Anal. Chem. 2007, 79, 8694-8698
Determination of Fluorescence-Labeled Asparaginyl-Oligosaccharide in Glycoprotein by Reversed-Phase Ultraperformance Liquid Chromatography with Electrospray Ionization Time-of-Flight Mass Spectrometry Takamasa Kurihara, Jun Zhe Min, Toshimasa Toyo’oka,* Takeshi Fukushima, and Shinsuke Inagaki
Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNSCl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (∼3 fmol, signal-to-noise ratio of 5 (S/N ) 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESITOF-MS. The detection limit of the PSC-labeled DisialoAsn by selected-ion chromatography was 58 fmol (S/N ) 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLCESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis. The importance of oligosaccharides in biological systems has been recognized, and many research papers concerning the structural elucidation have been published. High-performance liquid chromatography (HPLC),1-4 capillary electrophoresis,5,6 and polyacrylamide gel electrophoresis7 have been developed to * To whom correspondence should be addressed. Tel: +81-54-264-5656. Fax: +81-54-264-5593. E-mail:
[email protected]. (1) Hase, S. J. Chromatogr., A 1996, 720, 173-182. (2) Lamari, F. N.; Kuhn, R.; Karamanos, N. K. J. Chromatogr., B 2003, 793, 15-36. (3) Hase, S. Methods Enzymol. 1994, 230, 225-237. (4) Guile, G. R.; Rudd, P. M.; Wing, D. R.; Prime, S. B.; Dwek, R. A. Anal. Biochem. 1996, 240, 210-226. (5) Koller, A.; Khandurina, J.; Li, J.; Kreps, J.; Schieltz, D.; Guttman, A. Electrophoresis 2004, 25, 2003-2009.
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achieve highly sensitive detection. HPLC is one of the important means for the determination of oligosaccharides. However, the trace analysis is very difficult because oligosaccharides do not possess a strong absorption in the UV-vis region and fluorescence (FL) for sensitive detection. Glycoamidase F (PNGase F) is generally used for releasing asparaginyl-oligosaccharides from glycoproteins, which are linked to the Asn-X-Ser/Thr motif through the N-glycosidic linkage. To determine the oligosaccharide structure, β-glycosylamine, liberated from a glycoprotein containing asparaginyl-oligosaccharides with the enzyme,8,9 is further converted to reducing oligosaccharides with the addition of acetic acid. The reduced oligosaccharides were then labeled with various fluorogenic and chromophoric reagents. The derivatization using a suitable labeling reagent is a key step in the oligosaccharide analysis. There are a significant number of precolumn labeling methods using fluorogenic and chromophoric reagents, e.g., 2-aminopyridine,10-12 4-aminobenzoic acid ethyl ester,13 1-phenyl-3-methyl-5-pyrazolone,14 2- (and 3)-aminobenzoic acids,15,16 2-aminobenzamide,4 and 1-aminopyrene-3,6,8-trisulfonic acid,5,6 for the analysis of asparaginyloligosaccharides. The methods possessing excellent UV absorption or FL properties are certainly good, and subpicomole sensitivities can be achieved. All of these reactions are usually based upon reductive amination, Schiff base formation, and reduction.17,18 Since the chromophore and fluorophore are introduced to the reducing terminus, the structure of the original (6) Suzuki, H.; Muller, O.; Guttman, A.; Karger, B. L. Anal. Chem. 1997, 69, 4554-4559. (7) Davies, M. J.; Hounsell, E. F. Biomed. Chromatogr. 1996, 10, 285-289. (8) Plummer, T. H., Jr.; Elder, J. H.; Alexander, S.; Phelan, A. W.; Tarentino, A. L. J. Biol. Chem. 1984, 259, 10700-10704. (9) Trimble, R. B.; Tarentino, A. L. J. Biol. Chem. 1991, 266, 1646-1651. (10) Hase, S.; Ikenaka, T.; Matsushima, Y. Biochem. Biophys. Res. Commun. 1978, 85, 257-263. (11) Kon, A.; Takagaki, K.; Kawasaki, H.; Nakamura, T.; Endo, M. J. Biochem. 1991, 110, 132-135. (12) Araki, Y.; Andoh, A.; Fujiyama, Y.; Hata, K.; Makino, J.; Okuno, T.; Nakanura, F.; Bamba, T. J. Chromatogr., B 2001, 753, 209-215. (13) Wang, W. T.; LeDonne, N. C., Jr.; Ackerman, B.; Sweeley, C. C. Anal. Biochem. 1984, 141, 366-381. (14) Honda, S.; Akao, E.; Suzuki, S.; Okuda, M.; Kakehi, K.; Nakamura, J.; Anal. Biochem. 1989, 180, 351-357. (15) Kakehi, K.; Funakubo, T.; Suzuki, S.; Oda, Y.; Kitada, Y. J. Chromatogr., A 1999, 863, 205-218. (16) Anumura, K. R.; Dhume, S. T. Glycobiology 1998, 8, 685-694. 10.1021/ac071140v CCC: $37.00
© 2007 American Chemical Society Published on Web 10/13/2007
oligosaccharide can not be retained after the labeling reaction. Furthermore, the derivatization procedure is time-consuming and typically requires a tedious purification step for the labeled oligosaccharides. To avoid this complicated pretreatment, Kamoda et al.19 reported the direct labeling of the β-glycosylamine with Fmoc-Cl. After labeling the oligosaccharides, the original oligosaccharides could possibly be recovered. Although the recovery is an excellent feature of this method, fractionation of the labeled oligosaccharides is required before MALDI-TOF-MS analysis for the structural elucidation. As an alternative detection method to maintain the original structure, we reported a chemoenzymatic FL labeling by a transglycosylation reaction utilizing endo-β-N-acetylglucosaminidase (Endo-M).20-23 Endo-M is an enzyme not only for the hydrolysis of the diacetylchitobiose core of the oligosaccharide chain but also for the transfer of the intact complex oligosaccharide to suitable acceptors. Although the labeling proceeded without a side reaction under mild conditions, the transglycosylation yield was relatively low.20 Hence, we investigated the chemical derivatization with several FL reagents, which are widely used for labeling of an amino functional group.24 The aim of the present research is the development of a reliable determination method for asparaginyl-oligosaccharides by UPLC-ESI-TOF-MS after labeling with a suitable fluorescent reagent.25 An application of the proposed method for glycoproteins is also described in this paper. EXPERIMENTAL SECTION Materials and Reagents. Disialo-Asn and Disialo-Asn-Fmoc were kindly donated by Otsuka Chemicals (Tokushima, Japan). 4-(N,N-Dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBDF), 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 9-fluorenylmethyl chloroformate (Fmoc-Cl), dansylchloride (DNS-Cl), and 2,3naphthalenedialdehyde (NDA) were purchased from Tokyo Kasei (Tokyo, Japan). 1-Pyrenesulfonyl chloride (PSC, Molecular Probes), fluorescein-5-isothiocyanate (FITC, Merck), and 3-chlorocarbonyl6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (DMEQ-COCl, Dojindo Lab., Kumamoto, Japan) were also used as received (Figure 1). Ammonium formate, trifluoroacetic acid, glacial acetic acid (AcOH), methanol, and acetonitrile were of special reagent grade (Wako Pure Chemicals, Osaka, Japan). Deionized and distilled water was used for all the experiments. All other chemicals were of analytical-reagent grade and used without further purification. Spectrofluorometer. The FL excitation and emission spectra were measured by a Hitachi F-3010 spectrofluorometer using a (17) Lamari, F. K.; Kuhn, R.; Karamanos, N. K. J. Chromatogr., B 2003, 793, 15-36. (18) Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W. Glycoconjugate J. 1998, 15, 737-747. (19) Kamoda, S.; Nakano, M.; Ishikawa, R.; Suzuki, S.; Kakehi, K. J. Proteome Res. 2005, 4, 146-152. (20) Min, J. Z.; Toyo’oka, T.; Kato, M.; Fukushima, T. Chem. Commun. 2005, 3484-3486. (21) Min, J. Z.; Toyo’oka, T.; Kawanishi, H.; Fukushima, T.; Kato, M. Anal. Chim. Acta 2005, 550, 173-181. (22) Min, J. Z.; Kurihara, T.; Toyo’oka, T.; Kato, M.; Fukushima, T. Biomed. Chromatogr. 2007, 21, 852-860. (23) Min, J. Z.; Kurihara, T.; Toyo’oka, T.; Fukushima, T.; Inagaki, S. J. Chromatogr., A 2007, 1160, 120-127. (24) Kurihara, T.; Min, J. Z.; Toyo’oka, T.; Fukushima, T.; Kato, M.; Inagaki, S. 126th Annual Meeting for Pharmaceutical Society of Japan, 2006; abstract Vol. 2, p 47. (25) Kurihara, T.; Min, J. Z.; Toyo’oka, T.; Fukushima, T.; Inagaki, S. 127th Annual Meeting for Pharmaceutical Society of Japan, 2007; abstract Vol. 3, p 65.
Figure 1. Structures of FL labeling reagents. Table 1. UPLC-FL and -ESI-TOF-MS Conditions column: Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm i.d. × 100 mm) temperature: 40 °C flow rate: 0.2 mL/min mobile phase: A, 10 mM ammonium formate in H2O B, CH3CN isocratic (B conc): 0 ∼20 min (15%) detector: FL (ex 347 nm; em 385 nm) detector: TOF-MS (waters LCT Premier XE) capillary voltage: 2100 V (ESI+), 1900 V (ESI-) sample cone voltage: 120 V desolvation temperature: 300 °C source temperature: 120 °C desolvation gas flow: 650 L/h cone gas flow: 50 L/h
Table 2. Optimal Reaction Conditions of Disialo-Asn with FL Reagents FL reagent
temp (°C)
reaction time (min)
NDA
37
1
DMEQ-COCl Fmoc-Cl PSC
37 37 50
1 60 120
DNS-Cl NBD-F FITC DBD-F
50 50 50 60
120 10 180 180
buffer (pH) 0.25 M NaCN in 0.1 M Borax (pH 9.8) 0.1 M K2CO3 (pH 11.5) 0.1 M Borax (pH 8.4) 0.1 M triethylamine (pH 11.7) 0.1 M NaHCO3 (pH 9.3) 0.1 M Borax (pH 8.0) 0.1 M triethylamine (pH 11.7) 0.1 M Borax (pH 9.3)
1-cm quartz cell. The wavelengths were recorded without spectral correction. UPLC-FL and -ESI-TOF-MS. The UPLC-ESI-TOF-MS system consisted of an Acquity ultraperformance liquid chromatography system and a Micromass LCT Premier XE mass spectrometer (high-sensitivity orthogonal time-of-flight instrument; Waters Corp.) equipped with an electrospray ionization (ESI) source. An Acquity UPLC BEH C18 column (1.7 µm, 100 mm × 2.1 mm i.d., Waters) was used as the analytical column. The column was maintained at 40 °C. An RF-10AXL FL detector, equipped with an 8-µL flow cell (Shimadzu, Kyoto, Japan), was directly connected between the column outlet and the MS instrument. The TOF-MS was operated in the positive and negative ion modes. The optimized conditions for the UPLC separation and FL and MS detection are shown in Table 1. General Reaction Procedure for Disialo-Asn with FL Reagents. Five-microliter aliquots of 0.5 mM Disialo-Asn in water were vigorously mixed with 50 µL of 5 mM FL reagents in Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
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Figure 2. Reaction of Disialo-Asn with PSC.
Figure 3. Time courses of Disialo-Asn with FL reagents (O) PSC at 50 °C; (b) Fmoc-Cl at 37 °C. Table 3. FL Maximal Wavelengths of Disialo-Asn Derivatives and the Detection Limits (S/N ) 5) for FL and MS Detections wavelength (nm)
LOD (S/N ) 5) (fmol)
FL reagent
ex
em
FL
MS (ESI-)
PSC Fmoc-Cl DNS-Cl NDA DMEQ-COCl FITC NBD-F DBD-F
347 265 320 401 408 442 431 468
385 313 537 464 478 520 556 535
3 48 2870 121 19 1520 44 2580
58 61 164 304 315 739 746 1950
acetonitrile and 20 µL of various buffer solutions (pH 8.0-11.7). After the reaction at 37-60 °C for 1-180 min, the reaction solutions were diluted 50 times with water, and then 10 µL each was subjected to the UPLC-FL system. The recommended reaction conditions are listed in Table 2. The FL maximal wavelengths and the detection limits of the FL-labeled Disialo-Asn are also listed in Table 3. Determination of Asparaginyl-Oligosaccharides in Ovalbumin by Proposed Procedure. The mixture of glycopeptides in ovalbumin was isolated from the enzyme digestion using Pronase E, according to the procedure of Huang et al.26 Briefly, ovalbumin dissolved in water was thoroughly digested by an excess amount of Pronase E in tris-HCl buffer (pH7.4) containing calcium chloride. The soluble fraction was freeze-dried, redissolved in 0.1 M AcOH, and centrifuged. The clean solution underwent gel permeation chromatography utilizing Sephadex G-25 and Bio-Gel P-2 columns. The fraction containing the oligosaccharides was used for derivatization with PSC. A 10-µL aliquot of the asparaginyl-oligosaccharide mixture (2.5 mg/mL) was reacted with 20 µL of 5 mM PSC in acetonitrile at 50 °C for 120 min in the presence of 10 µL of 0.1 M triethylamine (pH 11.7). The reaction mixture was diluted twice with water, and then a 30-µL aliquot was subjected to the UPLC system. The separation
Figure 4. UPLC-ESI-TOF-MS obtained from PSC-labeled Disialo-Asn. Panel A: SIC in ESI+ mode. Panel B: SIC in ESI- mode. Panel C: MS spectrum in ESI+ mode. Panel D: MS spectrum in ESI- mode. The flow rate was 0.3 mL/min. The other conditions are the same as those in Table 1. 8696 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
and detection conditions were the same as those listed in Table 1. RESULTS AND DISCUSSION Derivatization of Disialo-Asn with FL Reagents. Eight FL reagents,27 i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, FmocCl, and DMEQ-COCl (Figure 1), which are reactive to an amino functional group in the asparagine residue of N-linked oligosaccharides, were tested as the candidate chemicals for the labeling. The labeling is a one-pot reaction and requires no complicated pretreatment. Figure 2 shows the reaction of Disialo-Asn (a representative asparaginyl-oligosaccharide) with PSC as an example. The results of the time course study with PSC and FmocCl are depicted in Figure 3. The suitable reaction conditions are varied and dependent upon the reagent used. The optimal reaction conditions are shown in Table 2. Relatively clean chromatograms were obtained from all reagents tested (data not shown). A highly sensitive detection at the femtomole level of Disialo-Asn was also obtained after the labeling of these reagents (Table 3). Therefore, the tested reagents were essentially adaptable for the determination of the asparaginyl-oligosaccharides. The detection limit (signal-to-noise ratio of 5, S/N ) 5) of PSC-labeled Disialo-Asn on the chromatogram by UPLC-FL was the lowest (∼3 fmol). Thus, PSC seems to be a very suitable fluorescent reagent for labeling of the asparaginyl-oligosaccharides. Separation and Detection of PSC-Labeled Disialo-Asn. HPLC is an efficient tool for the separation of an oligosaccharide mixture. However, the retention and good separation of oligosaccharides by reversed-phase chromatography are fairly difficult, because intact oligosaccharides are hydrophilic and polar compounds. Separation by reversed-phase chromatography of the oligosaccharides followed by its detection seemed to be improved after derivatization with a suitable hydrophobic reagent.22,23 Indeed, retention on the resins in the column after PSC labeling was stronger than the nonlabeled oligosaccharides. The FL label is suitable for the selective and sensitive detection of oligosaccharides; however, no structural information could be obtained from the FL detection. On the other hand, TOF-MS analysis is an efficient means to obtain structural information, because an exact m/z value corresponding to the molecular mass is easily obtained. The labeling also increased the MS sensitivity as compared to the MS of the unlabeled intact oligosaccharide. Thus, an ESI-TOFMS instrument was directly connected after the FL detector for the determination of the PSC-labeled oligosaccharides. Furthermore, the UPLC system was also used for rapid separation instead of conventional HPLC. Figure 4 shows the selected-ion chromatograms (SIC) and MS spectra obtained from the PSC-labeled Disialo-Asn. In the ESI+ mode, in addition to H+ ions, ammonium ions were observed. The appearance of ammonium adducts seems to be due to the ammonium formate used as the mobile phase for the separation of the labeled Disialo-Asn. In ESI-, only Hions were detected. mode. In addition, the sensitivity was also higher than that in the ESI+ mode. Therefore, the negative ion mode is recommended for the determination of the oligosaccharide label. As shown in Table 3, the sensitivity of TOF-MS detection was lower than that of FL detection. However, information from the m/z values seems to be one of the important features (26) Huang, C.-C.; Mayer, H. E., Jr.; Montgomery, R. Carbohydr. Res. 1970, 13, 127-137. (27) Yamaguchi, M; Ishida, J. In Modern derivatization methods for separation sciences: Reagents for FL detection; Toyo’oka, T., Ed.; John Wiley & Sons: Chichester, 1999; pp 99-165.
Figure 5. Typical UPLC-FL and -ESI-TOF-MS obtained from PSClabeled asparginyl-oligosaccharides in ovalbumin. Panel A: FL chromatogram (ex 347 nm, em 385 nm). Panel B: SIC in ESI- mode. Panel C: MS spectra in ESI- mode.
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Figure 6. Comparison of detection sensitivity between PSC label and Fmoc label.
for structural elucidation of the oligosaccharides. Based upon these observations, the UPLC-ESI-TOF-MS system was adopted for the determination of the asparaginyl-oligosaccharides after FL labeling with PSC. Determination of Asparaginyl-Oligosaccharides in Ovalbumin. As one of the applications, we tried to detect the N-linked oligosaccharides liberated from ovalbumin by Pronase E, which releases the oligosaccharides containing an Asn as the terminal residue. Fmoc-Cl and DNS-Cl have been developed for FL labeling of a NH2 group in the terminal Asn, which has been used for determination of the hydrolysis activity of endo-β-N-acetylglucosaminidases, such as Endo-M.28,29 As shown in Table 3, the detection sensitivity of the PSC method was higher than those methods using Fmoc-Cl and DNS-Cl. Thus, detection of the asparaginyl-oligosaccharides in ovalbumin was carried out by the proposed procedure using PSC. The method utilizing Fmoc-Cl was also tested as the reference for determination of the asparaginyl-oligosaccharides in ovalbumin, because the structural elucidation of the oligosaccharides in glycoproteins has not been performed using the fluorophores until now. Figure 5A shows the chromatogram obtained from FL detection at 385 nm (ex 347 nm). The PSC-labeled oligosaccharide fraction was easily identified from the FL chromatogram. When the ESI+ mode was adopted for detection of the PSC-labeled oligosaccharides in ovalbumin, ammonium ions were identified in addition to the Disialo-Asn derivative. On the other hand, [M + HCOOH - 2H]2- was detected in the ESI- mode. Panels B and C in Figure 5 show the typical SIC and MS spectra obtained from a couple of oligosaccharides in the ESI- mode. Judging from the MS spectra, the oligosaccharides were efficiently detected as bivalent ions. Fifteen oligosaccharides in ovalbumin, i.e., (Man)3(GlcNAc)5 (M3N5), (Man)3(GlcNAc)5(Gal) (M3N5G1), (Man)3(GlcNAc)6 (M3N6), (Man)3(GlcNAc)6(Gal) (M3N6G1), (Man)3(GlcNAc)7 (M3N7), (Man)3(GlcNAc)8 (M3N8), (Man)4(GlcNAc)2 (M4N2), (Man)4(GlcNAc)4 (M4N4), (Man)5(GlcNAc)2 (M5N2), (Man)5(GlcNAc)3 (M5N3), (Man)5(GlcNAc)4 (M5N4), (Man)5(GlcNAc)5 (M5N5), (Man)5(GlcNAc)5(Gal) (M5N5G1), (Man)6(GlcNAc)2 (M6N2), and (Man)7(GlcNAc)2 (M7N2), were identified from the proposed procedure using PSC. A rapid detection within 20 min was performed using the UPLC system. M3N7 and M4N2 could not be identified from the method using Fmoc-Cl. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Furthermore, the sensitivity of the present method was higher than that of the Fmoc-Cl method (Figure 6). Judging from the results, the proposed method utilizing (28) Yamamoto, K.; Kadowaki, S.; Watanabe, J.; Kumagai. H. Biochem. Biophys. Res. Commun. 1994, 203, 244-252. (29) Inazu, T.; Kobayashi, K. Synlett 1993, 869-870.
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PSC seems to be useful for on-line oligosaccharide analysis. Although each separation of PSC-labeled oligosaccharides is still insufficient, the separation and the sensitivity seem to be improved utilizing another type of column and nanoLC system. In comparison with the off-line MALDI-TOF-MS analysis, the proposed online separation and ESI-TOF-MS detection may be suitable for rapid determination of oligosaccharides. The determination of asparaginyl-oligosaccharides in various glycoproteins, such as fetuin, has been proceeding in our laboratory. Four oligosaccharides in fetuin are currently identified using the proposed procedure. CONCLUSION This paper describes a method for the determination of asparaginyl-oligosaccharides in glycoproteins by combination of FL labeling and UPLC-ESI-TOF-MS detection. As one of the applications of the present method, determination of oligosaccharides liberated from ovalbumin with Pronase E was attempted. The structures of 15 asparaginyl-oligosaccharides were successfully identified using the proposed procedure. Although the analytical run time might still be long, in spite of the use of UPLC, a high-throughput determination will be performed by optimization of the elution conditions and the column. Although the sensitivity of the present method may be lower than that of MALDI-MS, online separation and detection without fractionation seems to be an advantage. The chromatographic separation and the ESI-TOFMS sensitivity were improved after derivatization. Consequently, the chemical derivatization method using PSC seems to be applicable for the structural elucidation of N-linked oligosaccharides in glycoproteins. The key step of the proposed procedure is the releasing of the asparaginyl-oligosaccharides from the target glycoprotein. If the asparaginyl-oligosaccharides are efficiently liberated from the enzyme digestion, the determination seems to be promising. The presented method might be useful for the detection of O-linked oligosaccharides, because Ser and Thr are also labeled with PSC. Furthermore, the method may be applicable to identify short-chain glycopeptides, because PSC labels an NH2 group of the terminal amino acid residue in the glycopeptide sequence. The structure of the labeled glycopeptide could be resolved by MSn analyses. We are currently investigating the resolution of N-linked oligosaccharides in various glycoproteins. The efforts also involve the determination of O-linked oligosaccharides and short-chain glycopeptides digested from proteases, such as trypsin. ACKNOWLEDGMENT The authors thank Otsuka Chemical Co. for the generous gifts of Disialo-Asn and Disialo-Asn-Fmoc. The present research was supported in part by a Grant-in-Aid for Scientific Research, and Global COE program from the Ministry of Education, Science, Sports and Culture of Japan.
Received for review May 31, 2007. Accepted July 30, 2007. AC071140V
