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Originally published In Press as doi:10.1194/jlr.M600296-JLR200 on October 5, 2006

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Journal of Lipid Research, Vol. 48, 96-103, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

Masahiro Ito1,*, Yuki Matsumuro*, So Yamada*, Tomonori Kitamura{dagger}, Saki Itonori{dagger} and Mutsumi Sugita{dagger}

* Department of Bioscience and Bioinformatics, College of Information Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan
{dagger} Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, 2-5-1 Hiratsu, Otsu, Shiga 520-0862, Japan

Published, JLR Papers in Press, October 5, 2006.

1 To whom correspondence should be addressed. e-mail: maito{at}is.ritsumei.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galß1-4(Fuc{alpha}1-3)GlcAß1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

Supplementary key words glucuronic acid • chemical structure • ceramide moiety • 1H-nuclear magnetic resonance • matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry

Abbreviations: MALDI-TOF MS, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry; PSD, postsource decay; UGL, uronic acid-containing glycosphingolipid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosphingolipids have been characterized in a range of animal phyla, including arthropods, and have been shown to participate in important functions such as cellular development (1–6). Comparison of protostome and deuterostome glycolipids shows dramatic structural differences even between species with closely related glycolipid expression. In particular, the structures of acidic glycolipids are unique. The sialylated glycosphingolipids, called gangliosides, have been widely distributed within the Echinodermata and in deuterostomes. For example, although gangliosides have not been detected in protostomia, other acidic glycosphingolipids containing uronic acid or inositol phosphate have been characterized (7–9).

Ascidians, which belong to the phylum Urochordata, are often referred to as protochordates because during the larval stage they possess chordate characteristics, most notably the tail contains a notochord and a dorsal hollow nerve cord. After a free-swimming stage, the simple tadpole-like larvae attach to a substrate and undergo metamorphosis that includes tail loss and rearrangement of the internal organs. Subsequently, in the adult form, the similarities to chordates are lost. In addition, ascidians are good model organisms for understanding vertebrates (and their development) because their cell lineage can be traced (10, 11) and a large number of genome-related data have been accumulated, including genome sequences, expressed sequence tag sequences, and gene expression patterns (12–15). However, very few studies have been directed to ascidian glycolipids, with the exception of some neutral glycolipids (16–18).

In this article, we describe the structural characterization of a novel acidic glycosphingolipid containing a uronic acid residue (UGL-1) from the ascidian Halocynthia roretzi. UGL-1 exhibits unique structural features, such as Galß1-4(Fuc{alpha}1-3)GlcAß1-1Cer. The chemical structure of UGL-1, which has a uronic acid with a fucosyl residue bond at the reducing end, is the first example to our knowledge.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and purification of acidic glycolipids
Specimens of H. roretzi were obtained from Hiroyo Fisheries, Ltd., at Miyagi in Japan, and the cellulose crust was removed, suspended in acetone, and dried naturally. The dry H. roretzi (830 g) were homogenized using a mixer and extracted twice with chloroform-methanol (2:1, v/v) for 2 h and once with chloroform-methanol (1:1, v/v) overnight at room temperature. The extracts were combined and concentrated by a rotary evaporator at 40°C. The crude lipid fraction was subjected to mild alkaline hydrolysis to eliminate glycerolipids. The resulting precipitate of alkali-stable product was applied to a DEAE-Sephadex A-25 (Amersham Pharmacia Biotech) column (column size, 3.4 cm x 22 cm; acetate form). The acidic glycosphingolipid fraction was eluted with 5 volumes of 0.05 M ammonium acetate in methanol, altered to an acetyl derivative by the addition of pyridine/anhydride at 20°C for 18 h, and then applied to a Florisil (magnesium silicate; Nacalai Tesque) column (column size, 1.8 cm x 100 cm). The acidic glycosphingolipid fraction was eluted with 3 volumes of 1,2-dichloroethane-methanol (3:1, v/v), deacetylated, and neutralized by the addition of an appropriate amount of HCl.

The acidic glycosphingolipid fraction was rechromatographed on an Iatrobeads (silica gel 6RS-8060; Mitsubishi Kagaku Iatron, Inc.) column (column size, 1.2 cm x 120 cm) with a two-step linear gradient in chloroform-methanol-water (80:20:1; 650 ml) to chloroform-methanol-water (50:50:5; 730 ml). The fractions were collected in test tubes, checked by TLC, and assessed directly by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

TLC
For TLC separation, TLC-silica gel 60 plates (E. Merck) were developed with chloroform-methanol-water (60:40:10, v/v/v) as the running solvent. The glycosphingolipids were visualized by spraying with orcinol/H2SO4 reagent for sugars, 5% H2SO4/ethanol reagent for organic substances, and ninhydrin reagent for free amino groups, followed by heating. Detection was performed with Dittmer-Lester reagent for phosphorus, azure-A reagent for sulfate, and resorcinol reagent for sialic acid.

MALDI-TOF MS
MALDI-TOF MS analysis of the purified UGL-1 was performed using an Applied Biosystems/Voyager-DE STRTM Biospectrometer with a nitrogen laser (337 nm) and an acceleration voltage of 20 kV, operating in the reflector positive and negative ion modes. Samples were analyzed in delayed extraction mode, postsource decay (PSD), using an acceleration voltage of 20 kV, a pulse delay time of 100 ns, and a grid voltage of 75%. The resolution of timed ion selector for the precursor ion was set at 80. The matrices used were 2,5-dihydroxybenzoic acid (Wako Pure Chemical Industries, Ltd.) and coumarin 120 (7-amino-4-methyl-cumarin; Sigma Chemical Co.). External mass calibration of MALDI-TOF MS was provided by the [M–H]– ion of a glucuronic acid-containing ceramide octasaccharide (at m/z 1,940.0) prepared from the fresh water bivalve Hyriopsis schlegelii (19) in the reflector negative ion mode, the [M+Na]+ ions of ceramide trisaccharide (at m/z 1,064.7) and pentasaccharide (at m/z 1,471.1) prepared from the green-bottle fly Lucilia caesar (20, 21) in the reflector positive ion mode, and the [M+Na]+ ion of angiotensin I (at m/z 1,296.7) from Sigma Chemical Co. in the PSD positive ion mode.

Sugar and ceramide analysis
UGL-1 was hydrolyzed by microwave exposure with 0.1 M NaOH/methanol for 2 min followed by 1 M HCl/methanol for 45 s (22). The fatty acid methyl esters of unnecessary reaction products, such as O-methyl ethers, were removed by three extractions in 0.5 ml of n-hexane and then analyzed using the Shimadzu GC-18A gas chromatograph with the Shimadzu HiCap-DBP5 (0.22 mm x 25 m) programmed at 2°C/min from 170°C to 230°C. The other products were neutralized with silver carbonate and then N-acetylated with pyridine-acetic anhydride in methanol at room temperature for 30 min. The N-acetylated products were trimethylsilylated with pyridine-hexamethyldisilazane-trimethylchlorosilane (9:3:1, v/v/v) at 60°C for 30 min (23). An aliquot of the residues was analyzed by gas chromatography as described above with the capillary column programmed at 2°C/min from 140°C to 230°C. Sphingoids prepared from glycolipids by methanolysis with 1 M aqueous methanolic HCl at 70°C for 18 h were converted into O-trimethylsilyl derivatives and then analyzed by GC on the same capillary column using a temperature program of 2°C/min from 210°C to 230°C.

Linkage analysis
For the determination of sugar linkages, 200 µg of each purified glycolipid was partially methylated with NaOH and methyl iodide in DMSO for 2 min (24), acetolyzed and hydrolyzed with 300 µl of HCl/water/acetic acid (0.5:1.5:8, v/v/v) in a microwave oven for 20 s, and then reduced with NaBH4 and acetylated with acetic anhydride-pyridine (1:1, v/v) at 100°C for 12 min. The obtained reaction products were analyzed by GC and GC-MS (GCMS-QP; Shimadzu) on the same capillary column programmed at 4°C/min from 140°C to 230°C for GC and from 80°C (2 min) to 180°C (20°C/min) to 240°C (4°C/min) for GC-MS.

Reduction of the carbonic acid group on uronic acid
For the reduction of uronic acid, 100 µg of UGL-1 was treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (20 mg/ml) in water and 0.01 M HCl (pH 4.7–5.0) for 2 h, which was then shifted to pH ~ 8 with ammonium hydroxide, and then stirred with 10 mg of sodium borohydride and several drops of butanol at 55°C for 18 h. The reaction was stopped with several drops of acetic acid, and then the obtained reduced sample was dialyzed and concentrated.

Partial acid hydrolysis
To remove a fucose residue, partial acid hydrolysis was carried out by treatment of the reduced sample (200 µg) with 0.1 M HCl at 100°C for 30 min. The degradation products were isolated by Iatrobeads column chromatography. The obtained reaction products were analyzed by GC and GC-MS under the same conditions described above.

1H-NMR spectroscopy
The purified glycolipid was dissolved in 0.60 ml of [2H6]DMSO + 0.05% (v/v) tetramethylsilane (Wako Pure Chemical Industries, Ltd.) containing 2% 2H2O (Nacalai Tesque, Inc.), and chemical shift was referenced to trimethylsilyl ({delta}H 0.00 ppm) as the internal standard. 1H-NMR spectra of purified UGL-1 (1.0 mg) was recorded on a 400 MHz spectrometer (JEOL, Ltd.; A-400) in the NMR tube (~5 mm; Kusano Science Co.) at an operating temperature of 60°C. The obtained NMR spectra data were analyzed with 1D, 2D Application software (http://nakamura-2.ees.hokudai.ac.jp/nmr/nmr.html).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and TLC analysis of acidic glycolipids from H. roretzi
Total acidic glycosphingolipid of the ascidian H. roretzi as obtained by DEAE-Sephadex A-25 and Florisil column chromatography comprised a mixture of several major compounds (Fig. 1 ). The one species was separated by Iatrobeads column chromatography, yielding a uronic acid-containing glycosphingolipid, called UGL-1. TLC analysis of UGL-1 showed positive reactions with orcinol/H2SO4 reagent (Fig. 1) and negative reactions with Dittmer-Lester, ninhydrin, azure-A, and resorcinol reagents (data not shown).


Figure 1
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  		Fig. 1. TLC of purified uronic acid-containing glycosphingolipid-1 (UGL-1) from H. roretzi. TLC was performed using chloroform-methanol-water (60:40:10, v/v/v) and visualized by orcinol/H2SO4 staining. Lane T, whole acidic glycolipids from H. roretzi; lane 1, UGL-1 obtained from H. roretzi.

 
Ceramide analysis
Characterization of the ceramide moiety was performed by CG and GC-MS analyses. Glycolipids were subjected to the same procedures of fatty acid determination (see Materials and Methods). However, for sphingoid analysis, methanolysis of UGL-1 afforded only small amounts of sphingoids, most likely owing to the attachment of glucuronic acid, which may interfere with acidic cleavage. When UGL-1 was reduced by sodium borohydride before methanolysis, three long-chain bases were detected and analyzed by GC and GC-MS, as with acetylated derivatives.

The main fatty acids of UGL-1 were C16:0 and C18:0; the minor fatty acid component h24:0 was also present. The sphingoids were composed entirely of C16-, C17-, and C18-phytosphingosines in UGL-1 (Table 1 ).


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  		TABLE 1. Ceramide moiety of UGL-1

 
Sugar analysis of UGL-1
To determine the sugar components of UGL-1, compositional analysis of UGL-1 was first carried out by the methanolysis method using GC. The composition of UGL-1 was determined to be fucose-galactose in a ratio of 1:1 (Table 2 ). Although UGL-1 was fractionated into the acidic fraction by ion-exchange column chromatography, UGL-1 showed negative reactions with Dittmer-Lester, azure-A, and resorcinol reagents on TLC. From the results of sugar, TLC (shown above), and PSD fragments from MALDI-TOF MS analysis (shown below), the acidic carbohydrate of UGL-1 was estimated to contain a uronic acid residue.


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  		TABLE 2. Sugar components of UGL-1

 
After UGL-1 was reduced with sodium borohydride (see Materials and Methods), sugar compositional analysis was carried out by the methanolysis method using GC. When sugar composition compared reduced UGL-1 with intact UGL-1, the reduced UGL-1 showed an increased glucose residue. The sugar composition of reduced UGL-1 was found to be fucose-galactose-glucose in a ratio of 1:1:1 (Table 2). Therefore, the sugar composition of UGL-1 was identified as fucose-galactose-glucuronic acid in a ratio of 1:1:1.

MALDI-TOF MS analysis
UGL-1 was characterized by several molecular masses in reflector negative and positive ion modes using MALDI-TOF MS (Fig. 2 , Table 3 ). The two highest peaks in each mode were found at m/z 1,152.7 and 1,154.7 in negative ion mode (Fig. 2A) and at m/z 1,176.5 and 1,178.6 in positive ion mode (Fig. 2B). The differences in m/z values between positive and negative ion modes correspond to molecular masses of 23.8 and 23.9 Da, which are attributable to the molecular mass of Na plus H.


Figure 2
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  		Fig. 2. Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of H. roretzi UGL-1. UGL-1 obtained after Iatrobeads column separation was monitored as [M–H]–, [M+Na–2H]–, and [M+Na–2H]– ions in the reflector negative ion mode (A) and as [M+Na]+, [M–H+2Na]+, and [M–2H+3Na]+ ions in the reflector negative ion mode (B) by MALDI-TOF MS with the coumarin 120 matrix in the positive ion mode. These results are summarized in Table 3.

 

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  		TABLE 3. Summary of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry analysis of UGL-1

 
The putative structure of the purified UGL-1 was confirmed by MALDI-TOF MS analysis in the negative ion mode (Fig. 2A). Peaks at m/z 1,126.6, 1,140.7, 1,152.7, 1,154.7, and 1,168.7 were detected as major peaks, in addition to those at m/z 1,112.6, 1,124.6, 1,138.6, 1,166.7, 1,180.7, and 1,182.7. Small peaks at m/z 1,108.6, 1,110.6, 1,122.6, 1,136.6, 1,146.6, 1,150.6, 1,160.6, and 1,174.6 were also detected from UGL-1. Based on the difference of ceramide composition in UGL-1, these major peaks formed six islands or three groups. For example, in the case of peaks at m/z 1,112.6, 1,126.6, 1,140.7, 1,154.7, 1,168.7, and 1,182.7, the difference of each molecular mass is ~14 Da. This value is similar to the molecular mass of CH2. Furthermore, it was similar in the case of peaks at m/z 1,110.6, 1,124.6, 1,138.6, 1,152.7, 1,166.7, and 1,180.7.

Based on consideration of molecular weight comprising the sugar component and the ceramide moiety, peaks detected as major peaks were determined with [M–H]– and [M+2Na–3H]– ions. On the other hand, small peaks were determined with [M+Na–2H]– and [M–H]– ions. These ionizations were identified by MALDI-TOF MS analysis using a saturated solution of 2,3-dihydroxybenzoic acid in 40 mM aqueous LiCl as the matrix (25). The putative structure of the purified UGL-1 was also identified by MALDI-TOF MS analysis in the positive ion mode (Fig. 2B). The summary of MALDI-TOF MS analysis is shown in Table 3.

The MALDI-TOF MS analysis of PSD fragments was carried out for several main peaks of UGL-1. The PSD fragment analysis of precursor mass at m/z 1,176.5 is shown in Fig. 3 . In the case of UGL-1, from the difference of ceramide composition, the former was characterized by [M+Na]+ molecular masses of m/z 1,163.3, 1,177.8, and 1,192.3 (Fig. 3). Fragment ions of [ceramide+Na]+ at m/z 677.5, 692.6, and 707.7, which were derived from the ceramide moiety, showed the same heterogeneous pattern. The fragment ion at m/z 507.0 of [Hex(dHex)HexA–H2O+Na]+, which was derived from the sugar component, was included with UGL-1. These data also showed that UGL-1 had trisaccharide carbohydrate moieties with different ceramide moieties.


Figure 3
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  		Fig. 3. Postsource decay fragments from MALDI-TOF MS analysis of H. roretzi UGL-1. UGL-1 was monitored with 2,5-dihydroxybenzoic acid in the postsource decay positive ion mode. The precursor mass was set at m/z 1,176.5. The resolution of timed ion selector for the precursor ion was set at 80, which would allow a mass window of ~30 Da.

 
Linkage analysis
To determine sugar linkages, UGL-1 was prepared as partially methylated alditol acetates for analysis of substitution positions by GC and GC-MS (Table 4 , Fig. 4 ). Methylation analysis of UGL-1 showed the presence of 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol (1Fuc) and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol (1Gal) (Fig. 4A). Here, as with methanolysis analysis, the glucuronic acid residue was not detected by partial methylated alditol acetate analysis.


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  		TABLE 4. Methylation analysis of UGL-1

 

Figure 4
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  		Fig. 4. Linkage analysis of H. roretzi UGL-1. Intact UGL-1 (A), reduced UGL-1 (B), and reduced and defucosed UGL-1 (C) were monitored by GC. 1Fuc, 1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol; 1Gal, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol; 1,3,4Glc, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl-N-acetylglucitol; 1,4Glc, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol.

 
UGL-1 was reduced by sodium borohydride and prepared as partially methylated alditol acetates for analysis of substitution positions by GC and GC-MS (Fig. 4B). As a result, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl-N-acetylglucitol (1,3,4Glc) was detected in reduced UGL-1. Furthermore, to determine the linkage position of the two reducing terminal sugar residues (galactose and fucose) to glucuronic acid, methylation analysis was performed with reduced UGL-1 after defucose processing by partial acid hydrolysis. Methylation analysis of reduced UGL-1 showed the presence of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol (1Gal) and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol (1,4Glc) (Fig. 4C).

Anomeric configuration analysis
To determine the anomeric configuration of the sugar residues, UGL-1 was subjected to 1H-NMR spectroscopy (Fig. 5 ). In the anomeric region of the spectrum for UGL-1, the following anomeric proton resonances were observed: at 4.21 ppm (J1,2 7.5 Hz), demonstrating ß-glucuronic acid residue; at 4.30 ppm (J1,2 6.4 Hz), demonstrating ß-galactose residue; and at 5.15 ppm (J1,2 2.1 Hz), demonstrating {alpha}-fucose residue. Furthermore, the H5 proton resonance of the {alpha}-fucose residue was observed at 4.55 ppm (J1,2 6.3 Hz).


Figure 5
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  		Fig. 5. 1H-NMR spectrum of H. roretzi UGL-1. Anomeric protein regions of UGL-1 are indicated.

 
These results suggested that the chemical structure of UGL-1 was Galß1-4(Fuc{alpha}1-3)GlcAß1-1Cer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
From the ascidian H. roretzi, we propose the chemical structure of the novel UGL-1, characterized as Galß1-4(Fuc{alpha}1-3)GlcAß1-1Cer (Fig. 6 ).


Figure 6
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  		Fig. 6. Chemical structure of UGL-1. R1, sphingoid; R2, fatty acid. For the combination of R1 and R2, see Table 3.

 
Glycosphingolipids are widely found as cellular constituents of marine and terrestrial animals, and they are believed to possess several interesting biological activities, including modulation of growth and regulation of differentiation. It is also commonly acknowledged that this bioactivity depends essentially on the nature of the functionalized structure of the molecule, including the carbohydrate moiety. In invertebrates, glucuronic acid-containing glycosphingolipids have been characterized from flies, Lucilia caesar (7) and Drosophila melanogaster (26), and in arthropods located at the top of the protostomia. From the Echinodermata, located downstream of the Urochordata, invertebrate deuterostomes, gangliosides, and sulfatide have been characterized (27–31), and characterization of glucuronic acid-containing glycosphingolipids has not yet been reported. On the other hand, in vertebrates, glucuronic acid-containing glycosphingolipids and sulfated glucuronyl glycosphingolipids have been characterized from the HNK-1 epitope in humans and mice (32, 33). HNK-1 has not yet been identified from ascidians, although recently it was identified from the lancelet in the Urochordata, similar to ascidians, and from the sea urchin in the Echinodermata (34). It is very interesting in terms of evolution and function that the novel structures of glucuronic acid-containing glycosphingolipids were characterized in invertebrate ascidians, which is nearest to the vertebrates.

Acidic glycosphingolipids, gangliosides, and sulfated glucuronic acid-containing glycosphingolipids are located in the outer leaflet of the plasma membrane, being expressed at the surface of various cell types and widely distributed in vertebrates, especially in the nervous system of mammals (35, 36). As ascidians are classified as invertebrates, which are close to vertebrates, we think that the analysis and comparison of the cellular localization of UGL-1 represent a shortcut for understanding the function of glycosphingolipids.

The presence of a neutral glycosphingolipid (sulcaceramide) has been reported in the ascidian Microcosmus sulcata (18). The sulcaceramide is a structural analog of UGL-1 that replaced glucuronic acid with glucose, Galß1-4(Fuc{alpha}1-3)Glcß1-1Cer. The biosynthetic pathways of ascidian glycosphingolipids are still little known. The structural characterization of the ascidian glycosphingolipids leads us to propose several biosynthetic pathways involving the addition of glucose or glucuronic acid to ceramide or the conversion of the 6-position group on glucose and glucuronic acid. The significance of such a unique acid glycolipid on the cell surface remains to be elucidated. However, glucuronic acid-containing glycosphingolipids have been reported from the bacterium Sphingomonas paucimobilis (37, 38). Glycosphingolipids in S. paucimobilis have been characterized as GalA{alpha}-Cer and Man{alpha}1-2Gal{alpha}1-5GlcN{alpha}1-4GlcA{alpha}1-1Cer and contained no lipopolysaccharide-like molecules. These findings on the cellular lipids of S. paucimobilis prompted the investigation of the cell surface structure of this Gram-negative bacterium and the physiological roles of the glycosphingolipids (39).

Thus, the finding in H. roretzi of a novel acidic glycosphingolipid, characterized by a unique sugar portion, suggests that glycosphingolipids also play an essential biological role in ascidians. In the near future, we expect an increasing amount of activity in this field on account of the recent improvements in spectroscopic and isolation techniques; it is now possible to perform conclusive structural determination studies on very small quantities of pure compounds, like those usually obtained from the extracts of most ascidians.


   ACKNOWLEDGMENTS
 
This work was supported in part by a Grant-in-Aid for Young Scientists B and a High-Tech Research Center Project for Private Universities matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to M.I.

Manuscript received July 10, 2006 and in revised form September 22, 2006 and in re-revised form September 28, 2006 and in re-re-revised form October 3, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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