Correspondence to: Francesco Ramirez, Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York University, One Gustave L. Levy Place, Box 1020, New York, NY 10029. Tel:(212) 241-1757 Fax:(212) 722-5999 E-mail:ramirf01{at}doc.mssm.edu.
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Abstract |
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The Tight skin (Tsk) mutation is a duplication of the mouse fibrillin 1 (Fbn1) gene that results in a larger (418 kD) than normal (350 kD) protein; Tsk/+ mice display increased connective tissue, bone overgrowth, and lung emphysema. Lung emphysema, bone overgrowth, and vascular complications are the distinctive traits of mice with reduced Fbn1 gene expression and of Marfan syndrome (MFS) patients with heterozygous fibrillin 1 mutations. Although Tsk/+ mice produce equal amounts of the 418- and 350-kD proteins, they exhibit a relatively mild phenotype without the vascular complications that are associated with MFS patients and fibrillin 1deficient mice. We have used genetic crosses, cell culture assays and Tsk-specific antibodies to reconcile this discrepancy and gain new insights into microfibril assembly. Mice compound heterozygous for the Tsk mutation and hypomorphic Fbn1 alleles displayed both Tsk and MFS traits. Analyses of immunoreactive fibrillin 1 microfibrils using Tsk- and species-specific antibodies revealed that the mutant cell cultures elaborate a less abundant and morphologically different meshwork than control cells. Cocultures of Tsk/Tsk fibroblasts and human WISH cells that do not assemble fibrillin 1 microfibrils, demonstrated that Tsk fibrillin 1 copolymerizes with wild-type fibrillin 1. Additionally, copolymerization of Tsk fibrillin 1 with wild-type fibrillin 1 rescues the abnormal morphology of the Tsk/Tsk aggregates. Therefore, the studies suggest that bone and lung abnormalities of Tsk/+ mice are due to copolymerization of mutant and wild-type molecules into functionally deficient microfibrils. However, vascular complications are not present in these animals because the level of functional microfibrils does not drop below the critical threshold. Indirect in vitro evidence suggests that a potential mechanism for the dominant negative effects of incorporating Tsk fibrillin 1 into microfibrils is increased proteolytic susceptibility conferred by the duplicated Tsk region.
Key Words: elastic fibers, microfibrils, Tsk, Marfan syndrome, extracellular matrix
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Introduction |
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Morphologically distinct networks of elastic fibers are present in the extracellular matrix of virtually every organ and are particularly abundant in tissues subjected to periodic stress, such as the respiratory and cardiovascular systems (
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Fibrillins 1 and 2 display superimposable multidomain structures that consist mainly of calcium-binding epidermal growth factor-like (cbEGF)1 repeats interspersed by a novel cysteine-rich (8-cys) motif, present only in the fibrillins and latent transforming growth factor-ßbinding proteins (
Two lines of mutant fibrillin 1 mice that were created by homologous gene targeting have recently refined and extended this pathogenic model () contains a mutation that combines a structural defect of fibrillin 1 with reduced gene expression. As a result, the mg
allele produces 510% of the normal amount of fibrillin 1 with an internal deletion of 272 amino acids (
product. On the other hand, homozygous mg
animals die of MFS-like vascular complications within the first month of postnatal life as a result of substantial fibrillin 1 deficiency. The second line of mice (mgR) contains a mutation that produces 1520% the amount of wild-type fibrillin 1 (
The Tsk mutation is a genomic duplication within the mouse fibrillin 1 (Fbn1) gene that results in the production of normal levels of a mutant protein with 984 additional amino acids and a predicted molecular mass of 418 kD (Fig 2; allele. According to the dominant negative model of MFS mutations, the Tsk gene product should negatively affect the function of the wild-type molecules and its antimorphic effect should cause vascular complications and the premature death of Tsk/+ mice. Paradoxically, Tsk/+ mice have a normal life span and no evidence of vascular complications (
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Here, we report studies of the Tsk mutation using genetic crosses between the various Fbn1 alleles, cell culture models, and antibodies specific for the Tsk protein. Our results exclude the assembly of distinct homotypic fibrillin 1 polymers in Tsk/+ mice and, instead, suggest the following model (see Fig 1 d). Tsk fibrillin 1 molecules participate in the initial stage(s) of microfibril assembly but cannot properly polymerize into long microfibrils unless they are in the presence of wild-type molecules. Copolymerization of Tsk and wild-type fibrillin 1 in Tsk/+ mice decreases the amount of functional microfibrils below the threshold level of bone overgrowth and lung emphysema, but not of vascular abnormalities. We also present indirect evidence suggesting that the Tsk duplication destabilizes the mutant product, thus, rendering the protein more sensitive to proteolysis than the wild-type molecule.
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Materials and Methods |
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Mice
The Tsk/+ mice used in this study were previously backcrossed onto the C57BL/6J background ( and mgR mutations were maintained in the heterozygous state in the mixed C57BL/6J x 129Sv-ter/+ background (
Cell Cultures
Primary dermal fibroblasts were prepared from newborn skin explants of Tsk/+, mgR/+, mg/+, Tsk/mg
, Tsk/mgR, and +/+ mice, whereas Tsk/Tsk cells were prepared from 9-d postcoitum embryos. Mouse fibroblasts and human amnion, epithelial-like WISH cells (ATCC CCL-25; American Type Culture Collection) were maintained in DME supplemented with 10% FBS and antibiotics (Gibco Laboratories). Aside from DNA genotyping, cell lines were characterized by protein analysis of metabolically labeled, conditioned medium that was immunoprecipitated and fractionated on SDS-PAGE (see below).
Antibodies and Immunoblotting
pAb8368 was raised against the peptide M-A-E-Y-Q-A-L-C-S-S-G-P-G-M-T-S-A-G-T-K synthesized on a Milligen 9050 peptide synthesizer using standard Fmoc chemistry. The peptide was deprotected, purified by reverse-phase chromatography and the amino acid sequence was confirmed by Edman degradation; the peptide was coupled to keyhole limpet hemocyanin using the COOH-terminaladded lysine residue, and injected into a rabbit in the presence of Freund's adjuvant. Serum was prepared and tested by ELISA against the immunizing peptide. Antibody specificity was established by Western blot analysis. Conditioned medium of fibroblasts cultured 48 h in DME without serum was harvested, briefly centrifuged to clear cell debris, and precipitated with 10% TCA (
Histological and Ultrastructural Analyses
Mouse tissues were processed as previously described ( and Tsk/mgR mice were ~2 wk old and were analyzed together with age-matched wild-type littermates. Samples of adult Tsk/+ and +/+ mouse skin were subjected to collagenase extraction and molecular sieve chromatography to isolate intact microfibrils according to the protocol of
Immunoprecipitation and Fibrillin 1 Proteolysis
For immunoprecipitation, subconfluent cells were grown for 24 h in Met/Cys-free DME supplemented with 50 µCi/ml of [35S]Cys/Met mixture (ICN Biomedicals;
Indirect Immunofluorescence
Early passages of primary culture fibroblasts were plated on glass coverslips at a density of 175,000 cell/ml and cultured for 120 h. Cells were washed with PBS, fixed at 20°C with cold methanol for 20 min and rinsed with PBS. The primary (pAb9543, pAb8368, or mAb201) and secondary antibodies (fluorescein isothiocyanate anti-rabbit [Sigma Chemical Co.], or Cy3 anti-rabbit [Jackson ImmunoResearch Laboratories]) were applied for 1 h at room temperature (
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Results |
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Analysis of Compound Heterozygous Mutant Mice
Tsk/+ mice are readily recognizable from wild-type littermates for the tightness of the skin, as early as 57 d of age, and, by 3 mo, they display a characteristic hump in the shoulder region. Skin tightness correlates with excessive matrix accumulation and marked hyperplasia in the loose connective tissue (Fig 3 a), whereas the hunched posture is a consequence of bone overgrowth ( alleles can be summarized as follows: connective tissue hyperplasia is only associated with the Tsk allele; dissecting aneurysm is only associated with the mgR and mg
alleles; and bone overgrowth and lung emphysema are common traits of the Tsk, mgR, and mg
alleles (Table 1).
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Heterozygous fibrillin 1 mutant mice were crossed to examine the segregation of the distinguishing and common traits in the compound heterozygous Tsk/mgR and Tsk/mg mice. Compound heterozygous Tsk/mgR mice die within the first 3 wk of life of the same vascular complications as mgR/mgR animals, which are absent in the parental Tsk/+ line (Fig 3e and Fig f). Moreover, Tsk/mgR animals exhibit the skin abnormalities of the Tsk/+ mice, which are absent in mgR/mgR mice (Fig 3 c). These results indicate codominant expression of the mutant Tsk and mgR allele; the same conclusion was reached with compound Tsk/mg
heterozygotes (Table 1). Additionally, Tsk/+ animals are more severely affected than mg
/+ mice, in spite of the fact that the two mutant mice make comparable amounts of wild-type fibrillin 1 (Table 1). This last observation raised the possibility that the Tsk protein may participate in heterotypic assembly, thus, exerting some antimorphic effect on the normal gene product.
Microfibrillar Assembly Differs in Tsk/Tsk, Tsk/+, and +/+ Cell Cultures
Metabolically labeled polypeptides from the media of cultured fibroblasts with different combinations of Fbn1 alleles were immunoprecipitated using polyclonal antibodies (pAb9543) that recognize the NH2-terminal half of fibrillin 1 (Fig 2). Since pAb9543 epitopes are present in both wild-type and mutant fibrillin 1 and in both human and mouse proteins, the pAb9543 antibodies will be referred to as -Fbn. As expected, SDS-PAGE analysis of the
-Fbn immunoprecipitates correlated normal size (350 kD), shorter (331 kD), and longer (418 kD) fibrillin 1 proteins with their respective Fbn1 alleles (Fig 4 a). It also confirmed the presence of comparable amounts of mutant and wild-type fibrillin 1 (418 and 350 kD, respectively) in the Tsk/+ sample, and the relatively low levels of mg
and mgR products (331 and 350 kD, respectively) in the compound heterozygous mutant cell cultures (Fig 4 a).
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Next, the -Fbn antibodies were used to examine the microfibrillar meshwork assembled in vitro by hyperconfluent fibroblasts. Consistent with quantitation of fibrillin 1 expression in Fig 4 a, the amount of
-Fbn immunofluorescent material deposited after 120 h of culture was noticeably less in mgR/+ than +/+ cultures (Fig 5, compare a and b). A less abundant meshwork was also observed in the Tsk/+ sample, and even less in the Tsk/mgR cell cultures (Fig 5, compare c and d). The same relative decrease in the amount of ordered meshwork was observed with the mg
/+ and Tsk/mg
cells (data not shown). Taken together, the results suggested that Tsk fibrillin 1 contributes poorly to the assembly of the microfibrillar meshwork that is normally observed in hyperconfluent +/+ fibroblast cultures. To test this postulate, the analysis was repeated with an antibody that recognizes only Tsk fibrillin 1.
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The Tsk mutation is an in-frame duplication of exons 1740 and, as a result, the mutant sequence differs from the normal counterpart by interrupting the continuity of the second 8-cys motif of fibrillin 1, which is normally encoded by exons 16 and 17. In turn, this creates a novel peptide with a single cysteine residue (the segment encoded by exon 17) immediately adjacent to the 24th cbEGF motif of fibrillin 1, which is the repeat encoded by exon 40 (Fig 2; -Tsk) was tested by a Western blot analysis of proteins from conditioned media of +/+, Tsk/+, and Tsk/Tsk cell cultures. Unlike
-Fbn, which recognized both the normal 350-kD and mutant 418-kD products, the
-Tsk antiserum showed specificity only for the longer species (Fig 4 B). The specificity of
-Tsk was further supported by the lack of immunostaining of +/+ fibroblast cultures (Fig 6 b). These results indicated that the Tsk peptide sequence is available for antibody binding in native Tsk fibrillin 1 and unavailable in native wild-type fibrillin 1; this, in turn, suggested that the sequence present in the native wild-type fibrillin 1 is probably folded in a manner that prevents antibody binding. Parallel immunostaining of Tsk/+ cell cultures with
-Fbn and
-Tsk antisera visualized both quantitative and morphological differences. Whereas the
-Fbn antibodies revealed an elaborate network of immunoreactive microfibrils, the
-Tsk antiserum yielded a reduced amount of immunoreactive material (Fig 6c and Fig d). The same results were obtained with fibroblasts from the compound heterozygous Tsk/mgR and Tsk/mg
mutant mice (data not shown). Surprisingly, both antibodies also revealed an immunoreactive meshwork in Tsk/Tsk cell cultures (Fig 6e and Fig f). However, in this case, the pattern of the immunoreactive material was more diffuse, less linear, and more punctate than the one generated by the
-Fbn antiserum with +/+ fibroblasts (Fig 6 a). The morphological differences between the wild-type and mutant fibrils were more apparent when the immunostaining patterns of the +/+ and Tsk/Tsk cultures were compared at a higher magnification (Fig 6g and Fig h).
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To validate the immunostaining data at the ultrastructural level, microfibrils were purified from the matrix elaborated by hyperconfluent +/+ and Tsk/Tsk cells and examined using two distinct electron microscopic techniques, rotary shadowing, and negative staining (Fig 7). In Tsk/Tsk preparations, the former technique visualized microfibrils that were fewer and significantly shorter than +/+ microfibrils, and which also display irregular arms and globular domains (Fig 7, compare a to cf). The gentler technique of negative staining revealed that the shorter Tsk/Tsk microfibrils have beads of different sizes and irregular periodicity (Fig 7, compare b to gk). The immunofluorescence and ultrastructural data suggested the following two points. First, Tsk fibrillin 1 molecules by themselves can initiate polymerization, but are incapable of elaborating in vitro the same complex meshwork of microfibrils as wild-type proteins; second, Tsk fibrillin 1 molecules are partially incorporated into the meshwork assembled by the wild-type proteins (Fig 1 d).
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Wild-type Fibrillin 1 Drives Partial Polymerization Of Mutant Tsk Fibrillin 1
We next tested whether Tsk fibrillin 1 could copolymerize with wild-type fibrillin 1 by assaying cocultures of normal and mutant cells. Recently, we found that human amnion, epithelial-like WISH cells secrete fibrillin 1 proteins into the medium but fail to assemble a microfibrillar network, either because the molecules are present at less than optimal concentration or because the cell line lacks other factors required for fibrillin 1 microfibril assembly. However, coculturing WISH cells together with mouse dermal fibroblasts leads to copolymerization of the human and mouse fibrillin 1 proteins (Sakai, L.Y., unpublished data). This same approach was employed here to test whether the microfibrillar pattern of Tsk/Tsk matrix could be rescued by wild-type human fibrillin 1 secreted by WISH cells.
WISH and Tsk/Tsk cells were cultured separately or together, and each sample was immunostained with antibodies specific for human fibrillin 1 (mAb201 or -hFbn), human and mouse fibrillin 1 (pAb9543 or
-Fbn), or Tsk fibrillin 1 (pAb8368 or
-Tsk). As expected, no extracellular immunostainable microfibrillar meshwork was observed in the WISH sample with
-hFbn antibodies, and only diffuse and irregular staining was seen in the Tsk/Tsk sample using the
-Fbn antiserum (Fig 8, a and b). Coculturing the two cell types led to the formation of an elaborate meshwork of microfibrillar bundles in which both
-hFbn and
-Tsk antibodies were colocalized with confocal microscopy (Fig 8, di). Finally, immunoelectron microscopy of the extracellular microfibrils elaborated by the cocultured cells confirmed that the immunostainable meshwork is made of microfibrils containing both human WISH fibrillin 1 and mouse Tsk fibrillin 1 (Fig 9e and Fig f). The morphology of the rescued Tsk microfibrils displayed features of the Tsk/Tsk microfibrils, such as abnormal globular domains and irregular periodicities between globules; unlike Tsk/Tsk microfibrils, however, these abnormal structures were embedded within seemingly normal microfibrils (Fig 9). Therefore, the results of the coculture experiments demonstrated copolymerization between human wild-type and mouse mutant fibrillin 1, and suggested that Tsk monomers participate in the initial steps of fibrillin 1 polymerization. Implicitly, the experiments confirmed that protein concentration and/or cell typespecific factors contribute to the in vitro assembly of fibrillin 1 microfibrils.
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Tsk Fibrillin 1 Is More Susceptible to Proteolysis
Rotary shadowing of microfibrils prepared from Tsk/+ skin has allegedly visualized two morphologically distinct beaded structures in equal amounts (
Indirect immunofluorescence on Tsk/+ skin with -Fbn and
-Tsk antibodies demonstrated that 418-kD fibrillin 1 molecules are integrated into the microfibrillar network. However, the images also suggested that the junctional epitopes of Tsk protein may not be as abundant as the epitopes recognized by
-Fbn, particularly in the mature, deeper portions of the dermis (Fig 10). Immunoelectron microscopy confirmed the presence of mutant fibrillin 1 in the microfibrils of Tsk/+ skin samples (Fig 11).
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The last experiment was designed to assess the stability of the Tsk protein using an in vitro assay that compared the resistance of wild-type and mutant molecules to a broad substrate protease. To this end, immunoprecipitates of metabolically labeled fibrillin 1 from cultured Tsk/+ cells were incubated in vitro with increasing amounts of plasmin. SDS-PAGE analysis of the digested products documented the more rapid disappearance of the mutant 418-kD product with respect to the wild-type 350-kD species (Fig 12).
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Discussion |
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Our current understanding of the role of fibrillin 1 microfibrils in connective tissue physiology is based primarily on the study of human mutations in MFS and homologous gene targeting in the mouse (
According to the pathogenic model of fibrillin 1 mutations in MFS, one would have predicted a full antimorphic effect of the Tsk mutation leading to the collapse of the elastic lamellae in the aortic wall and the early demise of Tsk/+ animals (
The first line of evidence relies on the phenotypic features of mutant mice with different combinations of Fbn1 alleles. Tsk/+ mice produce nearly the same amount of normal fibrillin 1 as mg/+ mice and, yet, they are more severely affected. The most likely explanation for this apparent discrepancy is that the greater number of Tsk molecules, compared with mg
products, may exert a partial antimorphic effect on the wild-type fibrillin 1 proteins. In this respect, the Tsk allele should be viewed as an antimorphic mutation with partial penetrance.
The second line of evidence in support of our pathogenic model of the Tsk mutation was derived from cell culture experiments. Immunofluorescence analysis of fibrillin 1 microfibrils elaborated by various cell cultures demonstrated decreased immunoreactivity in the following order: +/+, mgR/+ or Tsk/+, and Tsk/mgR. These results indicate that Tsk fibrillin 1 cannot substitute for wild-type fibrillin 1 in this assembly assay, and imply that the mutant protein may be assembled into microfibrils more slowly than the wild-type counterpart. In contrast to the extended linear microfibrils found in wild-type cultures, immunofluorescence of Tsk/Tsk cultures demonstrated a punctate pattern of fibrillin 1 in somewhat focal aggregates. Consistent with these images, electron microscopic analyses revealed short strings of beaded fibrils composed of Tsk fibrillin 1. Compared with wild-type fibrillin 1 microfibrils, the Tsk/Tsk aggregates exhibited clear irregularities in the size and shape of the globular beads, as well as in the distances between beads. These data strongly suggest that Tsk fibrillin 1 can polymerize into microfibril-like structures with abnormal morphologies.
The third and final supporting evidence was based on a novel coculture assay, which documented the ability of Tsk fibrillin 1 to interact with the wild-type counterpart. The resulting fibrillin 1 microfibrils were long and composed of both wild-type human fibrillin 1 and mutant mouse fibrillin 1, thus, demonstrating that Tsk and wild-type molecules can copolymerize into a single microfibril (Fig 1 d). The microfibrils exhibited focal areas of abnormalities similar to those found in Tsk/Tsk fibrils. Interestingly, abnormal microfibrils were not seen in Tsk/+ cell cultures or tissues. We believe that the reason for this apparent discrepancy reflects intrinsic differences of the two experimental systems. Fibroblasts synthesize and secrete the mutant and wild-type products together before they are assembled extracellularly and a number of factors may influence this coupled process. In the coculture system, the two populations of wild-type and mutant molecules are secreted separately and, subsequently, are assembled by the fibroblasts. Therefore, the cocultured system should be viewed more as a biochemical binding assay to test homopolymerization versus heteropolymerization rather than a physiological model of microfibril assembly. With this reservation in mind, the coculture data indicate that Tsk fibrillin 1, when copolymerized with wild-type fibrillin 1, can participate in microfibril elongation.
The comparison of the mutant Fbn1 mice has also raised an interesting structural consideration. The phenotypic severity of Tsk and mg homozygotes is dramatically different. Tsk/Tsk embryos produce a homogenous population of longer molecules in normal abundance; mg
/mg
mice produce a homogeneous population of shorter molecules in a much lower abundance. However, Tsk/Tsk embryos die in utero at the peri-implantation stage, and mg
/mg
embryos complete development (
/mg
fibrillin 1 microfibrils (
The Tsk duplication begins with exon 17, which encodes the COOH-terminal 18 amino acid residues of the second 8-cys domain (-Tsk antibodies was observed at the dermal-epidermal junction, an area of the skin which is thought to represent the more immature stroma compared with the connective tissue of the deeper dermis.
In conclusion, we propose that incorporation of Tsk fibrillin 1 along with wild-type fibrillin 1 renders all microfibrils more susceptible to proteolysis. According to this model, Tsk/+ animals are more severely affected than mg/+ mice because of the relatively higher ratio of mutant to wild-type molecules in the former compared with the latter. This model is in line with the conclusion of a recent study on the consequences of mutations in cbEGF domains in fibrillin 1 (
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Footnotes |
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1 Abbreviations used in this paper: cbEGF, calcium-binding epidermal growth factor-like; cys, cysteine; Fbn1, mouse fibrillin 1 gene; MFS, Marfan syndrome; Tsk, Tight skin mutation.
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Acknowledgements |
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The authors are much indebted to Drs. S. Jimenez and L. Siracusa (Jefferson Medical College, Philadelphia, PA) for continued support, Dr. K. Kasturi (Mount Sinai School of Medicine, New York, NY) for useful suggestions, and Dr. D. Rifkin (New York University, Medical Center, New York, NY) for critically reviewing the manuscript. They also wish to thank Ms. N. Charbonneau, S.F. Tufa, and S. Lee for excellent technical assistance and Ms. K. Johnson for typing the manuscript.
This work was supported by grants from the National Institutes of Health (AR42044 and AR32564), Shriners Hospital for Children, the National Marfan Foundation, and the Dr. Amy and James Elster Research Fund (all except Shriners [which is to L. Sakai] are to F. Ramirez).
Submitted: 13 October 1999
Revised: 14 June 2000
Accepted: 15 June 2000
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References |
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