transient expression of TnBV1 in Sf21 cells, without baculovirus infection, also resulted in a slight, yet significant, decrease in the number of viable cells calculated by trypan blue exclusion, demonstrating that TnBV1 alone is sufficient to cause this effect (data not shown). Surprisingly, DNA cleavage leading to TUNEL staining was also detected in High Five cells transiently expressing TnBV1 (Fig. 3
, Table 3
), showing that this phenotype was not unique to Sf21 cells. Tn-368 (Hink, 1970
), the Trichoplusia ni parental cell line from which the High Five cell line was derived, is the cell line that is typically used to propagate the p35 del virus, as this virus does not cause apoptosis when infecting this cell line (Clem et al., 1991
). The Tn-368 cell line has been demonstrated to be resistant to a variety of apoptotic stimuli (Clem & Miller, 1993
; Clem et al., 1994
) and, to our knowledge, this is the first published report of an apoptotic-like response in a T. ni-derived cell line.
Definitive categorization of the PCD pathway engaged by TnBV1 expression in insect cells is difficult. Expression of TnBV1 in insect cell lines caused nuclear and cytoplasmic shrinkage, caspase activation, DNA fragmentation as indicated by TUNEL staining, and membrane permeabilization as defined by trypan blue staining. The absence of blebbing and apoptotic bodies in infected and transfected insect cells, however, prevents the characterization of the TnBV1 effects as classical apoptosis. Apoptosis-like PCD, in various systems, shows degrees and combinations of apoptotic features; chromatin condensation is usually less compact and is also caspase-independent (Leist & Jäättelä, 2001).
Similarly, caspase activation and the absence of cytoplasmic vacuolation prevented the categorization of TnBV1 effects into non-apoptotic PCD pathways, such as necrosis, autophagy and paraptosis. Although TUNEL staining has been described in autophagic Manduca sexta fat-body cells (Muller et al., 2004), DNA fragmentation is not typical of non-apoptotic PCD. Caspase-dependent autophagy has been documented in Drosophila melanogaster salivary glands, where baculovirus p35 expression inhibited cell death (Kimura et al., 2004
), supporting the complexity of PCD occurring in various systems. Finally, multiple death pathways can be triggered by a single protein, such as the tumour necrosis factor receptor (TNFR1) (Laster et al., 1988
; Leist & Jäättelä, 2001
). Depending on the cell type, TNFR1 signalling induces classical apoptosis, necrosis or a cytolytic phenotype that resembles apoptosis, but does not include DNA fragmentation. To delineate further the PCD features present in TnBV1-expressing cells, staining and electron microscopy could be carried out in future experiments to examine both chromatin-condensation patterns and the appearance of the organelles.
Despite effects in cell culture, expression of TnBV1 did not affect the virulence of AcMNPV in H. virescens fourth-instar larvae following injection of the budded form of the virus into the haemolymph (Table 4). Several factors could be causing this apparent discrepancy between the in vitro cell line and in vivo bioassay data. Production of budded virus from Ac-TnBV1 infection of Sf21 cells was similar to that caused by wild-type AcMNPV (data not shown). This is probably due to the fact that budded virus is abundantly produced from 12 to 20 h p.i. (Miller, 1997
), before the expected onset of TnBV1 expression from the polyhedrin promoter. Any apoptosis-like effects of infected cells following this period would not affect the spread of infection by budded virus in the infected insects. Also, we did not test Ac-TnBV1 in H. virescens cell lines to demonstrate apoptosis-like PCD. Therefore, the possibility that TnBV1 expression does not have an effect on cells derived from this caterpillar requires further investigation. However, recent experimental data indicate that granulocytes of H. virescens larvae undergo apoptosis, starting 6 h after T. nigriceps parasitization (M. Eguileor & F. Pennachio, unpublished data).
Detecting apoptosis of individual cells/tissues in whole infected caterpillars is difficult, but has recently been reported by using TUNEL staining (Clarke & Clem, 2003). It would be interesting to apply this approach to Ac-TnBV1-injected H. virescens. Another measure of the effect of TnBV1 expression on the productivity of Ac-TnBV1 versus wild-type AcMNPV infection would be to reintroduce polyhedrin under its own promoter in Ac-TnBV1 and then assess the production of infectious occlusion bodies from each viral infection in H. virescens cadavers. If infected cells are undergoing apoptosis-like PCD in vivo, thereby truncating infection within the very late phase, fewer occlusion bodies (and/or containing fewer virions, making them less infectious) would be expected to arise from Ac-TnBV1- than from wild-type-injected caterpillars. At present, the role of TnBV1 expression in parasitized H. virescens larvae remains elusive and needs to be demonstrated more directly in vivo. However, this might be difficult, as the TnBV1 protein may interact with other TnBV proteins during parasitism to exert its function. The timing, level and localization of expression of TnBV1 within the host caterpillar might also need to be replicated faithfully to study its function.
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ACKNOWLEDGEMENTS |
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Received 16 December 2004;
accepted 20 December 2004.