ARTICLE |
Correspondence to: Mary Ann Stepp, Dept. of Anatomy and Cell Biology, The George Washington University Medical Center, 2300 I Street NW, Washington, DC 20037.
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Summary |
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In the unwounded cornea, tenascin-C localizes to a short stretch of the basement membrane zone at the corneoscleral junction or limbus. To determine whether the function of the limbus is affected by the absence of tenascin-C, mice possessing a deletion of tenascin-C and strain-matched wild-type mice are used in corneal debridement wounding experiments. The expression of integrins (3,
9, and ß4) in the tenascin-C knockout corneas is evaluated by producing polyclonal cytoplasmic domain antipeptide sera and performing immunofluorescence microscopy. In addition, we evaluate the localization of several other proteins involved in wound healing, including fibronectin, laminin ß1, nidogen/entactin, and VCAM-1, in both the tenascin knockout and wild-type mice. There are no differences in healing rate, scarring, or neovascularization after corneal debridement wounds.
9 integrin is expressed at the limbal border of unwounded tenascin-C knockout animals and is upregulated during migration only after the larger wounds. At 8 weeks after larger wounds, the localization of
9 again becomes restricted to the limbal border. Results show that tenascin-C is not required for development or maintenance of the corneal limbus or for normal re-epithelialization of corneal epithelial cells after debridement wounding.
(J Histochem Cytochem 48:363375, 2000)
Key Words: tenascin-C, wound healing, integrins, re-epithelialization, cornea
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Introduction |
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The pattern of expression of the extracellular matrix protein tenascin-C during development and wound repair strongly suggests that it has an important role in the life of an organism. It is upregulated during embryonic development and wound healing and is found around areas of tumor formation and inflammation (
Tenascin-C is a large modular glycoprotein consisting of a single fibrinogen-like domain and multiple EGF and FN-type III domains. The tenascin-C mRNA is subject to tissue-specific alternative splicing due to the inclusion or exclusion of a variable number of the exons encoding the FN-type III domains. As a result, multiple tenascin-C isoforms are generated, with distinct sizes ranging from 180 to 330 kD. In the corneas of humans and mice, the sizes of the tenascin isoforms produced range from 180 to 230 kD (
Cells can adhere directly to tenascin-C via binding to members of the integrin family of receptors including 2ß1,
8ß1,
9ß1,
vß3, and
vß6 (
ß heterodimers to interact extracellularly with a variety of molecules within the ECM and/or with other receptors on adjacent cell surfaces. Intracellularly, integrins bind to the internal cytoskeleton of the cell and regulate a variety of signal transduction events (
In addition to mediating attachment of cells to matrix, tenascin functions in anti-adhesion (
Despite many studies documenting tenascin-C as a molecule whose expression correlates with organogenesis, wound healing, and cancer, tenascin-C knockout mice develop normally and have the same fecundity and physical appearance as wild-type mice (
Although tenascin-C is not required for the development of a viable mouse, inactivation of tenascin-C clearly alters cellular behavior and tissue architecture in cells and tissues that normally express it. During studies of the developing mouse eye, we observed that 9 integrin expression and localization became increasingly restricted to a subset of epithelial cells in the basal cell layer of a region called the corneoscleral junction or limbus. Tenascin-C also localized to the basement membrane zone beneath the
9-positive cells and in the underlying stroma (
9tenascin-C interactions may be involved in mediating adhesion and/or proliferation of the
9-positive limbal epithelial basal cells. The limbus is important to the health of the cornea because it is the location at which the corneal epithelial stem cells reside (
9 expressed and the numbers of cells that are
9-positive increases as the limbal basal cell population is forced to proliferate and TA cells migrate towards the center of the cornea after an epithelial injury (
9 integrin therefore is expressed not only on the stem cells themselves but also on the TA cells. Tenascin-C localization is retained at the basement membrane zone beneath the epithelial cells at the limbal border during re-epithelialization. In addition, an increase in the distribution of tenascin-C occurs within the corneal stroma at later times after epithelial cell migration is complete (
Although it has been shown that the corneal epithelial stem cells are restricted to the basal cell compartment at the limbus, it is not at all clear what keeps them there. It has been postulated that basement membrane specializations exist at the border between the limbus and peripheral cornea that function as a barrier which the stem cells do not normally cross (
To determine whether 9tenascin-C interactions function to maintain the cornealscleral junction, we used the tenascin knockout mouse, generated by
9 integrin ligands besides tenascin-C to determine whether there is evidence of compensation for the lack of tenascin by changes in expression of osteopontin or VCAM-1.
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Materials and Methods |
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Animals
Tenascin-C-deficient mice were generated by
Wound Model
All experiments on mice were conducted in compliance with the recommendations of the Association for Research in Vision and Ophthalmology and with the GWU IACUC. Manual debridement wounds were created on the corneas of 8-week-old tenascin-C knockout mice and their wild-type counterparts as described previously (
Immunohistochemical Analysis
The frozen corneas were sectioned (10 µm) and stained with the primary antibodies to the basement markers laminin ß1, entactin, and perlecan (A. Ljubimov; Cedars-Sinai Medical Center, Los Angeles, CA) as well as J-18 (laminin 5; J. Jones, Northwestern University Medical School, Chicago, IL). Corneas were also stained using antibodies to tenascin (Mtn-12; T-3413, Sigma, St Louis, MO), VCAM-1 (429:MVCAM.A, #01810D, Pharmingen, San Diego, CA), osteopontin (C. Giachelli; U. of Washington, Seattle, WA), and fibronectin (R.O. Hynes; Mass. Inst. of Tech./HHMI, Cambridge, MA) and with antibodies to the integrins 3,
9, and ß4. The polyclonal integrin sera were generated using standard procedures (Covance Laboratories; Vienna, VA) from the human amino acid sequences encoded by the C-terminal cytoplasmic domains of the three integrins. The
3 peptide was EMK SQP SET ERL TDD Y, the
9 peptide was RYK EII EAE KNR KEN EDS WDW VQK, and the ß4 peptide was KKK TTS GSL STH MDQ QFF QT. For
3 and ß4, the generation of these sera involved injection of two rabbits each with a KLH-conjugated peptide. The longer peptide comprising the complete cytoplasmic domain of
9 was used for generation of
9 sera after four rabbits injected with a shorter peptide failed to generate sera useful for immunofluorescence. Of four rabbits injected with the longer peptide, only one generated a serum that recognizes
9 on human or mouse tissues. Biochemical and immunohistochemical characterization of these sera are described in Results. For double labeling, a rat anti-mouse ß4 monoclonal antibody (346-11A; #09491D; Pharmingen) was used along with the rabbit
9 polyclonal sera. The images were viewed and captured using an Olympus BX60 or with confocal microscopy using the BioRad MRC 1000 program. All images were imported into Adobe Photoshop 4.0.
Immunoblot Analyses
Immunoblotting was carried out as described previously (3 and
9 sera, mouse liver was used as a source of protein extracts. For ß4, mouse corneal epithelium was used. Tissue extracts were run unreduced on 7.5% SDS-PAGE gels and proteins transferred to the nitrocellulose membranes. Membranes were cut into several strips after blocking with blocking buffer consisting of 5% milk in Tris-buffered saline containing 0.1% Tween-20. Strips were incubated separately with dilutions of each of the three integrin sera. Peptide inhibition was performed by preincubating diluted sera with 50 µg/ml peptide for 30 min to 1 hr before adding to the nitrocellose strips. Specificity of peptide inhibition was determined by preincubating sera with two peptides: (a) the peptide used to generate the serum, and (b) one of the integrin peptides from a different integrin
- or ß-chain. HRP-conjugated donkey anti-rabbit IgG was used as secondary antibody and proteins were detected by ECL Western Blot Detection reagent (Amersham; Arlington Heights, IL). BioMax film (Kodak; Fisher, Rochester, NY) was used to obtain images, which were then scanned and processed using Adobe Photoshop.
Tenascin RT-PCR
To confirm the absence of tenascin-C mRNA in tissues obtained from tenascin-C KO mice, total RNA was isolated from liver obtained from either tenascin-C KO or wild-type GRS/A mice. Mice were sacrificed by lethal injection, liver tissues rapidly frozen and powdered in liquid nitrogen, and total RNA extracted using RNAzol according to the manufacturer's instructions (Tel-Test; Friendswood, TX). Total RNA per well of 25 µg was used for RT-PCR studies with tenascin primers. Primers for mouse G3PDH (Life Technologies/Gibco; Gaithersburg, MD) and ß-actin (Clontech; Palo Alto, CA) served as controls. The primers for tenascin were made against the region flanking the translation start site of the mouse tenascin gene to direct the amplification of a tenascin-C rtDNA product of 834 bp (
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Results |
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Localization of Tenascin-C in the Unwounded Cornea Is Restricted to the Periphery
A rat monoclonal antibody recognizing mouse tenascin-C was used on the pigmented inbred GRS/A wild-type mice and the GRS/A tenascin-C KO mice. As expected, tenascin-C localization was high at the limbal region (Fig 1A) and was absent in the central cornea (Fig 1B) in mice containing the intact tenascin-C gene. No tenascin-C was observed in the KO mouse cornea in the limbal area (Fig 1C) or central cornea (Fig 1D). To further confirm that tenascin-C was not expressed in the tissues of the knockout mice, RT-PCR was performed using tenascin-C primers derived from the sequence of the mouse tenascin-C gene flanking the translational start site (Fig 1E). RNAs isolated from the tenascin-C KO and wild-type mouse liver are capable of amplifying control primers for G3PDH and ß-actin, but only the wild-type liver RNA amplifies the tenascin-C primers. These data show that neither the tenascin-C protein nor its mRNA is detected in these tenascin-C KO mice.
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Polyclonal Sera Against 3,
9, and ß4 Were Generated and Characterized for Use in Immunofluorescence Studies
Peptide antibody reagents were generated and characterized first by immunofluorescence and their specificity confirmed by immunoblotting. Data on the characterization of these sera by blotting are shown in Fig 2. For 3,
9, and ß4, a single band of the appropriate kD was observed for each serum (Fig 2, Lanes 2, 5, 8, respectively) which was not present when sera were preincubated with the peptide used for their production (Fig 2, Lanes 4, 7, and 10, respectively). Peptide blocking was specific because addition of the
9 peptide to either the
3 or ß4 serum did not interfere with its ability to interact with
3 or ß4 (Fig 2, Lanes 3 and 9), nor did the addition of the
3 peptide interfere with the
9 binding (Fig 2, Lane 6). Using immunofluorecence microscopy, all three of these newly prepared sera were tested against other sera known to be specific against each of these integrins. Identical results were obtained in the corneas of mice, rats, and humans. Although data from rats and humans are not presented, mouse data are described below.
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Localization of Several Epithelial Integrins and Basement Membrane Markers Is Not Altered in Corneas of Tenascin-C KO Animals
To determine whether the differentiation of the epithelial cells of the cornea is altered by the disruption of tenascin-C gene expression, we localized 3, ß4, and
9 integrins in the unwounded tenascin-C KO cornea using immunofluorescence (Fig 3). Compare to the wild-type GRS/A animals, the localization of
3 and ß4 in the tenascin-C KO animals was identical, and therefore data from only the tenascin-C KO tissues are shown. The localization of
3 was restricted primarily to the basal and basolateral membranes of the basal cells (Fig 3A). ß4 was more abundant at the basal cell basal membrane (Fig 3B), where it is known to be present in hemidesmosomes. However, ß4 was also present at the lateral membranes between the basal cells. Like
3 and ß4, the localization of
9 was similar in the tenascin-C KO and wild-type animals.
9 was localized exclusively to the epithelial cells at the corneoscleral junction between the limbus and the peripheral cornea (Fig 3C) and was not detected in the central cornea (Fig 3D). Therefore, tenascin-C expression at the corneoscleral junction is not a prerequisite for the localization of
9 integrin to this site. To further evaluate the tenascin-C KO cornea, the localization of several basement membrane proteins was evaluated, including perlecan, laminin ß1, laminin
1, entactin/nidogen, and laminin-5. Shown in Fig 3E and Fig 3F are the results for entactin/nidogen and laminin ß1, respectively. The basement membrane zone is linear and continuous and there is no evidence of any abnormalities in the tenascin-C KO animals. Taken together, the localization of integrins and basement membrane markers indicates that the tenascin-C KO cornea is similar to other wild-type control, unwounded corneas.
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Tenascin-C is a component of the anatomic border formed at the corneoscleral junction in the normal cornea and has been postulated to play a structural role (9-positive cells to the limbal border in the unwounded tenascin-C KO cornea suggests that the anatomic border normally formed by tenascin-C is not a requirement for the formation of a normal limbal junction. Given the ability of tenascin-C to mediate either adhesion or anti-adhesion, depending on the cell type and nature of the substrate (
9-positive, early transit-amplified cells to migrate past this border after being forced to proliferate in response to wounding.
Despite the Lack of Tenascin-C, 9-positive Cells Are retained at the Limbal Border After Epithelial Cell Migration Has Been Induced by Debridement Wounding
Both smaller wounds, which close within 24 hr without a significant increase in cell proliferation, and larger wounds, which take 23 days to re-epithelialize and require cell proliferation during migration, were made on the corneal surface of the tenascin-C KO mice and tissues were evaluated for the rate of wound closure, for signs of neovascularization and scarring, and for the localization of integrins. These types of wounds remove only the epithelial cells and leave the basement membrane intact and native (
The rate of wound closure was determined after the removal of a 1.5-mm area of epithelial cells from the corneal surface of a total of 42 tenascin-C KO and 30 GRS/A wt mice at 12, 18, 24, and 48 hr. For large wounds, a total of 43 tenascin-C KO and 30 GRS/A wt were evaluated at 1, 2, 3, 6, and 12 days after initial injury. We observed no difference in the length of time to wound closure between KO and wild-type strains. In addition, there was no increase in scarring or neovascularization within the first 6 weeks post wounding. Although there were no differences between the wild-type and KO GRS/A mice, large wounds were generally observed to take 3 days to close in the GRS/A strain of mice rather than 2 days, which is standard for 8-week-old Balb/c mice. This difference indicates that variation in healing rates exists among different mouse strains.
Immunofluorescence studies performed on tissues at various times after wounding indicated that both the tenascin-C KO and GRS/A wt mouse corneas showed similar changes in integrin localization during healing. The data presented in Fig 4 and Fig 5 are from tenascin-C KO animals. After smaller wounds, the localization of 9 to the basal cells at the limbus was retained at 12 hr (Fig 4A) and 18 hr (Fig 4C) and was absent at the leading edge at both time points (Fig 4B and Fig 4D).
3 localized to the basolateral membranes and at the basal surface of the basal cells at the leading edge at both 12 and 18 hr after injury (Fig 4E and Fig 4F, respectively). ß4 appeared to increase in abundance in the cytoplasm and on the membranes of cells at the leading edge and back towards the periphery, and the relative abundance of ß4 appeared higher at 18 hr (Fig 4H) compared to 12 hr (Fig 4G). After small debridement wounds in the rat, we have shown, using a biochemical approach, that ß4 expression increases during migration and decreases as soon as migration is complete (
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Thus far, the tenascin-C KO mouse cornea has responded similarly to wild-type corneas to debridement wounding. Unlike the small wound, in which a wedge of cells makes up the leading edge, in the large wound a single-cell layer sheet of cells makes up the leading edge, with all of its cells derived from proliferation of transit-amplifying cells from the limbus. Past studies have shown that 9 is expressed later during migration after these larger wounds and during restratification (
9 is upregulated after the large wounds or whether there are also changes in ß4 expression and localization after the larger wounds. In Fig 5, we used confocal microscopy to evaluate simultaneously the localization of
9 and ß4 integrins after large wounds in dual-labeling experiments conducted on tissues from tenascin-C KO mice; similar results were obtained from wild-type mice. In Fig 5A, at 1 day after wounding, a gradient of ß4 is observed, with ß4 becoming more abundant away from the leading edge. In Fig 5A, note also that at the tip of the leading edge (arrowhead), a portion of the epithelial sheet made up of three or four cells has loosened and has come off the stroma. This is a common occurrence after larger wounds, indicating that the sheet is loosely adherent and more so at the leading edge. ß4 appears to be localized primarily to the basal surface of the migrating sheet of cells.
9 localization at 1 day after large wounds also increases towards the periphery, with the three or four cells at the very tip of the leading edge expressing little if any
9. The normal restriction of
9 within the limbus is lost at 24 hr after larger wounds; earlier times were not evaluated.
By 2 days, both 9 and ß4 were expressed abundantly, as shown in Fig 5B in a typical section taken from a region behind the leading edge during migration. Although Fig 5A and Fig 5B show a single layer of migrating epithelial cells expressing both
9 and ß4, the staining for ß4 remains most abundant at the basal surface, where the cells are in contact with the underlying matrix.
9 is most abundant at the basolateral cell surfaces and within the cytoplasm. Comparing the localization of
9 and ß4 during migration after the large wound (Fig 5) with their localization after the smaller wounds (Fig 4A4D) reveals important differences. ß4 appears to increase in amount at longer times after both types of injury and to be abundant at both basal and nonbasal cell membranes in response to both small and large wounds.
9 localization shows more dramatic differences; only after the larger wounds is
9 present on actively migrating corneal epithelial cells in the central cornea. By 8 weeks after injury in the TN KO mouse, migration and restratification are complete and the expression of
9 in the central cornea is again restricted to the cells within the limbus; there is little if any
9 in the central cornea (Fig 5C). The pattern shown is identical to that observed in control mice. To the left of the arrow in Fig 5C, the
9-positive conjunctival and limbal basal cells are seen to express a low level of ß4 at their basal surfaces. To the right of the arrow towards the center of the cornea, cells possess less
9 and more ß4. The inset shows the transition zone between limbus and peripheral cornea at a higher magnification. Note that cells at this site display dramatic differences in
9 and ß4 localization and expression. Taken together, these data show that the ability of the epithelial cells of the cornea to heal in response to manual debridement wounds is not affected by the lack of tenascin-C.
VCAM-1 Expression in Healing Corneas Is also Comparable Between the Wild-type and the Tenascin-C KO Mice
Recent data show that, in the vasculature, 9ß1 integrin on leukocytes can function as a receptor for VCAM-1 on activated endothelia (
9 and VCAM-1 might be co-distributed in either normal or wounded wild-type and tenascin-C KO corneas, we evaluated these tissues for expression of VCAM-1 and present the data from tenascin-C KO animals in Fig 6. Similar data are obtained from wt animals. VCAM-1 was not expressed by the epithelial cells in the cornea (Fig 6A). However, VCAM-1 did appear to be expressed on the normal, unwounded fibroblasts in the stroma. At 18 hr after small wounds, the staining for VCAM-1 disappeared at the anterior surface of the central cornea (Fig 6B). By 48 hr, VCAM-1 was again present in this region (Fig 6C). At 2 days after the larger wounds, a marked loss of VCAM-1 staining in the stroma was observed over the entire corneal surface, with VCAM-1-positive fibroblasts visible only along the posterior aspect of the stroma (Fig 6D). By 6 days, a normal-appearing VCAM-1 staining profile was observed. In addition to tenascin-C and VCAM-1, another ligand for
9 has been demonstrated. A proteolytically cleaved fragment of osteopontin has been shown to mediate
9ß1 adhesion and migration (
9 ligand, tenascin-C, by the upregulation of the other known ligands, VCAM-1 and osteopontin.
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Discussion |
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Re-epithelialization Is Not Delayed in the Absence of Tenascin
Several years of studies had shown that tenascin-C is upregulated in response to epidermal wounding (
The larger-sized debridement wounds we perform induce tenascin-C expression both in the stroma and by the epithelial cells. This increase in tenascin-C occurs immediately after closure of the wound at 3 to 6 days (
9 and ß4 Integrin Expression Is Increased During Wound Healing in Normal and Tenascin-C KO Mice
We are unable to document any differences in integrin expression in the corneas of the tenascin-C KO mouse. Analysis of 9 localization in control and wounded corneas revealed no differences between control and tenascin-C KO animals. The localization of
9 varies in the KO animals after wounding in precisely the same way as in Balb/c and GRS/A mice. The same is true for
3 and ß4 integrins. There are no changes in
3 and the increased expression of ß4 occurs during migration after the small wound as it does in normal animals. For the larger wounds, we show that
9 is upregulated at 1 day after injury and continues to increase at Day 2 in both the tenascin-C KO and wild-type animals.
The absence of one member of a receptorligand pair has been shown to disrupt the localization of the other member of that pair. In the 8 integrin KO mouse, various ligands of
8ß1 integrin, including fibronectin, vitronectin, and tenascin, are reduced in expression and localization during development (
3 KO mice have disrupted localization of laminin basement membrane components (
9 in the absence of tenascin-C. Other
9 ligands (VCAM-1 and osteopontin) do not compensate for the lack of tenascin-C in the tenascin-C KO mouse.
We see no evidence of localization or expression of either VCAM-1 or osteopontin in the cells of the corneal epithelium of the tenascin-C KO or the wild-type animals, despite the abundant amount of 9 localized to these tissues in response to the large wound. Dynamic upregulation of expression and localization for
9 integrin after injury in both normal and tenascin-C KO animals is unlikely to occur in the absence of function. Our data suggest that the exact ligand or counter-receptor for epithelial
9 integrin remains to be determined. Given that after the large wound, the tenascin-C KO mouse corneal epithelial cells express none of the known ligands for
9, they would provide an excellent system to use to discover the nature of any additional
9 integrin ligandscounter-receptors.
Tenascin-C Is Not Required for Maintenance of Corneoscleral Junctions After Wounding
The importance of the corneoscleral junction in the maintenance of corneal health cannot be overestimated (
The extracellular matrix environment at the limbus is likely to play a role similar to that of the stromal microenvironment in the bone marrow. In the bone marrow, specific stromalstem cell interactions mediate the proliferation rate and retention of stem cells (4 integrin in the marrow causes the hematopoietic stem cells located there to migrate into the bloodstream. This observation has provided insight into hematopoiesis and has provided a method for obtaining large numbers of hematopoietic stem cells from patients. Tenascin-C is also important in the bone marrow; several hematapoietic precursor cells interact with tenascin-C in the marrow and this interaction stimulates stem cells to proliferate (
Although tremendous progress has been made in understanding how to obtain and manipulate the stem cells involved in hematopoiesis, we know little about regulation and maintainance of the stem cells needed for the corneal epithelium. Keeping their niche at the corneoscleral junction free from trauma is important, but we must also identify the proteins, both cell-associated and those present within the microenvironment at the limbus, that are required to maintain the health of these stem cells. Only then can we hope to develop culture methods and treatments to help those suffering from the loss of stem cells caused by damage to the limbus. Our studies demonstrate that one of the known components of this junction, tenascin-C, is not necessary for the limbus to retain its normal function in preserving the stem cell population after injury. Additional studies and the identification of the cellcell and cellsubstrate interactions necessary for limbal stem cell maintenance and function are necessary to address the problem of corneal stem cell deficiency.
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Acknowledgments |
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Supported by NIH RO1 EYO 8512-09 (to MAS).
We wish to thank Drs H.P. Erickson and M. Kusakabe for providing the GRS/A tenascin-C and the GRS/A wild-type mice, Dr Robyn Ruffner and the GWU Center for Microscopy and Image Analysis for assistance with the photography and imaging, and Dr Bernard Zook and the staff at the GWUMC Animal Research Facility for caring for the mice.
Received for publication July 21, 1999; accepted October 8, 1999.
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