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* Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, and Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada
Correspondence: Address reprint requests to Bernd Grohe, E-mail: bgrohe{at}uwo.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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).
One of the urinary proteins thought to play an important role in oxalate stone formation is osteopontin (OPN). In vitro studies have shown that OPN is a potent inhibitor of COM growth (2,3), promotes the formation of calcium oxalate dihydrate (COD) rather than monohydrate (4), and inhibits the aggregation of COM crystals (5). In vivo, OPN is the major component of the organic matrix of oxalate-containing kidney stones (6–8).
Accumulating evidence suggests that OPN is part of the body's defense mechanism against pathological calcification (9). OPN expression is upregulated in animal models of stone disease (10–14). Although mice in which the OPN gene has been inactivated exhibit no overt phenotypic abnormalities (15,16), feeding such animals a diet containing ethylene glycol results in higher levels of kidney calcification than it does in wild-type littermates (17). Based on such observations, it has been proposed that OPN acts as an inducible inhibitor of COM formation in the urinary system (18).
OPN contains 
300 amino acids, including a conserved sequence of contiguous aspartic acid residues (19). This polypeptide chain undergoes extensive posttranslational modification, including glycosylation, phosphorylation, and sulfation. The exact pattern of modification depends upon the species and tissue in which the protein is synthesized. The bovine milk isoform of OPN contains 27 sites of serine phosphorylation, 1 of threonine phosphorylation, and 3 of O-linked glycosylation (20). Human milk OPN is phosphorylated at 34 serine and 2 threonine residues, and O-glycosylated at five sites (21). The rat bone isoform has at least 29 sites of phosphorylation, 4 sites of N-linked glycosylation, and 1 site of tyrosine sulfation (22). No detailed information is available about posttranslational modification of kidney OPN. There is some disagreement in the literature about the presence of secondary structure in OPN, but it seems likely that the protein has an extended, flexible conformation (23).
Although the functional significance of posttranslational modifications in OPN is not well understood, it appears that phosphorylation is required for the inhibition of biological crystal formation. Synthetic phosphopeptides corresponding to sequences in human OPN inhibit growth and aggregation of COM crystals to a much greater degree than the nonphosphorylated counterparts (24). Phosphorylation is also required for OPN to inhibit the formation of hydroxyapatite (25–28) and calcium carbonate (29) crystals.
The aim of our research program is to determine the features of both protein and crystal that are involved in the interaction between OPN and COM. In a recent study, we used scanning confocal interference microscopy (SCIM) to measure the growth of calcium oxalate crystals in specific directions in the presence and absence of poly-L-aspartic acid (poly-asp) (30). SCIM has great potential for the study of crystal growth because of its high spatial resolution, sensitivity to parallel reflecting planes such as crystal growth steps, and ability to produce time-resolved image sequences without physical contact. In this study, we combine SCIM with fluorescence imaging to visualize the adsorption of OPN and synthetic polypeptides to COM crystals. Using this approach, we demonstrate novel face- and edge-specific interactions that may be responsible for effects on the COM growth habit observed in vitro and in vivo.
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MATERIALS AND METHODS |
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). Poly-L-glutamic acid (poly-glu) sodium salt (10.25 kDa) and poly-asp sodium salt (9.15 kDa) were obtained from Sigma-Aldrich (Oakville, Canada). Alexa Fluor-488 carboxylic acid (AlexaFluor-488) was obtained from Invitrogen Canada-Molecular Probes (Burlington, Canada). Native rat bone OPN was extracted and purified as described by Goldberg and Sodek (31). The molecular weight of 37622 for OPN was determined by mass spectrometry (matrix-assisted laser desorption ionization-time of flight, Bruker Reflex III, Bruker Daltonics, Billerica, MA).
Fluorescence labeling and solution preparation of polypeptides
Poly-asp, poly-glu, and OPN were labeled with the Alexa fluorochrome according to the manufacturer's recommendations. Briefly, 5 µl of AlexaFluor-488 in dimethylformamide (10 µg/µl; high performance liquid chromatography grade, 99.9%) was added to 200 µl of polypeptide in phosphate-buffered saline (2.5 mg/ml) and 20 µl of 1 M disodium carbonate (Na2CO3), pH 8.3, and incubated for 1 h at room temperature (23°C). Unconjugated label was removed by dialysis against 4 changes of 2l of Tris-buffered saline, pH 7.4, for 4 h each using 3.5-kDa dialysis tubing (Spectra/Por 3, Spectrum Laboratories, Rancho Dominquez, CA). After freeze-drying, the samples were stored at –20°C. All chemicals were supplied by Sigma-Aldrich except Na2CO3, which was purchased from Merck (Darmstadt, Germany). Amino acid analysis (Alberta Peptide Institute; University of Alberta, Edmonton, Canada) was carried out using norleucine as an internal standard to determine the yield of labeled peptides. The masses obtained were used to prepare aqueous stock solutions of 1 mg/ml labeled poly-asp, poly-glu, and OPN. In addition, aqueous stock solutions of 1 mg/ml and 50 µg/ml unlabeled poly-asp, poly-glu, and OPN were prepared, respectively.
Crystallization experiments
Crystallization of COM was initiated using the method previously described (30). For low supersaturation conditions (LSC), final concentrations were 1 mM calcium nitrate, 1 mM sodium oxalate, 10 mM sodium acetate, and 150 mM sodium chloride. For high supersaturation conditions (HSC), final concentrations were 2 mM calcium nitrate, 2 mM sodium oxalate, 10 mM sodium acetate, and 150 mM sodium chloride.
For scanning electron microscopy, 1-ml aliquots of these solutions were added to wells of tissue culture plates (24-well Falcon; Becton Dickinson, Franklin Lakes, NJ) containing freshly cleaved mica disks (diameter: 9.5 mm, V-1 grade, SPI Supplies, Toronto, Canada). If unlabeled poly-asp, poly-glu, or OPN was added to the wells, the volume of water was correspondingly reduced. After incubation (Ultra Tec WJ 501 S; Baxter Scientific Products, Mississauga, Canada) at 37°C for 30 min, the mica disks were rinsed with deionized water and air-dried.
Confocal microscopy was performed under LSC (see above), and 200-µl aliquots of calcium oxalate solution were added to glass-bottomed polystyrene dishes (35-mm diameter; glass bottom: grade No. 1.5, diameter: 10 mm; Mattek, Ashland, MA). Each dish was covered with laboratory film (Parafilm "M", Chicago, IL) to prevent evaporation and incubated at room temperature for 3 h. The dish was then placed on the heated (37°C ± 0.2°C) stage of a Zeiss LSM 410 confocal microscope (Carl Zeiss, Jena, Germany) and a 20-µl aliquot of a 1 µg/ml solution of poly-asp, poly-glu, or OPN was added to the solution. The protein or polypeptide was allowed to adsorb to the crystals for a period of 45 min before imaging. For time-course experiments, imaging started 4 min after addition of polyelectrolytes.
The final pH of all reaction solutions was between 6.65 and 6.75; even high polyelectrolyte concentrations (up to 500 µg/ml) did not significantly change the pH.
Scanning electron microscopy
A LEO 1540XB scanning electron microscope equipped with a Gemini field emission column (Carl Zeiss, Jena, Germany) was employed. Precipitates on mica substrates were investigated without metal coating, using an acceleration voltage of 1 kV and a working distance of 3.5 mm.
Scanning confocal microscopy
SCIM (30) was used to image the crystal-glass interface. Conventional scanning confocal microscopy (SCM) was used to make optical sections at higher levels of the crystal and to image fluorescence-labeled peptides. In both cases, a 63x oil-immersion objective and a 90/10 mirror as a beam splitter were used. All procedures were carried out in the dark to avoid the effects of scattered light on crystal imaging and to prevent the fluorochrome from bleaching. Before the first scan of every crystal, prealignments of the microscope were carried out and the focus adjusted to the interface between crystal and glass. If required, the focus was adjusted again during image acquisition. Crystals were scanned using a helium/neon laser (
= 632.8 nm). To excite the fluorochrome, a krypton/argon laser ( = 488 nm) was used. Images of crystals were obtained in a single scan (4 s) with the gain and offset selected to provide optimal contrast of the crystal. The pinhole was adjusted to <1 airy unit to provide maximum resolution. Fluorescence images were obtained using an LP 515 emission filter (Chroma Technology, Rockingham, VT) (the emission maximum for AlexaFluor-488 is at 519 nm). Gain and offset were adjusted to provide optimal contrast, and the pinhole was set to maximum aperture. Ten large (20 µm x 5 µm), well-formed COM crystals nucleated from a {010} face were scanned in optical sections every 500 nm along the z axis (corresponding to the 
010
directions of the crystal).
Image and data processing
Images were imported into Photoshop 7.0 (Adobe, San Jose, CA) and the red and green channels separated. The green-channel images were analyzed using the plot profile option in ImageJ (National Institutes of Health, Bethesda, MD). Fluorescence intensity was measured along lines corresponding to each crystal face, and the background intensity subtracted. Measurement started at the vertex between a {100} and a {121} face and proceeded clockwise such that the sequences of faces was (100), (
21), (121), (00), (12), (
). The resulting data were transferred to Origin 5.0 (Microcal; OriginLab, Northhampton, MA) and used to plot graphs of relative fluorescence intensity (raw data minus background noise) versus fractional perimeter length (obtained by dividing actual distances by the total length of the perimeter) for each section of each crystal. Using the "average multiple curves" option in Origin 5.0, a mean curve for all sections of each crystal was generated. Finally, the mean curves for all 10 crystals were again averaged.
Time-course experiments
Experiments to examine polyelectrolyte adsorption with time were carried out as described in the crystallization section for confocal microscopy (see above), except that investigations started subsequently after polyelectrolytes were added to crystal-containing solutions. For image acquisition the procedure described above under "Scanning confocal microscopy" was modified by scanning the fluorescent profile surrounding crystals several times during a time course beginning 4 min after polyelectrolyte addition to the crystals until the end of the elapsed time (80–100 min).
To determine the relative fluorescence intensity, green-channel images were analyzed using the plot profile option in ImageJ as described above under "Image and data processing". Measurements were made along lines of {100} and {121} faces and a spot representing {100}/{121} edges. The background fluorescence was then subtracted. From the measured intensities, mean values of {100}, {121}, and {100}/{121} intensities were calculated for every time segment of the time course. For the first 16 min of the time course, no above background fluorescence was detected.
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RESULTS |
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), all crystals formed appear to be COM. As shown in Fig. 1, COM crystals grown on mica disks are penetration twins, with a {100} twin plane perpendicular to the 100 directions. The faces developed are {010}, {100}, and {121}. Most crystals nucleate from a {010} face (Fig. 1 a), but some nucleate from a {100} face (Fig. 1 b). Under HSC (
), both COM and COD (not shown) morphologies are observed.
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100 directions, these crystals are oval in cross section, with an oval-shaped depression on the {100} faces (Fig. 2 a); the depressions are also visible in crystals viewed from the 010 directions (Fig. 2 b). Using HSC, OPN in the range 10–15 µg/ml promotes the formation of COD but does not have an appreciable effect on overall number and size of particles. The COM crystals generated are dumbbell-shaped, with more pronounced depressions on {100} faces than those grown at low supersaturation (Fig. 2 c).
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001 direction) to width (010 direction) is lower (compare Fig. 2, c and d). Another difference is that both ends of the COM crystals shown in Fig. 2 d exhibit a 72° angle, which is the angle formed by the {121} faces bounding the {100} faces of well-facetted COM of controls (see Fig. 1 b), whereas the ends of the crystals formed in the presence of OPN are rounded.
Using LSC, poly-glu, like poly-asp, results in the formation of COD, even at low polypeptide concentrations (1 µg/ml), whereas the generation of COM was rarely observed. Using HSC, addition of poly-glu (8–14 µg/ml) did not have an observable effect on overall number and size of particles, although the formation of COD crystals was promoted. COM crystals formed are roughly orthorhombic, with highly facetted faces (Fig. 2 e). At high magnification, these crystals exhibit features (steps and pits) containing angles characteristic of the control COM crystals (compare inset of Fig. 2 e with Fig. 1). It appears that the monohydrate phase forms superstructures organized by multiple twinning of COM both at {100} planes and along the 001 axis.
Adsorption of polyelectrolytes to COM
The COM crystals grown on glass-bottomed dishes under control (no additive) conditions were similar in growth habit to those grown on mica disks. These nucleated from the glass surface at either a {010} or a {100} face. For the most part, crystals nucleated from {010} were used for fluorescence imaging, as these provide better visualization of the {121} faces.
Fig. 3 shows combined SCM/fluorescence and fluorescence alone images of COM crystals to which labeled polyelectrolytes have been added. In the SCM/fluorescence images, crystals appear dark red or black and Alexa-labeled polyelectrolytes appear green. In the fluorescence alone images, Alexa-labeled polyelectrolytes appear white-gray. As the crystals shown in Fig. 3 all nucleated from a {010} face, optical sections through them are bounded by two {100} side faces and four {121} end faces. Note that whereas the {100} faces are perpendicular to the glass surface, the {121} faces are at an oblique angle to it (see Fig. 1 a). For OPN, fluorescence appears to be most intense at the edges between {100} and {121} faces (Fig. 3, a and b). For poly-asp, fluorescence is concentrated along the {121} faces (Fig. 3, c and d). For poly-glu, fluorescence is approximately uniform on the {100} and {121} faces (Fig. 3, e and f).
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For OPN, poly-asp, and poly-glu, fluorescence intensity was measured around the perimeter of the crystal in several optical sections for each of 10 crystals. To account for variations in perimeter length between different sections of the same crystal and between different crystals, the length of each crystal face was divided by the total length of the perimeter. Relative intensity (RI) was then plotted against fractional perimeter distance.
Fig. 4 shows plots of RI versus fractional perimeter distance for single COM crystals labeled with OPN, poly-asp, or poly-glu. In these plots, the profiles for each section of the crystal have been superimposed. Also indicated are the points on the perimeter at which one face ends and another begins. The profiles of each optical section of a crystal are very similar. For OPN, there are four peaks of RI, corresponding to the four edges between {100} and {121} faces (Fig. 4 a). Between these edges, the RI falls to close to zero. For poly-asp, RI is higher (15–25 arbitrary units) along the {121} faces and lower (5–10 arbitrary units) along the {100} faces (Fig. 4 b). Fluorescence resulting from poly-glu adsorption is fairly uniform around the perimeter, although slightly higher at {121} faces than {100} faces (Fig. 4 c). Note that the RI values associated with {121} faces are similar for poly-glu and poly-asp.
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The images and data in Figs. 3–6
were all obtained from crystals nucleated from a {010} face. Panels a–d of Fig. 7 show two crystals, one nucleated from {010} and the other from {100}, labeled with poly-asp. Panels a and b are composite (red-green) images taken at the glass-crystal interface and at the crystal-solution interface of the latter crystal, respectively. Panels c and d are green-channel (fluorescence) images corresponding to panels a and b, respectively. From the glass-crystal sections (a and c), poly-asp binding to the {121} faces can be seen in both crystals. From the crystal-solution sections (b and d), it is clear that there is negligible binding to the {100} face. Panels e and f of Fig. 7 are images of crystals nucleated from a {100} face and labeled with poly-glu and OPN, respectively. Poly-glu adsorption occurs on {010} (side) and {121} (end) faces, although slightly more on the latter. As noted above, this is likely to be because the {121} faces are not vertical. OPN adsorption is seen at the {121}/{100} edges, as it was for crystals nucleated from {010} faces (Fig. 3 a).
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60 min. The final amounts of fluorescence associated with {100}/{121} edges (Bmax = 5.26) was 100% higher than those associated with the {100} (Bmax = 2.66) and {121} (Bmax = 2.25) faces.
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DISCUSSION |
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,32), the effects of poly-asp and poly-glu on crystal formation are often studied (33–35). Crystals grown with poly-asp are generally similar in growth habit to those grown with OPN, particularly in that both show depressions on the surfaces that we believe to be the {100} faces. Poly-glu also has profound effects on the growth habit of COM. However, crystals grown with poly-glu generally lack the rounded edges and regularity of those grown with OPN or poly-asp.
In previous studies, Wesson and co-workers found that growth of COM in the presence of poly-glu resulted in smaller, dumbbell-like crystals (33). The term "dumbbell" was also applied to COM crystals grown in the presence of heparin (36). In the latter study, the indexing of the crystals agrees with our identification of the concave faces as {100}. It is apparent that the effects of polyanions on COM growth habit are highly dependent upon the experimental conditions used. However, the striking similarities between our OPN-induced dumbbells and the COM crystals found in human crystalluria (37) and in the urine of individuals suffering from intoxication with ethylene glycol, a metabolic precursor of oxalate (38), suggest that modification of the COM growth habit is a physiological effect of OPN in urine.
Specificity of synthetic polypeptide adsorption to COM
The interaction between synthetic polypeptides and COM was studied in multiple optical sections through crystals using a combination of SCM and fluorescence imaging. By quantifying the fluorescence associated with different faces and interfacial edges, we have shown that poly-asp adsorbs preferentially to {121} faces and poly-glu adsorbs nonspecifically to {100}, {010}, and {121} faces.
Previous studies on interactions between polyelectrolytes and COM crystals have mainly used in situ atomic force microscopy (AFM). In these studies, rates of movement of steps on crystal surfaces (or rates of filling of etch pits, which is essentially the same thing) are used to infer the site of binding of inhibitors. The rational is that binding to a particular crystal face will inhibit growth perpendicular to that face; that is, a substance adsorbing to {hkl} faces will inhibit growth in hkl directions (39).
AFM analysis of COM growth showed that poly-glu was more potent than poly-asp in inhibiting the movement of growth hillocks on {010} faces in both the 021 and 121 directions; however, poly-asp was more potent than poly-glu in inhibiting the movement of hillocks on {100} faces in 001 directions (35). These inhibitory effects were attributed to "pinning" of the steps formed by the edges of the hillock and the underlying crystal face.
Studies on the filling of etch pits on {100} faces of COM indicated that poly-asp interacts preferentially with {001} or {021} lattice planes, whereas poly-glu prefers {010} planes (34). A recent study using AFM has confirmed that aspartic acid-rich peptides inhibit growth of hillocks on {100} faces in 001 directions more effectively than in 021 directions (40). It is interesting that peptides of sequence (asp-asp-asp-ser)n were much better inhibitors than those of sequence (asp-asp-asp-gly)n, as OPN contains numerous sequences rich in both aspartic acid and serine.
As noted by Jung et al., pinning of a particular step could be caused by interactions with different lattice planes. For example, the poly-asp-mediated inhibition of step growth in the 001 direction on the (100) face of COM could result from the polypeptide adsorption to either the (001) face of the step, the (100) face itself, or both. These possibilities can be distinguished by studying the effects of inhibitors on the overall growth of the crystal. Using real-time SCIM imaging, we determined growth rates of COM crystals nucleated from {100} faces. It was found that poly-asp decreased growth in 001 directions but not in 010 directions (30). Because COM crystals of the growth habit studied will grow in 001 directions only by lattice-ion addition to {121} faces, adsorption of poly-asp to these faces would be expected to inhibit growth along 001 (see Fig. 1 c). Therefore, the face-specific adsorption of poly-asp to COM observed in this study is generally consistent with previous studies on the effects of poly-asp on crystal growth.
In the case of poly-glu, the pattern of adsorption to COM crystals and effects on growth habit are more difficult to correlate. Real-time SCIM analysis may be of value in determining whether poly-glu inhibits growth in all directions equally, as predicted by this study, or unequally, as suggested by the AFM studies cited above.
Specificity of OPN adsorption to COM
In a previous study, fluorescence microscopy was used to study the interaction between rhodamine-labeled staphylococcal protein A and COM crystals. It was shown that protein adsorbs to {110} planes and is incorporated into the crystal lattice (41). In this study, we use a similar technique to show that OPN adsorbs preferentially to the edges between {100} and {121} faces of COM crystals.
In situ AFM analysis by Qiu et al. (42) showed that human kidney OPN strongly inhibits movement of growth hillocks on {010} faces of COM, which grow in the 101 and 100 directions. (In the indexing scheme used by Qiu et al., 101 and 100 directions are equivalent to the 100 and 001 directions used here.) However, OPN had minimal effect on growth hillocks on {101} faces, which grow in 120 and 100 directions (equivalent to 021 and 001 directions in the indexing scheme used here). The different effects of OPN on growth of the {010} and {101} faces were attributed to the greater height of hillocks on the former, rather than face-specific binding of the protein.
Using a different AFM technique, the strength of interaction of carboxylate- or amidinium-functionalized tips with faces of COM crystals was shown to be {100} > {121} > {010}. Addition of bovine milk OPN resulted in an increased adhesion of the tips to {100} faces, suggesting that the protein is preferentially adsorbing to these faces (43).
If OPN preferentially adsorbs to {010} faces of COM, it should decrease growth of the crystals in 010 directions; if it preferentially adsorbs to {100} faces, OPN should decrease growth in 100 directions. In fact, as we have shown, the effects of OPN on COM growth habit are more complex than can be explained solely by inhibition of growth in a specific set of directions. We have also shown that the greatest degree of adsorption of OPN to COM is not associated with a particular face but with the edges between {100} and {121} faces. We hypothesize that this edge-specific binding is responsible for the dumbbell morphology of COM crystals grown in the presence of OPN and, indeed, COM crystals found in human urine.
The mechanism by which OPN adsorbs to {100}/{121} edges of COM is not immediately obvious. One possible explanation is that OPN is actually interacting with {021} faces lying between {100} and {121}. Such faces have been reported to develop in COM penetration twins (44) and are occasionally observed under the conditions used in this study, particularly at longer incubation times (>3.5 h). COM crystals with well-developed {021} faces have an overall shape similar to dumbbell crystals (compare Fig. 6 of Millan (44) with Fig. 2 c of this work). However, {021} crystals of COM would be expected to exhibit interfacial edges.
Another possibility is that these edges are sites of high surface energy (45). The acute angles formed by the {100} and {121} faces would support this possibility. Adsorption of OPN at edges rather than faces might well be related to the loss of edges in COM dumbbells.
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CONCLUSION |
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ACKNOWLEDGEMENTS |
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The studies reported here were supported by the Canadian Institutes of Health Research.
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FOOTNOTES |
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Submitted on December 1, 2006; accepted for publication April 27, 2007.
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