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1 Graduate School of Science and Technology,
2 Biosignal Research Centre, Kobe University, Kobe 657-8501, Japan
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Abstract |
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-tubulin, centrin-2 and kendrin as well as endogenous CG-NAP. When CG-NAP/D was dislocated from centrosomes by deleting the centrosome-targeting domain or by fusing with a membrane-targeting sequence, centrosome amplification was suppressed. CG-NAP/D interacted with exogenously expressed cyclin E, which co-localized at centrosomes. The immunoprecipitates of CG-NAP/D exhibited histone H1 kinase activity, suggesting the co-immunoprecipitation of active cyclin-cdk complexes. Furthermore, centrosome fractions prepared from cells expressing CG-NAP/D contained increased amount of cdk2 compared with those from control cells. Centrosome amplification by CG-NAP/D was suppressed by co-expression of a mutant cyclin E unable to interact with cdk2. These results suggest that CG-NAP/D causes centrosome amplification by anchoring excess amount of cyclin E-cdk2 to centrosomes and, possibly, CG-NAP participates in centrosome duplication by recruiting cyclin E-cdk2 to centrosomes in normal cell cycle. |
Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Recently, various molecules that affect the centrosome duplication have been discovered. Especially, suppression of centrosome duplication has been demonstrated by inhibition of cyclin E/cyclin A-cdk2 activity through the expression or microinjection of cdk inhibitor proteins p16, p21 and p27 (Hinchcliffe et al. 1999; Lacey et al. 1999; Matsumoto et al. 1999; Meraldi et al. 1999), or the treatment with cdk inhibitors such as roscovitine (Matsumoto et al. 1999), or the expression of a kinase-dead form of cdk2 (Meraldi et al. 1999). Cdk2 is activated by associating with cyclin E from late G1 to S phase or with cyclin A from S phase to prometaphase. Therefore, cyclin E-cdk2 and/or cyclin A-cdk2 are thought to trigger centrosome duplication in coordination with DNA replication. Candidates for centrosome substrates of cyclin E/cyclin A-cdk2 have been reported, such as nucleophosmin/B23 (Okuda et al. 2000), Mps1 (Fisk & Winey 2001) and CP110 (Chen et al. 2002). In spite of these observations, cyclin E and cyclin A have been shown to localize mainly in nucleus (Pines & Hunter 1991; Ohtsubo et al. 1995), and there has been almost no report of their localization at centrosomes except for Xenopus embryonic blastomeres (Hinchcliffe et al. 1999) and mitotic HeLa cells (Bailly et al. 1992). Other molecules that affect centrosome duplication have also been reported. They include centrosome-localizing kinases [Aurora A (Zhou et al. 1998), plk1 (Liu & Erikson 2002)], transcription factors [p53 (Fukasawa et al. 1996), E2F3 (Saavedra et al. 2003)] and ubiquitination enzymes; for example, Skp2 (Nakayama et al. 2000). How cyclin E/cyclin A-cdk2 and other molecules are targeted to and regulated at centrosomes remains to be elucidated. One possibility is that there is some centrosomal protein(s) that regulates the localization of various molecules involved in centrosome duplication and coordinates their actions.
A giant anchoring protein CG-NAP (Takahashi et al. 1999), also known as AKAP350 (Schmidt et al. 1999) or AKAP450 (Witczak et al. 1999), localizes to centrosomes throughout the cell cycle and the Golgi apparatus at interphase. CG-NAP has been found to anchor various molecules to these organelles such as protein kinase A (PKA), PKN, protein phosphatase 1, protein phosphatase 2A (Takahashi et al. 1999), phosphodiesterase 4D (Tasken et al. 2001), casein kinase 1
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(Sillibourne et al. 2002), cdc42 interacting protein 4 (Larocca et al. 2004) and Ran (Keryer et al. 2003a). CG-NAP also anchors immature hypophosphorylated PKC to Golgi/centrosomal area (Takahashi et al. 2000). Moreover, we found that CG-NAP anchors the -tubulin ring complex to centrosomes and is involved in the microtubule nucleation (Takahashi et al. 2002).
In this study, we examined whether CG-NAP and its deletion mutants affect the centrosome duplication in CHO-K1 cells, and found that the expression of CG-NAP/D containing the centrosome-targeting region led to the increase in the centrosome number by recruiting excess amount of cyclin E-cdk2 to centrosomes. Therefore, CG-NAP may be involved in centrosome duplication by targeting cyclin E-cdk2 to centrosomes.
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Results |
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We expressed various Flag-tagged CG-NAP deletion mutants in CHO-K1 cells to examine their effects on the number of centrosomes (Fig. 1A). Centrosomes were visualized by immunostaining of -tubulin, a well-established marker of centrosomes. Majority of the control cells (more than 90%) possessed one or two centrosomes (Fig. 1B, left), and the expression of CG-NAP/AB or CG-NAP/C (Fig. 1A) did not affect the centrosome number (our unpublished results). In contrast, more than 30% of the cells expressing the carboxyl-terminal fragment CG-NAP/D (amino acid residues 2875–3899, Fig. 1A) possessed three or more number of centrosomes (Fig. 1B, right, and 1C). Similarly, the expression of GFP-tagged CG-NAP/D resulted in centrosome amplification (Fig. 1C). When increasing amounts of CG-NAP/D vector (0, 0.3, 1 and 3 µg/dish) was transfected, the percentage of cells with amplified centrosomes was increased in accordance with the increase in the amount of the expression vector up to 3 µg (our unpublished results). The expression of full-length CG-NAP increased the number of centrosomes to a lesser extent than that of CG-NAP/D (Fig. 1C). Centrosome amplification was also observed in HeLa cells expressing CG-NAP/D: about 22% of the cells vs. 3% of the control cells.
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-tubulin ring complex (Takahashi et al. 2002), and co-localized with the amplified centrosomes (Fig. 2A,a–f). Endogenous CG-NAP also co-localized with the amplified centrosomes and its localization to the Golgi apparatus was not affected (Fig. 2A,g–i), suggesting that the centrosome amplification was not caused by displacing endogenous CG-NAP from centrosomes by CG-NAP/D. GFP-tagged centrin-2, a centriole marker, was co-localized with amplified centrosomes when co-expressed with CG-NAP/D (Fig. 2A,j–l). Furthermore, endogenous kendrin also co-localized with the amplified centrosomes (Fig. 2A,m–o). In order to examine whether the amplified centrosomes can function as microtubule organizing centres, microtubule regrowth assay was performed. After treatment with nocodazole to disassemble microtubules, CG-NAP/D-expressing cells were incubated in a fresh medium to allow microtubules to regrow. As shown in Fig. 2B, microtubule asters were assembled around the amplified centrosomes. These results suggest that the expression of CG-NAP/D causes the amplification of functional centrosomes rather than ectopic accumulation of -tubulin, although the possibility of centriole splitting cannot be ruled out (Di Fiore et al. 2003).
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To minimize the region of CG-NAP/D for centrosome amplification, we examined the effect of further deletions. We found that a mutant lacking the centrosome-targeting PACT domain (CG-NAP/D/dPACT, Fig. 1A), which distributed to cytoplasm (Fig. 3A,d–f), could not efficiently amplify centrosomes (Fig. 3B). To examine whether the centrosomal targeting of CG-NAP/D is important for the centrosome amplification, we constructed a membrane-bound form of CG-NAP/D by fusing Ki-Ras CAAX motif containing a farnesylation site (Leevers et al. 1994) to the carboxyl-terminus (CG-NAP/D-CAAX). CG-NAP/D-CAAX distributed throughout the cell, probably corresponding to various membranous organelles including plasma membrane (Fig. 3A,g–l). Cells expressing CG-NAP/D-CAAX had decreased number of centrosomes compared with those expressing CG-NAP/D (Fig. 3B). The suppression of centrosome amplification was not complete probably because a small portion of CG-NAP/D-CAAX was still detected at centrosomes in some of the cells (Fig. 3A,j–l, arrows). These results indicate that centrosomal localization of CG-NAP/D is important for its effect on centrosome amplification.
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Centrosome amplification is observed when cells are arrested at S phase by hydroxyurea in CHO cells (Balczon et al. 1995) or U2OS cells (Balczon et al. 1995; Liu & Erikson 2002). In addition, cytokinesis failure followed by cell cycle progression also leads to multiple centrosomes, which is characterized by the appearance of cells with more than 4 N DNA content (Nigg 2002). To examine whether the centrosome amplification of CG-NAP/D-expressing cells is caused by these mechanisms, we performed cell cycle analysis of CHO-K1 cells expressing GFP-tagged CG-NAP/D in comparison with the cells expressing GFP-CG-NAP/D/dPACT, which possessed normal centrosome number (Fig. 3B), as a negative control. Cell cycle analysis was carried out by laser scanning cytometry, which allows determining both the DNA content and the level of DNA condensation of each GFP-positive cell in a defined microscopic field as described in Experimental procedures. As shown in Fig. 4, CG-NAP/D-positive cells showed similar cell cycle distribution to CG-NAP/D/dPACT-positive cells, indicating that the centrosome amplification is not correlated with cell cycle arrest at S phase. Furthermore, the ratio of cells with more than 4 N DNA content in CG-NAP/D-expressing cells was less than 7%, which was much lower than that with amplified centrosomes (more than 30%, Fig. 1C). These results suggest that the centrosome amplification in CG-NAP/D-expressing cells is caused by some mechanisms other than cell cycle arrest or cytokinesis failure.
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Since CG-NAP has been shown to anchor various signalling molecules to centrosomes and the Golgi apparatus, we hypothesized that CG-NAP/D might anchor some molecule(s) that positively regulate centrosome duplication. We examined the interaction of CG-NAP/D with several candidate proteins by immunoprecipitation, and found that CG-NAP/D and cyclin E associated efficiently when they were co-expressed (Fig. 5A). Cyclin E also interacted with full-length CG-NAP (Fig. 5B). Next, we examined the subcellular localization of cyclin E expressed in CHO-K1 cells by immunofluorescence microscopy. When cells were fixed with methanol or paraformaldehyde, cyclin E was mainly localized to nucleus as reported (Ohtsubo et al. 1995) even when CG-NAP/D was co-expressed (our unpublished observation). To visualize centrosomal proteins more clearly, cells were briefly extracted with detergent prior to fixation. Then in some of the cells, weak staining of cyclin E was detected at centrosomes, which was confirmed by the co-localization with -tubulin (Fig. 5C, d–i). Furthermore, cyclin E was obviously detected at centrosomes when CG-NAP/D was co-expressed (Fig. 5C,a–c). These results suggest that a fraction of cyclin E is recruited to centrosomes by CG-NAP/D.
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-tubulin (Fig. 7A) and efficiently nucleated microtubules in vitro (our unpublished results), indicating that these fractions contained centrosomes (Moudjou & Bornens 1994). CG-NAP/D was detected in these centrosome fractions (Fig. 7A), as well as endogenous CG-NAP and kendrin as previously described (Takahashi et al. 2002). Endogenous cdk2 was detected in the centrosomes isolated from cells expressing control vector and, moreover, increased amount of cdk2 was detected in the centrosomes of cells expressing CG-NAP/D (Fig. 7B). Taken together, these results suggest that CG-NAP/D recruits excess amount of endogenous cyclin E-cdk2 to centrosomes.
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Next we examined whether centrosome amplification by CG-NAP/D is caused by recruiting cyclin E-cdk2 to centrosomes. CHO-K1 cells co-transfected with CG-NAP/D and cyclin E possessed slightly increased number of centrosomes compared with those transfected with CG-NAP/D alone (Fig. 8). To examine the effects of cyclin E-cdk2 more clearly, we constructed two kinds of cyclin E mutants (Fig. 9A). One mutant cyclin E/dNLS was designed to change the distribution of cyclin E from nucleus (wild-type cyclin E) to centrosomes as well as to cytoplasm by deleting the amino-terminal NLS sequence. The other mutant cyclin E/dNLS/R145A has an additional mutation of Arginine 145 to Alanine which disrupts the interaction with cdk2 (Jackman et al. 2002). Whereas cyclin E/dNLS interacted with cdk2 and showed histone H1 kinase activity to a similar extent to the wild-type cyclin E, the cyclin E/dNLS/R145A neither interacted with cdk2 nor showed kinase activity (Fig. 9B–D). Both cyclin E mutants associated with CG-NAP/D as wild-type cyclin E did (Fig. 9E). When cyclin E/dNLS was co-transfected with CG-NAP/D, the average centrosome number was increased more than that of the cells co-transfected with wild-type cyclin E and CG-NAP/D (Fig. 8). This result suggests that the cytoplasmic form of cyclin E interacted with CG-NAP/D at centrosomes more efficiently than wild-type cyclin E and enhanced the centrosome amplification. On the other hand, cells co-expressing cyclin E/dNLS/R145A and CG-NAP/D possessed decreased number of centrosomes compared with those expressing CG-NAP/D alone (Fig. 8), which may be attributed to the displacement of endogenous cyclin E-cdk2 on CG-NAP/D at centrosomes by cyclin E/dNLS/R145A. Centrosome number was not much affected by the expression of wild-type or mutant cyclin E alone (Fig. 8). These results suggest that CG-NAP/D increases centrosome number by recruiting cyclin E-cdk2 to centrosomes.
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Discussion |
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Unusual accumulation of cyclin E-cdk2 to centrosomes by CG-NAP/D may cause phosphorylation of centrosome substrates excessively or in an unregulated manner. For instance, nucleophosmin/B23 has been reported to dissociate from centrosomes upon phophorylation by cyclin E-cdk2 prior to the initiation of centrosome duplication, thus is thought to be a licensing factor for centrosome duplication (Okuda et al. 2000). Up-regulated phosphorylation of nucleophosmin has been observed in E2F3-null cells in which cyclin E up-regulation and centrosome amplification occurred (Saavedra et al. 2003). Mps1 has been reported to be stabilized on phosphorylation by cyclin E-cdk2 and to increase centrosome number when over-expressed (Fisk & Winey 2001; Fisk et al. 2003). Although we could rarely detect the interaction of CG-NAP with these substrates thus far (our unpublished results), cyclin E-cdk2 anchored to centrosomes by CG-NAP/D may readily act on these or other unidentified centrosome substrates. Such enhanced phosphorylation may lead to earlier start of centrosome duplication or defects in the termination of centrosome duplication. Either of these mechanisms may allow cells to overduplicate centrosomes during one cell cycle.
Keryer et al. (2003b) reported that the expression of the PACT domain (98 amino acid residues) of CG-NAP/AKAP450 caused the displacement of endogenous AKAP450 from centrosomes, delay in cytokinesis and polyploidy in HeLa cells. When we expressed CG-NAP/D consisting of 1028 amino acid residues containing the PACT domain in CHO-K1 cells, localization of endogenous CG-NAP at centrosomes and the Golgi apparatus was not affected (Fig. 2A,g–i) and the polyploidy was not significantly increased (Fig. 4). Since the deletion constructs, cell conditions and anti-CG-NAP/AKAP450 antibodies employed in their paper were different from ours, further study is required to explain these differences.
Endogenous cdk2 was detected in the centrosome fractions isolated from CHO-K1 cells (Fig. 7B), in which endogenous CG-NAP is also present (Takahashi et al. 2002). Although cyclin E was hardly detected in the centrosome fractions due to the reactivity of commercially available anti-cyclin E antibodies to cyclin E of CHO-K1 cells, cyclin E expressed in CHO-K1 cells was detected at centrosomes by immunofluorescence microscopy (Fig. 5C). We examined whether endogenous CG-NAP can anchor endogenous cyclin E-cdk2 by co-immunoprecipitation of cell lysates; however, we have not detected the interaction thus far, probably because endogenous cyclin E-cdk2 mainly localizes in nucleus and only a small portion may be present in the cytoplasm where CG-NAP and centrosomes are localized. Nevertheless, the interaction of full-length CG-NAP with cyclin E was observed when they were co-expressed (Fig. 5B) and, moreover, centrosome amplification was observed in cells expressing full-length CG-NAP (Fig. 1C). Therefore, CG-NAP may be a possible candidate for recruiting cyclin E-cdk2 to centrosomes in normal cell cycle to trigger centrosome duplication. The level of centrosome amplification by full-length CG-NAP was lower than that by CG-NAP/D (Fig. 1C). One possibility is that the amino-terminal region of CG-NAP associates with proteins that play negative-regulatory roles in centrosome duplication. Such proteins may include those suppressing earlier initiation of duplication and/or blocking reduplication (Wong & Stearns 2003). In the amino-terminal region, CG-NAP has been shown to interact with various molecules such as PKN, PKA, protein phosphatase 1 (Takahashi et al. 1999) and phosphodiesterase 4D (Tasken et al. 2001). Further study is required to elucidate the molecules anchored on CG-NAP that potentially regulate centrosome duplication to take place once per one cell cycle.
A recent report indicated that embryonic fibroblasts from cyclin E1/E2 knockout mice represent normal cell cycle and normal centrosome appearance (Geng et al. 2003). In those cells, the role of cyclin E-cdk2 might be compensated by other cyclin-cdk complexes. Actually, cyclin A has been proposed to be another candidate of cdk2 partner in centrosome duplication (Meraldi et al. 1999). We have found that CG-NAP/D also associated with cyclin A2 and cyclin B1 (our unpublished results), and thus CG-NAP can be involved in centrosome duplication even in the absence of cylin E by recruiting other combinations of cyclin-cdk complexes to centrosomes.
Centrosome amplification is a feature frequently seen in various cancer cells (Pihan et al. 1998; Lingle et al. 1998). Aneuploidy, which has been reported to be also common in cancer cells, could be caused by hyperamplified centrosomes through abnormal mitotic spindle formation and chromosome missegregation (Nigg 2002). It would be interesting to investigate the expression level and mutations of CG-NAP in cancer cells and assess the role of CG-NAP in the maintenance of genetic stability.
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Experimental procedures |
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Mammalian expression plasmids for CG-NAP deletion mutants were constructed by inserting the corresponding cDNA fragments into pTB701-Flag (Kuroda et al. 1996) or pEGFP-C1 (Clontech). Membrane-targeting form of CG-NAP/D was constructed by replacing the stop codon with a cDNA fragment encoding the carboxyl-terminal 17 amino acid residues of Ki-Ras containing CAAX motif (Leevers et al. 1994) by PCR using appropriate primers. cDNAs of cyclin E, centrin-2 and cdk2 were cloned by PCR using HeLa Marathon-Ready cDNA library (Clontech) with appropriate primers and subcloned into pTB701-HA (Kuroda et al. 1996), pEGFP-N3 (Clontech) and pcDNA3.1/Myc-His(+) (Invitrogen), respectively. Cyclin E/dNLS mutant was generated by deleting the first 40 amino acid residues containing the amino-terminal nuclear localization signal (NLS) sequence. Cyclin E/dNLS/R145A was generated by using QuickChange site-directed mutagenesis kit (Stratagene).
Cell culture and transfection
COS7 and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated foetal bovine serum, 50 units/mL penicillin, and 50 µg/mL streptomycin at 37 °C in a humidified 5% CO2 atmosphere. For CHO-K1 cells, Ham's F12 medium was used as a basal medium. COS7 cells were transfected with expression plasmids by electroporation using GenePulser II (Bio-Rad). CHO-K1 and 293T cells were transfected using TransIT LT-1 Transfection Reagent (Mirus).
Antibodies
Polyclonal antibodies to human CG-NAP (
EE) and human kendrin were previously described (Takahashi et al. 1999, 2002). EE was raised against the amino acid residues 423–542 of CG-NAP (Takahashi et al. 1999) and does not recognize CG-NAP/D. The following antibodies were purchased: anti--tubulin GTU88, anti--tubulin DM1A, rabbit anti-Flag, mouse anti-Flag clone M2 (Sigma); rat anti-hemagglutinin (HA) clone 3F10, mouse anti-HA clone 12CA5 (Roche Diagnostics); anti-cdk2 clone 55 (BD Biosciences); rhodamine or fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Chemicon International); and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).
Immunofluorescence microscopy
Cells grown on cover glasses were fixed with cold methanol at –20 °C for 5 min. To visualize centrosome proteins more clearly, cells were extracted with 0.1% Triton X-100 in a buffer consisting of 50 mM piperazine-1, 4-bis (2-ethanesulphonic acid)/KOH at pH 6.9, 5 mM MgCl2, and 1 mM EDTA prior to fixation. The cells were blocked with 5% (v/v) normal donkey serum or 10% (w/v) skim milk in Ca2+, Mg2+-free phosphate-buffered saline (PBS (–)) containing 0.1% Triton X-100, and incubated with the relevant antibody at room temperature for 2 h or at 4 °C overnight. Then the primary antibodies were visualized by incubation with appropriate secondary antibodies conjugated with either rhodamine or FITC at room temperature for 1 h. DNA was visualized by incubating with 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI). The fluorescence was observed under a fluorescence microscope (Zeiss) equipped with a CCD camera (Hamamatsu Photonics).
Centrosome counting
CHO-K1 cells transfected with expression plasmids were fixed after 48 h of incubation. In the case of co-transfection, pEGFP-C1 vector was used as a transfection marker. Centrosomes were visualized by staining with anti--tubulin antibody. At least 100 transfected cells were randomly selected and their centrosomes were counted. For cells transfected with pTB701-Flag vector as a control, at least 200 cells were randomly selected and their centrosomes were counted.
Microtubule regrowth assay
CHO-K1 cells transfected with green fluorescent protein (GFP)-tagged CG-NAP/D were treated with 20 µg/mL nocodazole at 37 °C for 2 h. Then the cells were washed with PBS (–) twice and incubated in a fresh medium at 37 °C for 15 min. The cells were fixed with cold methanol and microtubules were visualized with anti--tubulin antibody followed by rhodamine-conjugated secondary antibody.
Cell cycle analysis
For laser scanning cytometry analysis, CHO-K1 cells were transfected with GFP-tagged CG-NAP/D or CG-NAP/D/dPACT and incubated for 48 h on cover glasses. Then cells were fixed with cold ethanol at –20 °C for 5 min. After rehydrating with PBS (–), cells were treated with 50 µg/mL propidium iodide (PI) containing 200 µg/mL RNase at 37 °C for 30 min. Each cell was monitored for GFP fluorescence intensity, PI fluorescence intensity (for DNA content) and PI fluorescence peak (for DNA condensation) by laser scanning cytometer (LSC2, Olympus), which allows for discrimination between G2 and M phase cells. GFP-CG-NAP/D was mainly localized to centrosomes in the cells with moderate GFP fluorescence intensity, whereas it was accumulated in cytoplasm and/or nucleus in the cells with higher fluorescence intensity. The former cells were selected for further analysis. The same window of GFP fluorescence intensity was used for the selection of cells expressing GFP-CG-NAP/D/dPACT.
Immunoprecipitation and kinase assay
Cells were lysed in a lysis buffer consisting of 50 mM Tris-HCl at pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, 1.5 mM MgCl2, 10 µg/mL leupeptin and 1 mM dithiothreitol at 4 °C for 20 min. After centrifugation, the supernatants were incubated with appropriate antibodies at 4 °C for 2 h. Then protein A-Sepharose beads (Amersham Biosciences) were added and incubated for further 30 min. The beads were washed with the lysis buffer three times, and the bound proteins were analysed by Western blotting. For kinase assay, the above beads were washed three times with the lysis buffer and once with a kinase buffer consisting of 25 mM Tris-HCl, pH 7.5, 10 mM MgCl2 and 1 mM dithiothreitol. Then the beads were incubated with the kinase buffer containing 74 kBq of [-32P] ATP, 50 µM ATP and 4 µg of Histone H1 (Calbiochem) in the presence or absence of 10 µM roscovitine at 30 °C for 20 min. The reaction mixture was subjected to SDS-PAGE and processed for autoradiography.
Isolation of centrosomes
CHO-K1 cells were transfected with Flag-tagged CG-NAP/D or control vector and incubated for 48 h. Then, the centrosomes were isolated as previously described (Takahashi et al. 2002). In brief, the cells were treated with nocodazole and cytochalacin B and lysed in a buffer containing 0.5% (w/v) NP-40. After removing cell debris and nuclei by centrifugation, the lysates were subjected to discontinuous sucrose density gradient with 60 and 40% (w/v) solutions from the bottom and spun at 30 000 g for 1 h. Fractions were collected from the bottom, centrifuged and subjected to immunoblotting.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yonodayo{at}kobe-u.ac.jp
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References |
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Received: 25 August 2004
Accepted: 25 October 2004
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