Department of Microbiology and Immunology, University of Kentucky College of Medicine, MS 415 Chandler Medical Center, Lexington, KY 40536-0298, USA1
Microscopy Branch, Rocky Mountain Laboratories, NIAID, National Institutes of Health, 903 South 4th St, Hamilton, MT 59840, USA2
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, PO Box 2087, Foothills Campus, Fort Collins, CO 80522, USA3
Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1671, USA4
Author for correspondence: Brian Stevenson. Tel: +1 859 257 9358. Fax: +1 859 257 8994. e-mail: bstev0{at}pop.uky.edu
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ABSTRACT |
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Keywords: spirochaete, immunofluourescence, Erp proteins, bacterial surface proteins, protease resistance, Lyme disease
Abbreviations: IFA, immunofluorescent antibody
a Present address: Dept. of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
b Present address: Centocor Inc., 200 Great Valley Parkway, Malvern, PA 19355, USA.
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INTRODUCTION |
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B. burgdorferi contains many lipoproteins, and early studies of these and other spirochaetes utilized a variety of methods to study lipoprotein localization (Barbour et al., 1983a; Bledsoe et al., 1994 ; Cunningham et al., 1988
; Fuchs et al., 1992
; Jones et al., 1995
; Lam et al., 1994
; Luft et al., 1989
; Radolf, 1994
; Radolf et al., 1995
). More recently, it has become apparent that outer membranes of many spirochaetes, including B. burgdorferi, are relatively fragile, and some previously utilized techniques may yield inaccurate results (Cox et al., 1996
). Re-evaluation of spirochaetal membrane proteins indicated that some lipoproteins initially described as outer surface proteins appear to be primarily located in the inner membrane and/or inner leaflet of the outer membrane (Bledsoe et al., 1994
; Brusca et al., 1991
; Cox et al., 1996
; Radolf, 1994
; Radolf et al., 1994
). It is important, then, that proteins designated as surface exposed by potentially flawed methods be examined further by other techniques (Haake, 2000
).
All Lyme disease spirochaetes that have been examined contain large numbers of genes encoding members of the Erp protein family, encoded on multiple, homologous circular and linear plasmids (the cp32 family of plasmids). These proteins apparently perform functions that are unique to the infectious cycle of Lyme disease spirochaetes, since erp genes have not been identified in any other bacteria of the genus Borrelia (Stevenson et al., 2000a ). All erp genes hold several features in common, including well-conserved promoter DNA sequences, locations on a family of homologous plasmids and the encoding of highly charged lipoproteins with well conserved leader polypeptides (reviewed by Stevenson et al., 2000b
). Those Erp proteins examined were all found to be lipidated by bacteria (Akins et al., 1995
; Lam et al., 1994
; Wallich et al., 1995
). Laboratory animals infected by tick bites generally produce antibodies against Erp proteins within the first 24 weeks of infection (Akins et al., 1995
; Das et al., 1997
; Nguyen et al., 1994
; Stevenson et al., 1998a
; Suk et al., 1995
; Sung et al., 2000
; Wallich et al., 1995
), suggesting that Erp proteins play roles in the initial stages of mammalian infection. Reverse transcriptase-PCR studies have also demonstrated erp expression during this time period (Anguita et al., 2000
; Das et al., 1997
), as have analyses of bacteria grown in chambers implanted within the bodies of laboratory animals (Akins et al., 1998
). To date, 17 erp genes at 10 loci have been identified in B. burgdorferi strain B31. The coding regions of three bicistronic loci, erpAB, erpIJ and erpNO, are identical, so their protein products are indistinguishable and are referred to collectively as ErpA/I/N and ErpB/J/O. The other seven B31 loci encode Erp proteins with varying degrees of dissimilarity (Casjens et al., 1997
, 2000
; Stevenson et al., 1998a
, b
). It is assumed that the variations among Erp protein sequences confer some advantage to the bacteria, but the nature of that benefit remains to be elucidated (El-Hage et al., 1999
; Stevenson et al., 2000b
; Sung et al., 2000
).
The lipid moiety of Erp lipoproteins anchors them to a membrane, but prior to the current study it was not known with confidence to which membrane(s) or to which leaflet(s) the Erps are attached. One previous study examined the cellular localization of two proteins of the Erp family, the OspE and OspF proteins of strain N40, using immunofluorescent antibody (IFA) analyses of bacteria fixed with formaldehyde (Lam et al., 1994 ). While that study detected antibody binding to the fixed bacteria, suggestive of surface exposure, it has since been observed that fixation can disrupt the outer membrane, allowing subsurface proteins to bind antibodies (Cox et al., 1996
). Additionally, the polyclonal antisera used in the earlier study contain antibodies that recognize other proteins besides OspE and OspF (Marconi et al., 1996
; Stevenson et al., 1995
), so it is possible that the proteins detected by those IFA analyses were neither OspE nor OspF. Other studies demonstrated that vaccination with some recombinant Erp proteins provided partial protection against B. burgdorferi infection, suggestive of exposure to the external environment, yet vaccination with other Erp proteins failed to protect against infection, arguing against surface localization (Nguyen et al., 1994
; Wallich et al., 1995
). Since the cellular location of the B. burgdorferi Erp proteins was unclear, we examined these proteins through a variety of independent techniques. Together, these data indicate that Erp proteins are exposed to the external environment in the B. burgdorferi outer membrane and can therefore facilitate interactions between these bacteria and their hosts.
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METHODS |
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In vitro temperature shift experiments were conducted as previously described (Stevenson et al., 1995 ), using BarbourStoenerKelly II (BSK-II) (Barbour, 1984
) medium containing 6% rabbit serum (Sigma). Bacteria used for Erp protein localization analyses were grown at 35 °C to late-exponential phase (approx. 108 bacteria ml-1) in a commercially prepared BSK formulation, BSK-H (Sigma), from lot number 38H8425. For an as yet undetermined reason, cultivation in this particular lot of BSK-H medium causes B. burgdorferi strain B31 to constitutively synthesize high levels of Erp proteins (K. Babb, N. El-Hage, J. C. Miller, J. A. Carroll and B. Stevenson, unpublished results).
Antibodies.
All antibodies were generated in strict accordance with United States requirements for the experimental use of animals. Polyhistidine-tagged recombinant B31 Erp proteins were produced in Escherichia coli and purified as previously described (Miller et al., 2000b ; Stevenson et al., 1998a
). Polyclonal antisera were generated for the B31 ErpA/I/N, ErpB/J/O, ErpK, ErpL, ErpM and ErpX proteins by vaccinating New Zealand White rabbits with approximately 50 µg purified protein in Freunds complete adjuvant, followed by booster vaccinations 2 and 4 weeks later with the same dose of protein in Freunds incomplete adjuvant. Rabbit antisera directed against the B31 ErpP, ErpQ and ErpY proteins were similarly produced by Animal Pharm Services. All rabbits were exsanguinated 2 to 4 weeks after the final boost. For some studies, nonspecific antibodies were adsorbed from polyclonal antisera by incubating serum, diluted 1:200 in Tris-buffered saline/Tween 20 (20 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·05% Tween 20), for 1 h at 37 °C with lysates of E. coli expressing all other recombinant Erp proteins (Stevenson et al., 1998a
). Specificities of preadsorbed antisera were assessed by immunoblotting with all purified recombinant Erps.
A panel of monoclonal antibodies (mAbs) were produced from the spleen of a mouse infected with isolate B31 via tick bite, as previously described (Gilmore & Mbow, 1998 , 1999
; Mbow et al., 1999
). Briefly, B. burgdorferi B31-infected Ixodes scapularis nymphal ticks were fed to repletion upon Balb/cByJ mice, and mice were reinfested 1 month later with additional B. burgdorferi B31-infected ticks. Three weeks after tick feeding, spleen cells were fused with P3X63-Ag8.653 myeloma cells. Wells were screened by ELISA using a B31 lysate as antigen and cells from positive wells were cloned by limiting dilution. mAb specificities were determined by immunoblotting isolate B31 whole-cell lysates and recombinant Erp proteins. Unless otherwise stated, all mAbs used in the following experiments were undiluted hybridoma supernatants.
Two previously described mAbs that recognize other B. burgdorferi B31 proteins were used as experimental controls: B5, directed against the OspC protein (Gilmore & Mbow, 1999 ; Mbow et al., 1999
), and H9724, directed against the flagellar FlaB subunit (Barbour et al., 1986
) (provided by Tom Schwan, Rocky Mountain Laboratories, NIH, Hamilton, MT, USA). Two antibodies directed against non-borrelial proteins were used as additional controls in growth inhibition assays: mAb 83-12-5, directed against mouse CD8 (provided by Jerold Woodward, University of Kentucky, KY, USA), and polyclonal rabbit antiserum HTV, raised against the Yersinia pestis LcrV protein (provided by Susan Straley, University of Kentucky, KY, USA).
In situ protease treatment.
B. burgdorferi was grown to mid-exponential phase in BSK-H, pelleted by centrifugation for 10 min at 1000 r.p.m. in a Beckman GPR centrifuge with a GH3.8 swinging-bucket rotor, washed once with PBS plus 10 mM MgCl2 (PBS-Mg), and resuspended in the same buffer to a final concentration of 2x109 bacteria ml-1. Examination of bacterial suspensions by phase-contrast light microscopy did not indicate detectable lysis of the bacteria. Bacteria were then incubated at room temperature with a protease for 30 min, 1 h or 2 h, whereupon digestion was terminated by addition of an appropriate inhibitor followed by sample boiling. One of three different proteases were used in each experiment at the following final concentrations: 40 µg proteinase K ml-1 (Sigma), 40 or 100 µg trypsin ml-1 (Sigma) or 0·05 µg Pronase ml-1 (Boehringer Mannheim). Proteinase K was inhibited by addition of PMSF to a final concentration of 1·6 mg ml-1. Trypsin was inhibited by the addition of PMSF and pefabloc SC (Boehringer Mannheim) to final concentrations of 1·6 mg ml-1 and 0·3 mg ml-1, respectively. Pronase was inhibited by addition of PMSF, pefabloc SC and EDTA to final concentrations of 0·06 mg ml-1, 0·3 mg ml-1 and 0·5 mg ml-1, respectively. Control aliquots of bacteria were incubated in buffer for 2 h at room temperature without added protease, followed by addition of inhibitor and boiling as with protease-treated bacteria. Equal volumes of bacterial lysates were subjected to SDS-PAGE and the proteins transferred to nitrocellulose membranes. Susceptibility of individual proteins to protease digestion was assessed by immunoblotting with appropriate monoclonal or polyclonal antibodies, followed by incubation with protein Ahorseradish peroxidase conjugate (Amersham) and bound antibodies visualized by enhanced chemiluminescence (Amersham) (Miller et al., 2000b ). As experimental controls, lysates were also immunoblotted with mAbs directed against OspC [(located on the bacterial outer surface and thus susceptible to proteolysis (Fuchs et al., 1992
; Mathiesen et al., 1998
; Wilske et al., 1993
)] and FlaB [located in the periplasmic space and thus protected against protease digestion in intact bacteria (Bono et al., 1998
; Bunikis & Barbour, 1999
; Holt, 1978
)].
Additional control experiments were performed to assess the protease susceptibility of proteins when not in situ. Bacteria were treated with proteases as above, but with the addition of Triton X-100 to a final concentration of 0·1%, which generally disrupts B. burgdorferi outer membranes (Cox et al., 1996 ), and lysates were immunoblotted with Erp- and FlaB-directed antibodies. Purified recombinant Erp proteins were incubated for 30 min in either of the three proteases and immunoblotted with an appropriate antibody.
Immunofluorescence analysis of intact B. burgdorferi.
One millilitre of a mid-exponential phase culture, grown in BSK-H, was placed on a Biocoat poly-D-lysine-coated 12 mm round cover slip (Becton Dickinson) in the well of a 24-well culture plate (Bunikis & Barbour, 1999 ; Cox et al., 1996
). Bacteria were gently pelleted onto the cover slip by centrifugation for 10 min at 200 g in a Beckman GPC centrifuge holding a GH3.8 swinging-bucket rotor. Slides were then washed twice with PBS. Bacteria were incubated in 500 µl PBS containing 10% heat inactivated fetal bovine serum (Life Technologies) and 1% mouse serum (Sigma) for 1 h at room temperature. Cells were then washed with PBS, incubated overnight with 200 µl mAb B11 (anti-ErpA/I/N), B5 (anti-OspC) or H9724 (anti-FlaB), followed by incubation for 2 h with goat anti-mouse IgGOregon green conjugate (Molecular Probes) in PBS containing 1% mouse serum. The coverslips were washed with PBS, mounted to slides and viewed by epifluorescence microscopy. As controls for possible interactions with the secondary antibody, bacteria were also treated as above except without incubation with a primary antibody.
Electron microscopy.
Samples were washed in Hanks Balanced Salt Solution (HBSS) (Life Technologies) and 5 µl droplets were allowed to adhere to carbon/collodion-coated nickel grids for 60 min at room temperature. The samples were blocked with 3% BSA in HBSS for 30 min prior to a 60 min incubation with either mAb B11 (anti-ErpA/I/N) or H9724 (anti-FlaB). After washing with the blocking agent, the samples were labelled with a secondary gold-conjugated antibody (Ted Pella) for 60 min, Cells were washed, fixed with 2·5% glutaraldehyde and stained with 1% ammonium molybdate prior to being viewed at 80 kV on a Hitachi 7500 transmission electron microscope.
Growth inhibition by antibodies.
B. burgdorferi were grown in BSK-H to mid-exponential phase (approx. 107 bacteria ml-1) and 100 µl aliquots were placed in each well of a 96-well tissue culture dish (Becton Dickinson). One hundred microlitres of each polyclonal rabbit antiserum or mAb hybridoma supernatant, either undiluted or serially diluted in BSK-H, was added to the bacterial cultures. Plates were covered with Breathe-Easy gas-permeable adhesive seals (Diversified Biotech) (Bono et al., 2000 ) and incubated at 37 °C in a 5% CO2 environment. After 72 h, growth was monitored visually for colour changes in the mediums phenol red indicator, since a change from red to yellow indicates acidification due to bacterial growth (Sadziene et al., 1993
). Culture aliquots were also examined at that time by phase-contrast light microscopy for absence of bacterial motility and the formation of immobile bacterial aggregates (Bunikis & Barbour, 1999
; Cinco, 1992
; Coleman et al., 1992
; Hanson et al., 1998
; Luke et al., 2000
; Pavia et al., 1991
; Sadziene et al., 1993
).
As controls, bacteria were also cultivated as above with the OspC-directed mAb B5, the FlaB-directed mAb H9724, non-borrelial mAb 83-12-5, hybridoma culture medium, non-borrelial polyclonal rabbit antiserum HTV, normal rabbit serum (all undiluted) or BSK-H without any additions.
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RESULTS |
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Protease sensitivity of Erp proteins in situ
Protein surface exposure was first examined by incubation of intact bacteria with proteases, on the premise that exposed proteins will be degraded, while subsurface proteins are protected against proteolysis. Three different proteases were used in these studies, since some B. burgdorferi surface proteins are known to be resistant to certain enzymes (Bunikis & Barbour, 1999 ; Dunn et al., 1990
; Zückert et al., 2001
). Some B31 Erp proteins, such as ErpA/I/N, were completely degraded after short exposure times to all examined proteases, indicative of exposure to the external environment (Fig. 2
, Table 1
). The known outer surface protein OspC was also degraded by protease (Fuchs et al., 1992
; Mathiesen et al., 1998
; Wilske et al., 1993
). On the other hand, some proteins, such as ErpB/J/O, were not detectably degraded by proteinase K and only a portion of the molecules were digested by Pronase or 40 µg trypsin ml-1. Complete degradation of ErpB/J/O was only observed after 60 min incubation with 100 µg trypsin ml-1 (Fig. 2
). Even a 2 h incubation with the highest tested concentration of trypsin did not completely digest all ErpX molecules, although some proteolysis was apparent (Table 1
). The bacterial outer membranes remained intact during all in situ proteolysis treatments, since there was no detectable degradation of FlaB, a component of the periplasmic flagella (Fig. 2
). We conclude that all tested B31 Erp proteins are exposed to the environment and that some are resistant to degradation by certain proteases.
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IFA analysis and immunogold labelling of unfixed bacteria
Since fixation can disrupt B. burgdorferi membranes, unfixed bacteria were used for IFA studies to further assess Erp surface exposure. We observed that greater than 70% of examined bacteria bound ErpA/I/N-specific mAb B11, again indicating that this Erp protein is exposed on the outer surface of B. burgdorferi (Fig. 3). Bacteria did not fluoresce uniformly, but instead exhibited a punctate pattern, suggesting localization of ErpA/I/N at certain points along the bacterial cell. However, since B. burgdorferi B31 does not produce quantities of ErpA/I/N sufficient to be visualized by Coomassie brilliant blue staining of cell lysates (Stevenson et al., 1995
), the punctuate fluorescence pattern may simply be a consequence of low ErpA/I/N protein concentration. This may also be the reason why we did not observe fluorescence from all of the bacteria. Alternatively, since membrane proteins are generally free to move laterally in the membrane, this pattern may have been due to aggregation of proteins by the antibodies (Barbour & Hayes, 1986
; Barbour et al., 1983a). Less than 1% of bacteria incubated with the FlaB-specific mAb H9724 exhibited antibody binding, indicating that the outer membranes of the vast majority of examined bacteria remained intact during IFA processing. Additionally, no bacteria were detected by epifluorescence microscopy after incubation with only the fluorescence-tagged secondary antibody.
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Cultivation of B. burgdorferi in medium containing Erp-directed antibodies indicated that such antibodies can indeed inhibit bacterial growth. Addition of mAb B11, directed against ErpA/I/N, caused bacterial aggregation and inhibited growth (Table 2). This effect was specifically due to ErpA/I/N binding, since growth inhibition could be prevented by addition of recombinant ErpA/I/N protein at concentrations as low as 1·5 ng ml-1, the lowest level tested. Similar growth inhibition was also observed when cultivating bacteria in medium containing mAb B5, directed against the outer surface protein OspC. No inhibition of growth was detected in control experiments with the FlaB-directed mAb H9724, non-borrelial mAb 83-12-5 or unused hybridoma culture medium.
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DISCUSSION |
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Since mammals produce antibodies against Erp proteins within the first few weeks of infection (Akins et al., 1995 ; Das et al., 1997
; Miller et al., 2000b
; Nguyen et al., 1994
; Stevenson et al., 1995
, 1998a
; Suk et al., 1995
; Sung et al., 2000
; Wallich et al., 1995
), it is clear that Erps are made by the bacteria during that time and presumably perform a function(s) for the bacteria. The surface exposure of Erp proteins indicates that these proteins are positioned to interact with host cells, extracellular matrices, or other substances encountered by the bacteria during the natural infection cycle. An individual bacterium may contain many different Erp proteins, often with extensively variable sequences, which may permit the bacteria to interact with numerous host tissues. Alternatively, there are also conserved characteristics among Erp proteins, such as a high percentage of charged amino acid residues, that might permit Erps with different primary structures to interact with similar ligands (Stevenson et al., 2000b
). Several B. burgdorferi proteins have been identified that bind specific host tissues (Coburn et al., 1999
; Guo et al., 1995
; Parveen & Leong, 2000
) and similar techniques might be adapted to search for substances that bind Erp proteins.
The ability of Erp-directed antibodies to prevent B. burgdorferi growth in vitro suggests that similar antibodies could protect animals against B. burgdorferi infection. Previous studies involving vaccination of mice with each of three Erp proteins, OspE or OspF of strain N40, or pG of strain ZS7, found either partial or no protection against bacterial challenges (Nguyen et al., 1994 ; Wallich et al., 1995
). We have noted in the current study that antibodies from animals vaccinated with some Erp proteins also cross-reacted with other, similar Erp proteins, which could have contributed to the observed growth inhibition. Thus it may be possible that protection can be achieved by vaccination with an Erp that promotes cross-reactive antibody formation, or with a mixture of several different Erp proteins.
All B31 Erp proteins were sensitive to in situ treatment with at least one protease, with some Erps demonstrating resistance to digestion by certain enzymes (e.g. ErpB/J/O was uncleaved by proteinase K; Fig. 2). Since B. burgdorferi may be exposed to proteolytic enzymes during the bacterial infection cycle, resistance to proteases may be an important feature of borrelial surface proteins. There are several possible explanations for the ability of surface-exposed proteins to resist proteolysis. Although recombinant Erp proteins synthesized by E. coli and native proteins in Triton X-100-treated B. burgdorferi were susceptible to the tested proteases, proteins of intact B. burgdorferi might be folded in different manners, such that protease recognition sites are hidden. Some surface proteins of the related spirochaete Borrelia turicatae are resistant to certain proteases, apparently due to their secondary structures (Zückert et al., 2001
). Erp proteins in their native states may form multimers or interact with other membrane proteins that protect protease cleavage sites, similar to what has been postulated for the B. burgdorferi p66 (Oms66) and OspA proteins (Bunikis & Barbour, 1999
; Exner et al., 2000
). While B. burgdorferi does not contain lipopolysaccharides identical to those of enteric Gram-negative bacteria (Takayama et al., 1987
), borreliae and other spirochaetes contain non-proteinaceous membrane constituents that might serve similar functions (Beck et al., 1985
; Cinco, 1992
; Cinco et al., 1991
; Eiffert et al., 1991
; Schultz et al., 1998
; Wheeler et al., 1993
), and could interact with membrane proteins such as the Erps in manners that interfere with protease accessibility. Since a portion of ErpX molecules were not degraded by any of the protease treatments used in these studies, it is also possible that some Erps are located both on the bacterial outer surface and in the periplasmic space, as apparently are some other B. burgdorferi lipoproteins (Brusca et al., 1991
; Cox et al., 1996
; Radolf, 1994
). Additionally, the insensitivity of ErpB/J/O and other proteins to digestion by proteolytic enzymes raises the possibility that the results of studies on other B. burgdorferi membrane proteins may be flawed in assigning subsurface localization based solely upon their inability to be degraded in situ.
While these studies demonstrated that Erp proteins are located on the B. burgdorferi outer surface, many additional questions about these proteins now must be answered. Why were some proteins, such as ErpQ and ErpX, resistant to in situ protease degradation? If Erps are protected from proteolysis by interactions with other surface components, what are they? If the reason behind partial resistance is that a fraction of those Erp protein molecules had subsurface locations, why were those unequally distributed while others, such as ErpA/I/N and ErpY, were completely localized to the outer surface? Do Erp proteins interact with host components, and if so, what are they? We are continuing studies to address these and other questions about the intriguing Erp proteins and their roles in the biology of B. burgdorferi and the pathogenesis of Lyme disease.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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We thank Don Cohen, Jerry Woodward, Tom Schwan, Susan Straley, Chris Wulff-Strobel and Ela Skrzypek for providing antibodies and for assistance with IFAs, Julie Stewart for technical assistance, Jeff Hopkins for assistance in producing recombinant Erp proteins, Ralph Larsen and Patti Rosa for assistance with raising rabbit antisera, and Gary Hettrick for graphics assistance.
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Received 12 September 2000;
revised 27 November 2000;
accepted 15 December 2000.