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Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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(RAR) generates the transforming protein found in acute promyelocytic leukemias (APL) (de The et al. 1991; Kakizuka et al. 1991). PML belongs to a family of proteins characterized by their RBCC motif, which consists of a C3HC4-type zinc finger (RING finger), and one or two Cys-rich regions (B boxes) and a coiled-coil domain. PML is localized to nuclear dot-like structures known as PML nuclear bodies (PML-NBs) or PML oncogenic domains (Dyck et al. 1994; Weis et al. 1994). They contain a number of proteins including Sp100, suggesting that PML-NBs have more than one function. PML-RAR alters the PML-NB structure into numerous small speckles, but normal PML-NBs structure can be regained when cells are treated with all-trans RA. Over-expression of PML in cells is accompanied by growth inhibition, suggesting that PML acts endogenously to block cellular proliferation (Mu et al. 1994). A functional knockout of the Pml gene in mice enhances tumorigenesis and inhibits the differentiation of myeloid precursor cells (Wang et al. 1998a). In addition, PML is also involved in controlling cellular senescence (Ferbeyre et al. 2000; Pearson et al. 2000; Bischof et al. 2002). PML-induced senescence is characterized by an increase in the levels of the tumor suppressor proteins p21/p53 and p16/Rb. A hallmark for PML-induced senescence is the formation of a functional complex between p53, CBP and PML.
In the nucleus, PML is involved in multiple processes through its interaction with various proteins. PML regulates the trans-activation of RAR when bound to CBP (Zhong et al. 1999). PML is also involved in the Mad- and Rb-mediated transcriptional repression through its interaction with multiple co-repressors, including N-CoR (Khan et al. 2001a,b). Furthermore, PML has been shown to regulate apoptosis by interacting with Bax and p27Kip1 (Quignon et al., 1998; Wang et al., 1998b). In addition, PML inhibits neoangiogenesis by blocking the translation of the hypoxia-inducible factor 1 (HIF-1) by sequestering mammalian target of rapamycin (mTOR) into the nucleus (Bernardi et al. 2006). HIF-1 level is enhanced under hypoxic condition and leads to stimulation of neoangiogenesis. PML-RAR behaves in a dominant negative manner to regulate these processes. Furthermore, an isoform of PML, PML3, controls centrosome duplication by suppressing the activation of Aurora A (Xu et al. 2005). To perform all of these functions PML must be localized to the nucleus. However, PML is also present in the cytoplasm. Cytoplasmic PML (cPML) positively regulates TGF-β signaling by enhancing the accumulation of SARA and the TGF-β receptor in early endosomes (Lin et al. 2004). However, the role of cPML still remains largely elusive.
One characteristic of tumor cells is their high glycolytic activity (Gatenby & Gillies 2004; Kim & Dang 2006; Shaw 2006). This aids in the migration and survival of tumor cells under hypoxic conditions as observed in solid tumors (Kim et al. 1997; Reshkin et al. 2000). At least two transcription factors, Myc and HIF, have been shown to contribute to the increased glycolytic activity in tumor cells. Myc activates the transcription of virtually all known glycolytic enzymes and directly binds to numerous glycolytic gene promoters (Kim et al. 2004). HIF-1, which is stabilized under hypoxic condition, also induces the transcription of numerous glycolytic enzymes (Semenza et al. 1994, 1996). In addition to hypoxia, oncogenic events, including activation of the Src and H-Ras oncogenes, have also been linked to the stabilization of HIF in the presence of adequate oxygen (Jiang et al. 1997; Chen et al. 2001). Although some of the molecular players have been identified, the mechanism by which the glycolytic activity in the tumor cells is stimulated remains elusive.
A rate-limiting enzyme during glycolysis is pyruvate kinase (PK). This enzyme catalyses the production of pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP (Tanaka et al. 1967; Ibsen & Trippet 1974; Nowak & Suelter 1981). In mammals, four PK isozymes (M1, M2, L and R) exist and are expressed in various cell types (L-PK in liver and kidney, M1-PK in muscle and brain and R-PK in erythrocytes) (Tanaka et al. 1967). The M1 and M2 isotypes are derived from the M form by alternative splicing. M1 and M2 differ in the one exon. Of the 51 amino acids encoded by this exon, residues, 21 are distinct between M1 and M2 (Dabrowska et al. 1998). Interestingly, the M2 isoform of pyruvate kinase (PKM2) is predominantly expressed in tumor cells, including HeLa uterine cervical cancer cells and MCF-7 breast cancer cells, where the expression of the other isoforms is low (Takenaka et al. 1996; Dabrowska et al. 1998). PKM2 can exist as a tetramer or as a dimer and the tetramer : dimer ratio of PKM2 is critical for controlling its glycolytic activity in tumor cells. In its tetrameric form, PKM2 has a high affinity for its substrate PEP, while in its dimeric form it has low affinity (Eigenbrodt et al. 1992). Only the tetrameric form of PKM2, can form a glycolytic enzyme complex. When PKM2 is in its inactive dimeric form, glucose carbons are directed for the synthesis of nucleic acids and are preferentially catalyzed by transketolase (Glossmann et al. 1981; Eigenbrodt et al. 1992; Boros et al. 1997, 2000; Zwerschke et al. 1999; Cascante et al. 2000; Mazurek et al. 2001). The HPV 16-E7 oncoprotein directly bind to PKM2, and trigger the dimerization of PKM2 (Zwerschke et al. 1999). Fructose 1,6-bisphosphate (FBP), a glycolytic phosphometabolite, then stimulates the formation of tetramers from dimers (Boros et al. 1997).
Here, we have identified PKM2 as a cPML-interacting protein. We have demonstrated that the expression of cytosolic PML in tumor cells results in suppression of lactate accumulation and PKM2 activity under both normoxic and hypoxic conditions. Our findings suggest that cPML may play a role in glycolytic metabolism by modulating the activity of PKM2.
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Results |
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To identify PML-interacting proteins, we purified the PML complex from HeLa S3 cell line expressing FLAG- and HA-tagged PML (FLAG/HA-PML). The HeLa cells were disrupted and the PML complex was purified from nuclear extracts using both the anti-FLAG and anti-HA antibodies under relatively low salt conditions (100 mM NaCl). Several proteins immunoprecipitated with FLAG/HA-PML, which were not detected in the purified sample from control HeLa cells not expressing epitope-tagged protein (Fig. 1A). By mass spectrometry, we indicated the 120-, 115- and 110-kDa proteins to be sumoylated PML, while the 58-kDa interacting protein was PKM2.
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PML binds to PKM2 through its coiled-coil domain
To examine which domains of PML are required for PKM2 binding, we performed GST pull-down assays. Full-length PML expressed in 293T cells efficiently bound to GST–PKM2 (Fig. 2A) as did the RBCC mutant which lacked the region downstream of the coiled-coil domain. However, the PML mutant lacking the coiled-coil domain (
C-C) failed to bind to GST–PKM2. These results suggest that the coiled-coil region of PML is required for PKM2 binding.
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Since nuclear PML affects the transcription of various genes, over-expression or down-regulation of wild-type PML may affect glycolysis via indirect effect of nuclear PML. Further, there is no way to specifically down-regulate only cPML. To investigate whether cPML affects the PK activity and glycolysis without the effect of nuclear PML, thus, we created a PML mutant which only localizes into the cytosol. To generate this mutant, we replaced two lysine residues (Lys-487 and Lys-490) with alanines (PML-2KA), which were previously reported to be in the functional nuclear localization signal (Duprez et al. 1999). To study the subcellular localizations of this mutant, we generated MCF-7 breast cancer cells that stably express FLAG-PML-2KA by infecting virus. Confocal microscopic analysis of the cells indicated that PML-2KA displayed almost uniform cytoplasmic distribution with large punctate staining near the nuclear membrane (Fig. 2B). To avoid confusion, we have used the word ‘cPML’ only for wild-type PML localized in the cytoplasm, but not for the PML-2KA mutant in this study. Nuclear localization of this mutant was not evident, and PML-2KA and PKM2 co-localized in much of the cytosol. The perinuclear punctate structures also stained positive with an antibody against heat shock protein 70 (HSP70) (Fig. 2C). Since HSP70 is a marker of aggresomes (García-Mata et al. 1999), this perinuclear staining may be generated by the aggregation of PML-2KA through its coiled-coil region. In the GST pull-down assays, PML-2KA expressed in 293T cells efficiently bound to GST–PKM2 (Fig. 2A). Thus, PML-2KA is primarily localized in the cytoplasm and can efficiently bind to PKM2.
PML-2KA suppresses PKM2 activity and lactate accumulation in tumor cells
To analyze the significance of the interaction between PML-2KA and PKM2, we compared the PKM2 activities between the FLAG-PML-2KA-expressing MCF-7 cells and the control MCF-7 cells infected with the empty virus. After isolating cytoplasmic fractions from these two types of MCF-7 cells, PKM2 activity was measured at a saturating PEP concentration (5 mM). The PKM2 activity of the PML-2KA expressing cell lysates was 23% lower than that of control cell lysates (Fig. 3A). A similar reduction (19%) in PKM2 activity was also observed from lysates prepared from cells cultured under hypoxic conditions. By Western blotting, we confirmed that the expression level of FLAG-PML-2KA in MCF-7 cells did not change from normoxic to hypoxic conditions (Fig. 3B).
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Modulation of tetramer PKM2 activity by PML-2KA
PKM2 exists in tetrameric and dimeric complexes, and the tetramer : dimer ratio of PKM2 is critical for its glycolytic activity (Glossmann et al. 1981; Eigenbrodt et al. 1992; Boros et al. 1997, 2000; Zwerschke et al. 1999; Cascante et al. 2000; Mazurek et al. 2001). To address whether cPML affects the tetramer : dimer ratio of PKM2, cell lysates were prepared from control and PML-2KA-expressing MCF-7 cells and subjected to glycerol-gradient centrifugation. PK activity was then measured in fractions taken at different glycerol concentrations. The high and low molecular weight PKM2 activity peaks correspond to the tetrameric and dimeric forms of PKM2 (Fig. 4A). The total PK activity of tetrameric complexes from PML-2KA-expressing cells was slightly (11%), but significantly, lower than that of control cells (Fig. 4B). In contrast, there was no significant difference in the total PK activity of the dimeric form between the two cell types. This result suggests that cPML specifically inhibits the activity of tetrameric PK. We have examined the distribution of PML-2KA in the glycerol gradient shown in Fig. 4A using Western blotting. PML-2KA proteins were detected not only in the fractions containing tetrameric PKM2 but also other fractions (data not shown), suggesting that PML-2KA interacts with tetrameric PKM2 only with low affinity. Although we cannot directly examine whether tetrameric form specifically bind PML-2KA due to lack of such system, our result suggest that cPML inhibits the activity of tetrameric PKM2 by specific interaction with low affinity.
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The cPML-dependent suppression of PKM2 activity may not regulate the initial step of immortalization because PKM2 is predominantly expressed in tumor cells. Thus, cPML would be expected to affect the malignancy of the cancer cells which express PKM2. Most tumors, if not all, have increased glycolytic activity, and the growth of tumor cells is considered to be linked to the increased production of ATP during glycolysis. To test whether cPML can suppress tumor cell growth by inhibiting the PK activity, we used MCF-7 cells as the tumor cells expressing both PKM2 and PML, in which over-expression of PML-2KA would be expected affect the cell growth. Control MCF-7 cells and MCF-7 cells expressing PML-2KA were injected into SCID mice, and tumor size was measured. We found that these tumors had a similar rate of growth as mice injected with control MCF-7 cells (Fig. 5A). At 27-day after injection, the weight of tumors was also not significantly different between the two MCF-7 cell lines (Fig. 5B). These results indicate that inhibition of PK activity by the over-expression of PML-2KA has no effect on the growth of MCF-7 tumor cells. We examined the amount of PML in the cytoplasm of parental MCF-7 cells and MCF-7 cells expressing PML-2KA. Significant amounts of PML were detected in the cytoplasm, which appears to be higher than that of HeLa S3 cells shown in Fig. 1B, in parental MCF-7 cells, while further increased amounts of cytosolic PML were detected in MCF-7 cells expressing PML-2KA (Fig. 5C).
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Discussion |
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It has long been known that tumor cells have increased glycolytic activity (Gatenby & Gillies 2004; Kim & Dang 2006; Shaw 2006). In fact, inhibition of ATP citrate lyase (ACL), a key enzyme-linking glucose metabolism to lipid synthesis, was recently shown to suppress tumor cell growth (Hatzivassiliou et al. 2005). Based on these reports, we speculated that an over-expression of PML-2KA may also suppress tumor cell growth. However, we found that the over-expression of PML-2KA in MCF-7 cells did not inhibit cell growth after injection into SCID mice. This may suggest that the slight reduction in the rate of glycolysis by cPML may not be sufficient to inhibit tumor cell growth. Alternatively, cPML may affect tumor cell growth in a cell-specific manner. Since significant amounts of cPML were detected in parental MCF-7 cells, cPML might more effectively regulate proliferation of other types of cells which have lower amounts of cPML. Recently, Bernardi et al. (2006) demonstrated that nuclear PML suppresses HIF-1 translation by recruiting mTOR to PML-NBs, leading to the suppression of tumor cell growth under hypoxia. Thus, the over-expression of cPML may cause mTOR to be anchored in the cytoplasm, and counteract the tumor suppressor function of nuclear PML during hypoxia. If this is the case, it will be necessary to examine the effect of cPML on tumor cell growth under normoxic conditions.
Previously, a Rev-type NES in the C-terminal region of PML isoform I has been identified (Henderson & Eleftheriou 2000), however, this NES sequence is not retained in any other PML isoforms including isoform VI (Jensen et al. 2001). However, it has been reported that HIV infection can trigger the exportin-mediated cytoplasmic export of nuclear PML (Turelli et al. 2001). Furthermore, under certain pathogenic conditions including arenaviral infection, PML is localized in the cytoplasm (Kentsis et al. 2001). These results suggest that PML can shuttle between the cytoplasm and the nucleus and is functional in the cytoplasm. The detection of significant amounts of PML in the cytoplasm in the HeLa cells expressing FLAG/HA-PML (Fig. 1B) also supports this notion. Using the NIH3T3 cell lines expressing the EGFP-PML fusion proteins, we observed that the treatment with 2-deoxyglucose, an inhibitor of glycolysis, decreased the amount of PML localized in the cytoplasm (data not shown). Thus, the shuttling of PML between the cytoplasm and the nucleus could be regulated by specific signals, some of which may be linked to glycolysis. Further investigation into the specific signals which control the shuttling of PML may lead to a further understanding of the function of cPML.
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Experimental procedures |
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The chicken β-actin promoter containing plasmids to express FLAG tagged wild-type and C-C mutant PML were described previously (Khan et al. 2001a). PCR-based methods were used to construct plasmids that express FLAG/HA-PML, GST–PKM2 and various PML derivatives. Human PML-VI cDNA was cloned into the pOZ-FH-N vector (Nakatani & Ogryzko 2003) to generate retrovirus vectors that express FLAG- and HA-tagged PML. In the PML RBCC construct, the C-terminal domain (residues 395–555) was deleted. In the PML-2KA mutant construct, the two lysine residues in positions 487 and 490 which are required for the nuclear localization of PML were replaced by two alanine residues.
Purification and characterization of the PML complex
The retrovirus vector encoding FLAG/HA-PML was transfected into amphotropic packaging Phoenix A cells and medium containing the amphotropic virus was collected. HeLa S3 cells were transduced with a recombinant retrovirus that expressed a bicistronic mRNA encoding FLAG/HA-PML linked to the IL-2 receptor subunit. The transduced subpopulation was purified by repeated cycles of affinity cell sorting. Several HeLa S3 cell lines were generated and the one, which expressed a minimum amount of FLAG/HA-PML, was cultured to purify PML complexes. Cells from a 12-L culture were disrupted in a hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and a protease inhibitor cocktail (Roche, Basel, Switzerland)), and cell lysates were centrifuged at 25 000 g for 20 min. The pellets were resuspended in an extraction buffer (20 mM HEPES, pH 7.9, 25% glycerol, 100 mM NaCl, 1.5 mM MgCl2, 9.6 mM N-ethylmaleimide (Sigma, Seelze, Germany), 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT and protease inhibitor cocktail) for 30 min at 4 °C and cell extracts were centrifuged at 25 000 g for 30 min. PML complexes were purified from these cell extracts essentially as described by Nakatani & Ogryzko (2003) using anti-FLAG M2 monoclonal antibody (mAb)-conjugated agarose beads (Sigma) followed by anti-HA 3F10 mAb-conjugated agarose beads (Roche) in a wash buffer (20 mM Tris–HCl, pH 8.0, 0.1 M KCl, 5 mM MgCl2, 10% glycerol, 1 mM PMSF, 0.1% Tween 20, 10 mM β-mercaptoethanol, and protease inhibitor cocktail). Purified proteins were separated on a 4%–20% gradient SDS polyacrylamide gel and silver stained. Protein bands were excised and analyzed by mass spectrometry at the RIKEN Brain Science Institute Mass Spectrometry Facility.
Cell fractionation
To examine the subcellular localization of PML, cells were lysed in a hypotonic buffer and the centrifuged at 17 000 g for 10 min to collect the nuclear and cytosolic fractions. The nuclear pellet was lysed in C buffer (20 mM HEPES, pH 7.9, 25% glycerol, 100 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT and a protease inhibitor cocktail) and centrifuged at 17 000 g for 20 min. The resulting supernatant was used as the nuclear extract.
Co-immunoprecipitation assay
Cytoplasmic fractions from HeLa S3 cells were mixed with a co-immunoprecipitation buffer and the resulting solution contained 100 mM NaCl, 0.2 mM EDTA, 9.6 mM NEM, 0.1% NP-40 and 10% glycerol. This solution was incubated overnight with 2 µg of either the anti-FLAG M2 monoclonal antibody or the anti-PML Antibody (PM001, MBL). The following day, 100 µL of Protein G-Sepharose beads were added and rotated at 4 °C for 3 h. After washing 5 times with wash buffer (20 mM Tris–HCl, pH 8.0, 200 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 0.1% NP-40, 10% glycerol, 7 mM β-mercaptoethanol, 1 mM PMSF and a protease inhibitor cocktail), bound proteins were resolved on a SDS-PAGE, followed by Western blotting using an anti-PML monoclonal antibody (PG-M3, Santa Cruz, Santa Cruz, CA) and an anti-PK antibody (100–1178, Rockland, Gilbertsville, PA).
GST pull-down assays
GST pull-down assays were performed using GST–PKM2 and the 293T cell lysates containing HA-PML essentially as described (Nomura et al. 1999). 293T cells (2 x 106 cells per 100-mm dish) were transfected with the plasmids to express HA-PML and PML derivatives using Lipofectamine Plus (Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, cell lysates were prepared by mild sonication in NET buffer (20 mM Tris–HCl, pH 8.0, 1 mM EDTA, 0.5% NP-40, protease inhibitor cocktail) containing 150 mM NaCl. The binding and washing buffer contained 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 2.5 mM MgCl2, 0.5 mM DTT, 0.01% BSA, 0.5% PMSF and 0.025% NP-40. Bound proteins were separated by SDS-PAGE and analyzed by immunoblot analysis with an anti-HA monoclonal antibody.
Immunocytochemistry and imaging
A MCF-7 cell line which stably expressed FLAG/HA-PML-2KA and a control cell line tat possessed empty vector were generated by retroviral transduction as described above. The cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then incubated with the anti-PK, anti-FLAG and anti-HSP70 (K20, Santa Cruz Biotechnology) antibodies. FITC or Alexa Fluor 546-conjugated anti-goat or anti-mouse secondary antibodies (Molecular Probes, Eugene, OR or Chemicon, Temecula, CA) were added along with TOTO-3 iodide (Molecular Probes) to label chromatin. Confocal images were obtained using an LSM510 (Zeiss, Jena, Germany) laser-scanning microscope.
Determination of lactate concentration and glycolytic activity
MCF-7 cells were incubated in a hypoxia environment for 12 h by the oxidative absorber AnaeroPack for Cell (Mitsubishi Gas Chemical, Tokyo, Japan). The concentrations of lactate in cellular supernatants were determined as described (Bergmeyer 1985). Glycolysis assays were performed as described (Plas et al. 2001). Briefly, MCF-7 cells (1.2 x 106 cells) were resuspended in 500 µL of CO2-buffered Krebs buffer (Hepes–NaOH, pH 7.4, 115 mM NaCl, 2 mM KCl, 25 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, 0.25% BSA) lacking glucose for 30 min at 37 °C. Cells were centrifuged and resuspended in 230 µL of Krebs buffer containing 25 mM glucose supplemented with 20 µCi/mL [5-N-3H]glucose (Perkin Elmer, Waltham, MA) for 1 h at 37 °C. The supernatant was acidified with an equal volume of 0.2 N HCl, and placed in an open tube in stoppered scintillation vials (Perkin Elmer) containing 0.5 mL H2O. 3H2O was separated from [3H]glucose by the evaporative diffusion of 3H2O in a closed chamber. After equilibration for 48 h at room temperature, the 3H2O was measured by a liquid scintillation analyzer (Tri-Carb 2800TR, Perkin Elmer).
Determination of PKM2 activity
MCF-7 cells (approximately 7 x 106 cells) were washed 3 times with ice-cold PBS, and suspended in 200 µL of buffer containing 10 mM Tris–HCl (pH 7.5), 1.5 mM MgCl2, 20 mM NaCl, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail). The cells were then incubated on ice for 10 min and lysed with a glass Dounce homogenizer (B pestle). After two centrifugations at 17 000 g for 30 min each, PK activity was measured in the supernatant by a lactate dehydrogenase (LDH) coupled assay. The 200 µL reaction mixture was prepared on ice containing 100 mM Tris–HCl (pH 8.0), 100 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 0.2 mM β-NADH, 1.5 mM ADP, 5 mM phosphoenolpyruvate (PEP) and 0.6 units/mL LDH. The reaction was initiated by the addition of 0.5 µg of total cell extract. PK activity was calculated at 37 °C by monitoring the absorbance at 340 nm in an OPTImax microplate spectrometer (Molecular Devices, Union City, CA). Due to the slight pyruvate kinase contamination in commercial LDH, a blank (containing no cell extract) was subtracted from the activity measurement. A unit of PK activity is defined here as the amount of enzyme required to oxidize 1 µM of NADH per minute under these experimental conditions.
Glycerol gradient sedimentation analysis of PK activity
Whole cell extracts were prepared from MCF-7 cells cultured for 12 h under normoxic or hypoxic conditions. After three washes with ice-cold PBS, approximately 7 x 106 cells were suspended in 200 µL of homogenization buffer (20 mM KH2PO4/K2HPO4, 50 mM NaCl, 1 mM DTT, 1 mM
β-mercaptoethanol, 1 mM EDTA, 1 mM
-amino-N-caproic acid, 0.5 mM PMSF, 10% glycerol and a protease inhibitor cocktail), lysed with a Dounce homogenizer, and centrifuged twice for 15 min at 17 000 g. For glycerol gradient sedimentation, 300 µg of the cellular supernatant (500 µL) was loaded on the top of a 4-mL 10%–22% glycerol gradient (in homogenization buffer) and centrifuged for 13 h at 55 000 rpm (Beckman, SW55Ti rotor). After centrifugation, 150 µL fractions were collected from the top of the gradient. Each fraction was analyzed for PKM2 activity. Assay conditions were the same as above, except that the concentration of PEP was 2 mM.
Tumorigenesis assay
An in vivo tumorigenesis assay was performed as described by Karpanen et al. (2001). MCF-7 cells (1 x 107) were inoculated into the fat pads of the second (axillar) mammary gland of ovarectomized SCID mice. Tumor length and width were measured every 4 days thereafter.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: sishii{at}rtc.riken.jp
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References |
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Received: 4 September 2007
Accepted: 25 November 2007
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