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1 Department of Biochemistry and Molecular Biology, and 2 Department of Gynecology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan
3 Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
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Abstract |
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Introduction |
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Control of the nuclear localization of specific proteins is an important mechanism in the regulation of many signal transduction pathways. Transcription factors cannot function until they are translocated to the nucleus in response to specific signals (Vandromme et al. 1996). The translocation of proteins through nuclear pore complexes is mediated by a selective mechanism that is controlled by saturable receptors and specific signals, which are termed nuclear localization signals (NLSs) and nuclear export signals (NESs) (Mattaj & Englmeier 1998; Yoneda et al. 1999).
The best-characterized process of nuclear import has been shown to be mediated by a basic type NLS, referred to as ‘classical NLS,’ which contains one or two clusters of basic amino acids. The import of substrates containing the classical NLS is initiated by the formation of an NLS-dependent ternary complex with importin (also called karyopherin) 
/ß in the cytoplasm. Importin recognizes the NLS and binds to importin ß via its N-terminal sequences, which are rich in basic amino acids, and is referred to as the importin ß-binding (IBB) domain (Görlich et al. 1996a). Importin ß accounts for the targeting of the complex to nuclear pore complexes. Nuclear RanGTP terminates the import reaction. After translocation of the trimeric complex, which is composed of importin /ß and a karyophile with the classical NLS, through the nuclear pores, the direct binding of nuclear RanGTP to importin ß causes the dissociation of the complex, thus releasing the cargo in the nucleus (Rexach & Blobel 1995; Görlich et al. 1996b; Moroianu et al. 1996). NTF2 (nuclear transport factor 2) mediates the import of RanGDP into the nucleus. In addition to this classical import pathway, several alternate pathways have been identified, which involve importin ß or importin ß family members that bind directly to their cognate cargo proteins without the need for adaptor proteins such as importin (Chan et al. 1998; Nagoshi et al. 1999; Yoneda 2000; Kurisaki et al. 2001; Lee et al. 2003).
The Snail family is defined by a highly homologous DNA-binding domain situated at the carboxyl terminus. The domain consists of four to six zinc fingers of the C2H2-type. Outside the zinc-finger domain, there is much less conservation among the family members. Nonetheless, the presence of many basic residues at the amino-terminus suggests that this region might be involved in nuclear localization (Hemavathy et al. 2000). All the vertebrate members contain a conserved motif at their amino-terminus, the so-called SNAG domain. This domain was originally identified in the oncoprotein Gfi-1, which contains six zinc fingers (Grimes et al. 1996). The first 20 amino acids of Gfi-1 are essential for nuclear localization and are necessary and sufficient for transcriptional repression (Grimes et al. 1996). Although the amino-terminal 7 amino acids in the SNAG domain of Gfi-1, which are conserved among the vertebrate Snail family of proteins and Gfi-1 proteins, have been shown to be essential for the nuclear localization of Gfi-1, the NLS of Snail has not been determined. Furthermore, although the nuclear localization of Snail is one of the most crucial processes in the Snail-mediated gene regulation, the precise molecular mechanism for the nuclear import of Snail remains to be determined.
In this study, we show that the carboxyl-terminal zinc finger domain of Snail, which exhibits considerable sequence similarity among family members, is sufficient for the selective nuclear accumulation of Snail. The mechanism of nuclear import was also investigated using a recombinant Snail protein in an in vitro transport assay system and in living cells. Our results indicate that the nuclear import of Snail occurs through a direct interaction of the zinc finger domain with importin ß in a Ran-dependent manner.
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Results |
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We previously reported that the Snail protein, when tagged at its carboxyl-terminus with HA (Snail-HA), could be detected in the nuclei of stably transfected canine kidney epithelial MDCK cells (Ohkubo & Ozawa 2004). This suggests that Snail was selectively imported into the nucleus of the transfectants. However, because of the small size of the fusion protein (32 kDa), the possibility that Snail-HA passively diffused into the nucleus and was retained there cannot be excluded, since proteins smaller than 
40 kDa have been reported to enter the nucleus through the nuclear pore complex by passive diffusion (Görlich 1998). To verify the selective nuclear import of Snail, we constructed a full-length Snail fusion protein tagged with GFP at its carboxyl-terminus (Snail-GFP), which would not be expected to enter the nucleus by passive diffusion, and introduced it into MDCK cells. After the isolation of stable transfectants, the subcellular localization of Snail-GFP was examined. Snail-GFP had an apparent molecular mass of 60 kDa (data not shown) consistent with the estimated molecular mass of the fused proteins.
Like Snail-HA (Ohkubo & Ozawa 2004), Snail-GFP expressed in MDCK cells was detected in the nucleus (Fig. 1B). Interestingly, the expression of Snail-GFP in MDCK cells induced a morphological change; the cells became fibroblastic (Fig. 1A). The down-regulation of E-cadherin expression was also observed in MDCK cells expressing Snail-GFP (Fig. 1C). Thus, Snail-GFP retains the ability to induce epithelial-mesenchymal transition. Therefore, addition of the GFP tag at the carboxyl-terminus had no effect on protein activity. In a control experiment, GFP alone was distributed diffusely throughout both the cytoplasm and the nucleus (data not shown). The nuclear fluorescence observed is probably due to the passive diffusion of 30-kDa GFP. Stable transfectants did not show any morphological changes (not shown), nor was the down-regulation of E-cadherin expression observed (Fig. 1C). These results indicate that Snail-GFP is selectively translocated from the cytoplasm into the nucleus.
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To define the region of Snail that is required for nuclear translocation, we divided Snail molecule into three parts, the amino-terminal region containing the SNAG domain, the middle portion of Snail, and the carboxyl-terminal region with four zinc finger domains (Fig. 2). Expression vectors encoding the different Snail domains fused to GFP were introduced into MDCK cells, and stable transfectants were isolated. A GFP fusion protein in which the carboxyl-terminal region contained four zinc finger domains (Snail-C-GFP), was exclusively localized to the nucleus (Fig. 3A), suggesting that the region containing the zinc finger domain contains an NLS. On the other hand, constructs containing the amino-terminal region or the middle portion of Snail (Snail-N-GFP and Snail-M-GFP, respectively) both displayed an intracellular localization similar to GFP alone (Fig. 3A), indicating that these regions do not contribute to the intracellular localization of Snail. Immunoblot analysis of cells expressing these constructs, after fractionation into nuclear and cytoplasmic fractions, supported this conclusion, although some Snail-N-GFP was detected in the nuclear fraction (Fig. 3B). Treatment of the transfectants with Leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, had no effect on the distribution of these proteins (data not shown), suggesting that Snail is specifically imported and is efficiently accumulated within the nucleus of MDCK cells even without inhibition of CRM1-mediated nuclear export.
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In order to define the factors required for the nuclear import of Snail, we performed a digitonin-permeabilized cell-free transport assay. As shown in Fig. 6A, in the presence of a previously described ATP regeneration system, GST-Snail-GFP accumulated in nucleus in the presence of exogenous cytosolic extracts but not in their absence. These results were highly reproducible, and indicate that the nuclear import of Snail requires soluble transport factors.
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/ß-mediated import pathway is not involved in its nuclear import. As expected, we found that GST-Snail-GFP accumulated in the nucleus in the presence of importin ß (Fig. 6A). On the other hand, in the absence of Ran and NTF2, GST-Snail-GFP showed nuclear rim staining, which means its targeting to the nuclear pore complex (Fig. 6A). Furthermore, addition of dominant negative Ran (RanG19V) strongly inhibited nuclear import of Snail (data not shown), indicating that importin ß, Ran and NTF2 play a pivotal role in nuclear import of Snail. When the in vitro assays were performed with Snail-N, Snail-M, and Snail-C constructs, only Snail-C was transported into the nucleus by importin ß-mediated pathway (Fig. 6B). Furthermore, as shown in Fig. 7 lane 3, solution binding assays consistently demonstrated that GST-Snail-GFP binds directly to importin ß. In addition, this interaction is dependent on the NLS of Snail (Fig. 7 lane 12), confirming that the nuclear import of Snail occurs through the direct interaction of importin ß with the zinc finger domains.
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Discussion |
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In addition to transcriptional regulation, the activity of the transcription factors can be controlled by changing their cellular location. This permits rapid response to signals, resulting in a powerful modulation of the system (Beg et al. 1992; Vandromme et al. 1996; Engel et al. 1998; Zhang & Xiong 2001), suggesting that it is important to understand the molecular mechanism of the nuclear import of the transcription factors. In this report, we present data that indicates that the transcription factor Snail enters the nucleus by mechanisms involving importin ß, and that the four carboxyl-terminal zinc finger domains are responsible for its transport into the nucleus.
A previous mutational analysis of the Gfi-1 oncoprotein revealed that a possible NLS is located in the amino terminal SNAG domain, which is found in all the vertebrate members of the Snail family (Grimes et al. 1996). This signal, however, was not sufficient to cause the amino terminal region of Gfi-1, which lacks the carboxyl-terminal zinc finger domain, to localize to the nucleus. The authors proposed that an additional NLS is present in the zinc finger domain, and that the nuclear localization of the Gfi-1 protein is achieved by the concerted effects of an NLS within the SNAG domain and a secondary signal in the zinc finger domain. In contrast to these findings, we found that the carboxy-terminal zinc finger domain, which was stably expressed in MDCK cells as a GFP fusion protein was exclusively detected in nucleus (Fig. 3A). Consistent with our observation that, of the stable transfectants, only the Snail-C-GFP construct, but not the Snail-N-GFP or the Snail-M-GFP constructs, was localized in the nucleus, only GST-Snail-C-GFP was transported into the nucleus in vitro in the presence of importin ß (Fig. 6B). Thus the zinc finger domain of Snail is necessary and sufficient for the nuclear localization of the Snail protein.
In addition, our results revealed that all four zinc fingers are necessary for efficient nuclear localization, because deletion of any individual finger alone or in combination resulted in a decreased nuclear accumulation (Fig. 4). It is likely that all four fingers are required for the coordination of the structure of the carboxyl-terminal domain to interact efficiently with the nuclear import machinery and actually function as the NLS of Snail. The NLS of several zinc finger proteins such as Wilms’ tumor 1 (Wt1) (Bruening et al. 1996), mouse orphan receptor (TR2; Yu et al. 1998), EKLF/KLF1 (Pandya & Townes 2002; Quadrini & Bieker 2002), NGFI-A (Matheny et al. 1994), JAZ (Yang et al. 1999), and Zif268 (Shields & Yang 1997) are known to be localized to their zinc finger regions. However, these proteins appear to have different sequence requirements for nuclear localization. For example, the NLS of Wt1 was delimited to the first (of four) zinc fingers, whereas the second (of two) zinc fingers of TR2 was sufficient for nuclear localization (Bruening et al. 1996; Yu et al. 1998). In contrast, all three zinc fingers are necessary for the efficient nuclear localization of EKLF/KLF1 (Pandya & Townes 2002; Quadrini & Bieker 2002). Results obtained for NGFI-A and JAZ demonstrate that mutations that disrupt the tertiary structure of zinc fingers also abrogated nuclear localization (Matheny et al. 1994; Yang et al. 1999), although the nuclear import activity of EKLF/KLF1 was independent of zinc finger ternary structure (Pandya & Townes 2002). On the other hand, the disruption of any one of the three zinc fingers of Zif268 resulted in cytoplasmic mislocalization (Shields & Yang 1997). It should be noted that there remains a possibility that deletion of any zinc finger domains or disruption of the ternary structure of zinc fingers may cause a loss of DNA binding activity, resulting in a loss of nuclear retention. Therefore, the effects of deletion of zinc finger on the nuclear retention and the importin ß binding activity should be further examined to confirm definitely that all four zinc fingers are actually required for efficient nuclear localization of Snail. In this study, we found that Snail can be transported into the nucleus by pathway involving importin ß. Direct interactions of the zinc finger domain of EKLF/KLF1 with both importin (1 and 2) and importin ß were recently reported (Quadrini & Bieker 2002). No experimental evidence to show that the importin-mediated pathways actually transport EKLF/KLF1 was provided in this previous study, however. Thus, this is the first example to definitely demonstrate the nuclear import mechanism of the zinc finger proteins on a molecular basis.
It has been known that importin ß directly interacts with and carries several distinct proteins including importin and snurportin as adaptors and the human immunodeficiency virus Rev, cyclin B1, the sterol regulatory element-binding protein 2 and Smad3 as cargoes (Harel & Forbes 2004). Recent advance of crystal structural analysis has revealed that importin ß consists of 19 HEAT repeats and that each interacting protein uses a different binding site on importin ß and has a different binding mode. Thus, it appears that importin ß has the intrinsic flexibility for binding many types of cargoes or adaptors (Lee et al. 2003). This study has added another novel and interesting example of direct binding cargoes for importin ß, the zinc finger protein Snail. Since the structure of the zinc finger domains appears to be quite different from that of known cargoes’ NLS for importin ß, it will be of interest to know how importin ß recognizes such different type of ligands as Snail.
The NES of Snail was recently identified in residues 132–143 of mouse Snail (Dominguez et al. 2003). The activity of the Snail NES appeared to be variable among the cell lines tested. Snail preferred a nuclear location in some cell lines, whereas it had a marked preference for an extranuclear location in others (Dominguez et al. 2003). Snail protein was exclusively detected in the nucleus of MDCK cells used in the present study (Dominguez et al. 2003; Ohkubo & Ozawa 2004), suggesting that the Snail NES is less active in MDCK cells compared with other cells, in which the Snail protein is localized in the cytoplasm. One of the mechanisms that regulate the export of Snail appears to be phosphorylation (Dominguez et al. 2003). Phosphorylation of a Ser-rich sequence adjacent to the NES of Snail facilitates its export. Thus, it would be interesting to determine how the nuclear import and export of Snail are regulated so as to develop a more complete understanding of the function of Snail.
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Experimental procedures |
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Enzymes used in the cDNA construction were commercially available products. Leptomycin B (Kudo et al. 1999) was kindly provided by Dr Minoru Yoshida (RIKEN, Tsukuba, Japan). A mouse monoclonal anti-GFP antibody was purchased from Clontech and was used for immunoblotting.
cDNA construction
cDNA, encoding full-length human Snail, has been previously described (Ohkubo & Ozawa 2004). cDNAs encoding various Snail peptide fragments were prepared by PCR using human Snail cDNA as a template. All the constructs expressed in stable transfectants were cloned into an expression vector, pCAGGSneo (Niwa et al. 1991; a gift from Dr K. Yamamura, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan) at a site upstream of green fluorescent protein (GFP) in frame as described in Ozawa (2002). The nucleotide sequences of the primers used for PCR are described below. BamHI and EcoRV sites were created by PCR at the 5'-end and 3'-end of all the constructs, respectively. All PCR products were cloned into the BamHI and EcoRV sites of pBluescript II KS (+) vector (Stratagene) and their sequences were confirmed. The following primers were used: the Snail-N construct (5'-CGGGATCCACTATGCCGCGCTCTTT and 5'-ATCACTCTCCTGGAGCCGAAGG), the Snail-M construct (5'-CGGGATCCACTATGCCAATTGCCTGGGCCTC and 5'-ATCGTTGAAGGCCTTTCGAGCCT), the Snail-C construct (5'-CGGGATCCACTATGGCCTTCAACTGCAAATACTG and 5'-ATCGCGGGGACATCCTGAGCA), the Snail-C-
ZF1 construct (5'-TGCCATAGTGGATCCACTAGT and 5'-TGCGTCTGCGGAACCTGC), the Snail-C-ZF2 construct (5'-GCTCTAGAGTGTGGCTTCGGATGTG and 5'- GCTCTAGAGAAGCCCTTCTCCTGTC), the Snail-C-ZF3 construct (5'-AAAGGCTTCTCGCCAGTGTG and 5'-AAAGTACCAGTGCCAGGCGT), the Snail-C-
’ZF4 construct (5'-CGGGATCCACTATGGCCTTCAACTGCAAATACTG and 5'-ATCTGAGTGGGTCTGGAGG), the Snail-C-ZF2,3,4 construct (5'-ATCCAGCGTGTGGCTTCGGATG and 5'-ATCGGCTACCCCTACGACG), the Snail-C-ZF1,3,4 construct (5'-ATCGCCAGTGTGGGTCCGGAC and 5'-ATCGGCTACCCCTACGACG), the Snail-C-ZF1,2,4 construct (5'-AAAGGCCATAGTGGATCCACTA and 5'-AAACCCTTCTCCTGTCCCCAC), the Snail-C-ZF1,2,3 construct (5'-AAAGGCCATAGTGGATCCACTA and 5'-AAATACCAGTGCCAGGCGTGT), the Snail-C-ZF3,4 construct (5'-GTGGCGGCCGCTCTAGAA and 5'- ATCGAAGGGCTTCTCGCCAGTG), the Snail-C-ZF1,4 construct (5'-ATTTGCGGCCGCCATGCTGCCCTGCGTCTGCGG and 5'-ATCGTACTTCTTGACATCTGAGTG), the Snail-C-ZF1,2 construct (5'-ATTTGCGGCCGCCATGGGCGAGAAGCCCTTCTCC and 5'-ATCGCGGGGACATCCTGAG), the Snail-C-ZF2,4 construct (5'-GTGGCGGCCGCTCTAGAA and 5'-ATCGTACTTCTTGACATCTGAGTG), the Snail-C-ZF1,3 construct (5'-ATTTGCGGCCGCCATGCTGCCCTGCGTCTGCGG and 5'-ATCGCGGGGACATCCTGAG), the Snail-C-ZF2,3 construct (5'-GGGGTCAAGAAGTACCAGTGCCA and 5'-GGGCAGCGTGTGGCTTCG). The NotI and EcoRV sites were used for ligation of the constructs with the unique NotI and EcoRV sites, which is upstream of the GFP epitope sequence in the pCAGGSneo vector (Ozawa 2002). To construct glutathione S-transferase (GST)-tagged Snail-GFP (GST-Snail-GFP), cDNA encoding full-length Snail-GFP fusion protein was isolated from the pCAGGSneo vector by digestion with NotI and EcoRV and cloned into pGEX4T vector in frame. GST-Snail-GFP fusion proteins with the amino-terminal region of Snail (GST-Snail-N-GFP), the middle portion (GST-Snail-M-GFP), and the carboxyl-terminal portion (GST-Snail-C-GFP) were constructed in exactly the same manner.
Cells and transfection
MDCK cells and their transfectants were grown in Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum under a humidified atmosphere containing 10% CO2 at 37 °C. To obtain transfectants, appropriate expression vectors (5 µg each) were introduced into MDCK cells (5 x 105) by the calcium phosphate method (Ozawa et al. 1989). After selection in medium containing G418, single colonies were isolated and the expression of the constructs was determined by fluorescent microscopic observations of cells for GFP and immunoblot analysis using anti-GFP antibody. The transfected cells were cultured for 24 h in serum-containing medium before subcellular localization analysis. For analysis of the nuclear export of Snail constructs, transfected cells that had been cultured for 24 h were treated with or without 5 ng/mL leptomycin B in serum-containing medium for 2 h.
Immunoblotting
After washing with phosphate-buffered saline (PBS), cells (1 x 106) were boiled for 5 min in Laemmli SDS-PAGE sample buffer, run on 12% polyacrylamide gels, and then electroblotted on to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in PBS, incubated with primary antibodies for 2 h at room temperature, and finally with secondary antibodies. After washing the membranes with PBS containing 0.05% Tween 20, the protein bands were visualized with an ECL detection kit (Amersham Corp., Amersham, UK).
Subcellular localization analyses
To study the subcellular localization of GFP fusion proteins, GFP fluorescence in transfected cells was visualized with an Olympus fluorescence microscope. To quantify the degree of nuclear localization, cells were fractionated into nuclear and cytoplasmic fractions as follows. Cells were homogenized in 20 mM Tris-HCl buffer, pH 8.0, containing 1 mM phenylmethylsulfonylfluoride, and centrifuged at 5000 r.p.m. for 10 min. The precipitates correspond to the nuclear fractions and the supernatants correspond to the cytoplasmic fractions. Microscopic examination confirmed that the fractionation was valid.
Expression and purification of recombinant proteins
Glutathione S-transferase (GST)-tagged Snail-GFP (GST-Snail-GFP) was expressed and purified as previously described (Nagoshi et al. 1999) with minor modifications. Briefly, a pGEX vector containing GST-Snail-GFP construct was introduced into an Escherichia coli strain BL21. Expression was induced by the addition of 1 mM isopropyl-ß-D-thiogalactopyranoside and incubated for 16–24 h at 18 °C. Cells were harvested by centrifugation and resuspended in a high-salt buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5 mM EDTA) containing 1 mM PMSF, 1 mM DTT, and 2% Triton X-100. After sonication, the extract was clarified by centrifugation and filtered through a 0.45 µm membrane. The extract was applied to a glutathione-Sepharose (Pharmacia) column. After washing with high-salt buffer without Triton X-100 and buffer without Triton X-100 and NaCl, the protein was eluted from the Sepharose beads with Tris-HCl buffer, pH 8.0, containing 10 mM glutathione. HA-importin ß, NTF2 and Ran were expressed, purified as previously described (Imamoto et al. 1995; Sekimoto et al. 1996; Tachibana et al. 1996).
Microinjection
Microinjection was carried out as previously described (Sekimoto et al. 2004). Briefly, HeLa cells were grown in DMEM supplemented with 10% fetal calf serum and plated on coverslips 24 h before use. Purified GST-Snail-GFPs at 2 mg/mL were injected through a glass capillary into the cytoplasm of HeLa cells. To monitor the injection, fluorescent rhodamine isothiocyanate-labeled bovine serum albumin (RITC-BSA) was included in the protein solution. After incubation at 37 °C for 30 min, the cells were fixed with 3.7% formaldehyde in PBS. The injected GFP-tagged protein and RITC-BSA were detected by fluorescent microscopy (Axiophot II; Carl Zeiss, Inc.).
In vitro transport assay
Transport assays were performed as previously described (Sekimoto et al. 2004). Briefly, HeLa cells were plated on eight-well multitest slide (ICN, Costa Mesa, CA) 36–48 h before use. The cells were permeabilized for 5 min in ice-cold transport buffer containing 40 µg/mL digitonin. Each reaction mixture contained an import substrate (10 pmol) combined with cytosol or a combination of recombinant transport factors (each 10 pmol) in the presence or absence of an ATP regeneration system. After incubation at 28 °C for 30 min, the cells were fixed and the import of GST-Snail-GFP was detected by Axiophot microscopy.
Solution binding assay using recombinant proteins
Binding assays using recombinant proteins expressed in E. coli were performed as previously described (Sekimoto et al. 2004). Briefly, purified GST-Snail-GFP (5 µg) and HA-importin ß (5 µg) were mixed in 100 µL of transport buffer containing 0.1% Triton X-100 in the presence of anti-HA agarose (Sigma) at 4 °C for 2 h. After extensive washing, bound proteins were eluted by SDS-sample buffer, applied on SDS-PAGE and stained with Coomassie Brilliant Blue.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to the article. 
* Correspondence: E-mail: yyoneda{at}anat3.med.osaka-u.ac.jp.
mozawa{at}m.kufm.kagoshima-u.ac.jp |
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Received: 24 December 2004
Accepted: 1 February 2005
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