| Glycobiology | Pages |
Localization of Lewisx, sialyl-Lewisx and [alpha]-galactosyl epitopes on glycosphingolipids in lens tissues
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Localization of Lewisx, sialyl-Lewisx and [alpha]-galactosyl epitopes on glycosphingolipids in lens tissues
Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisx and sialyl-Lewisx epitopes and the absence of [alpha]-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisx containing neolactotetraosylceramide, respectively, which were found in primate lens, together with the [alpha]-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and [alpha]-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the [alpha]-galactosyl epitope are evolutionarily replaced by the Lewisx and sialyl-Lewisx epitopes in Old World monkeys and humans. Key words: ganglioside/glycosphingolipid/immunohistochemistry/lens/sialyl-Lewisx
Introduction
Lens tissues in vertebrates are composed of multiple layers of fiber cells and a monolayer of epithelial cells. The fiber cells accumulate throughout life, traveling from the equatorial region of epithelia to the lens nucleus, a process accompanying differentiation and maturation (Bloemendal, 1977). Humans and the diurnal primates have the flattest lenses among mammals (ratio of equatorial to axial diameter; flatness index, 2.75), with the exception of the spiny anteater (Duke-Elder, 1958; de Jong, 1981). In contrast, other mammals, including some nocturnal prosimian primates, have perfectly spherical or very nearly spherical lenses.
Our previous studies revealed that mammalian lenses contained several glycosphingolipids (GSLs) with various sugar chain backbones, including ganglio-, neolacto-, globo-, and isoglobo-series sugar chains, and that the [alpha]-galactosyl (Gal[alpha]1-3Gal-R), Lewisx (Lex, Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc-R) and sialyl-Lex (NeuAc[alpha]2-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc-R) epitopes were formed on the nonreducing terminal of neolactotetraosylceramide Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1ceramide (nLc4) (Ogiso et al., 1992, 1993, 1994a, 1995a,b). The [alpha]-galactosyl epitope is evolutionarily conserved in many types of cells of nonprimate mammals, prosimians, and New World monkeys, but not in those of Old World monkeys, apes, or humans (Galili et al., 1987a, 1988; Hendricks et al., 1990). In the embryonic rat, the expression of GSLs and glycoproteins with the [alpha]-galactosyl epitope was closely involved in the elongation of primary lens fibers (Ogiso et al., 1997).
A comparative study of lens GSLs confirmed the expression of the [alpha]-galactosyl epitope in several neutral and acidic GSLs in several nonprimate mammalian lenses (Ogiso et al., 1994a). Lex GSLs were restricted to Old World monkeys, including rhesus monkeys and Japanese monkeys, and humans, whereas sialyl-Lex GSLs were detected in rats, pigs, monkeys, and humans. However, the immunohistochemical detection of lens GSLs, particularly Lex- and sialyl-Lex-containing GSLs, has not been possible because of technical difficulties, for example, the extreme hardness and density of mammalian lens, the occurrence of folds and cracks in lens sections, and the detachment and substantial background staining of cryosections in the course of immunohistochemistry.
In this study, the localization of lens GSLs was immunologically examined in dog, pig, and Japanese monkey lenses. Ganglio- and globo-series GSLs were principally distributed from lens epithelial cells to the cortical and nuclear fibers, and neolacto-series GSLs were localized in differentiated fibers of the inner cortical and nuclear regions. The evolution-related expression of Lex and sialyl-Lex epitopes in place of the [alpha]-galactosyl epitope is also discussed in relation to the characteristics of morphology and accommodation in primate lenses.
Results
Distribution of neutral GSLs and gangliosides in dog, pig and Japanese monkey lenses
To examine the distribution of lens GSLs, dog, pig, and Japanese monkey lenses were dissected into three parts: anterior lens epithelia with capsule, and cortical and nuclear regions. The composition of neutral GSLs and gangliosides in each part was analyzed by high-performance thin-layer chromatography (HP-TLC; Figure 1). Aliquots of the GSL fractions, corresponding to approximately one lens for lens epithelia (lane 1), and one-third of one lens for the cortical and nuclear fibers (lanes 2 and 3), were applied to examine species-specific differences in their composition, content and distribution. Dog lens contained more neutral GSLs than gangliosides in the cortical and nuclear fibers, but pig lens expressed lower amounts of neutral GSLs and gangliosides in lens fibers, compared with in lens epithelia. In contrast, monkey lens contained higher amounts of neutral GSLs and gangliosides in the cortical and nuclear fibers. In lens epithelial cells, simple GSLs such as CMH, CDH, and GM3 were mostly observed in all the lenses. The content of neutral GSLs and gangliosides in the lens epithelia of dog and rhesus monkey was much lower than in lens fibers (Ogiso et al., 1994b).
Compositional changes in lens GSLs were immunologically evaluated by TLC-immunostaining. GM3 was widely expressed in dog, pig and Japanese monkey lenses (data not shown). The expression of GM1 was observed from lens epithelia toward the lens nucleus in monkey lens (Figure 2A), but GM1 was not recognized in dog and pig lenses as described previously (Ogiso et al., 1994a). In addition, no GD1b was detected in any of the lenses by polyclonal anti-GM1 antiserum, which is reported to cross-react with GD1b (62.5%; data from Iatron). Although GD3 was widely detected in all the lenses, GD3 appeared to be enriched in the cortical and nuclear fibers, except for the epithelial fraction of dog lens (Figure 2B). Two major bands were observed, in addition to a nonspecific band in dog lens, because monoclonal antibody (MAb) R24 reacts with GD3 lactone I (upper band) as well (Ando et al., 1989). On the other hand, CSLEX-1-positive sialyl-Lex gangliosides, which migrated below GD1a on TLC sheets (Ogiso et al., 1994a), were distributed in the cortical and nuclear fibers of pig and monkey lenses (Figure 2C).
Figure
Figure
Among neutral GSLs, globotriaosylceramide (Gb3) was stained intensively in lens fibers of dog and monkey lenses (Figure 3A). Griffonia (Bandeiraea) simplicifolia-IB4 (GS-I) lectin is reported to react with the nonreducing terminal of [alpha]-linked galactosyl residues, particularly the Gal[alpha]1-3Gal[beta]1-4GlcNAc-R epitope (Eckhardt and Goldstein, 1983). GSLs bearing the [alpha]-galactosyl epitope were detected by GS-I lectin in the cortical and nuclear fibers of dog and pig lenses (Figure 3B).
Figure
Immunohistochemical study of ganglio-, globo- and neolacto-series GSLs in Japanese monkey lens
To locate ganglio-series gangliosides, three MAbs to GM3, GM1, and GD3 were incubated with frozen sections (Figure 4). GM3 was widely distributed from a monolayer of anterior epithelial cells toward the lens nucleus in monkey lens. The expression of GM1 was also observed from lens epithelia toward the lens nucleus. GD3 was practically undetectable, probably due to low concentration of GD3, while GD3 was detected in the cortical and nuclear fibers by TLC-immunostaining (Figure 2B). Experiments with control ascites fluid showed no immunoreaction (data not shown).
Figure The distribution of Lex and sialyl-Lex epitopes was examined using MAbs Y12 and CSLEX-1, respectively (Figure 5). In contrast to anti-ganglioside antibodies, anti-Lex and anti-sialyl-Lex antibodies exclusively bound to the inner cortical fibers, in which a small number of cell nuclei could be observed (Figure 4D), and nuclear fibers. Lex epitope was also detected in the equatorial region of the lens and in the transition zone (Figure 5C). In 3-year-old monkey lens, however, immunoreaction to anti-Lex and anti-sialyl-Lex antibodies was restricted to the inner cortical fibers toward the lens nucleus (data not shown). Another neolacto-series GSL, nLc4, a precursor GSL of III3FucnLc4 and IV3NeuAcIII3FucnLc4, was slightly detected in the inner cortical and nuclear fibers (Figure 6A), in accordance with the distribution of Lex and sialyl-Lex epitopes (Figure 5). Binding of anti-nLc4 antibody was also observed in the equatorial region of the lens.
Figure
Figure On the other hand, a globo-series GSL, Gb3, was detected from the superficial cortical fibers to the nuclear region (Figure 6B). It was noted that intense immunoreaction was observed in the superficial cortical fibers beneath anterior lens epithelia and posterior subcapsular region.
Immunohistochemical study of globo- and neolacto-series GSLs in dog and pig lenses
Among nonprimate mammalian lenses, dog and pig lenses contained Gb3 and sialyl-Lex gangliosides, respectively (Ogiso et al., 1994a). The distribution of Gb3, nLc4, sialyl-Lex epitope, and [alpha]-galactosyl epitope was examined in dog and pig lenses to analyze their physiological roles in lens tissues. Immunoreaction to nLc4 and [alpha]-galactosyl epitope, probably IV3Gal[alpha]nLc4, was restricted to the lens fibers in dog and pig lenses (Figures 7, 8). Intense staining was recognized from the inner cortical fibers toward to the lens nucleus. However, anti-sialyl-Lex antibody appeared to be positive in the nuclear region of pig lens (Figure 7E,F). Anti-Gb3 antibody intensely bound to anterior lens epithelia of dog lens and weak immunoreaction was observed in the cortical and nuclear fibers (Figure 9A,B).
Figure
Figure
Figure
In addition, cell nuclei could be recognized even in the inner cortical fibers, where the cell-to-cell attachment appeared to change toward the lens nucleus (Figure 9C).
Immunoelectron microscopic study of Lex and sialyl-Lex epitopes in monkey lens
To further examine the localization of III3FucnLc4 and IV3NeuAcIII3FucnLc4 the ultrathin sections of monkey lenses were prepared as described in Materials and methods. The surfaces of elongated fibers, hexagonal in cross-section, have numerous ball-and-socket joints at their lateral edges. In the adult rhesus monkey the diameter of the ball is about 1 µm and the depth of the socket about 1.5 µm (Kuwabara, 1975). The ultrastructure of lens fibers was well preserved and apparently no cell organelles were found in the inner cortical fibers. Lex and sialyl-Lex epitopes were clearly detected as gold particles on cell membranes (Figure 10). Intense immunoreaction to both epitopes was observed on cell membranes in the inner cortical fibers, where both anti-Lex and anti-sialyl-Lex antibodies strongly bound as mentioned above (Figure 5). On the other hand, a slight number of gold particles were seen on cell membranes of superficial fibers (Figure 10A,C), but not on cell membranes of epithelial cells (data not shown).
Figure
Discussion
In epithelial cells attached to capsule of rhesus monkey and dog lenses, simple gangliosides with a Gal[beta]1-4Glc[beta]1-1ceramide core, GM3 and GD3, were principally detected, differing from those in lens fibers (Ogiso et al., 1994b). Gangliosides from monolayer cultures of epithelial cells were predominantly composed of GM3 and GD3, but no GSLs bearing Lex, sialyl-Lex, or [alpha]-galactosyl epitopes were detected. The current study provided additional evidence for the absence of Lex, sialyl-Lex, and [alpha]-galactosyl epitopes in epithelial cells of some mammalian lenses. Anti-sialyl-Lex-positive gangliosides were restricted in the cortical and nuclear fibers of pig and Japanese monkey lenses, and [alpha]-galactosyl GSLs were similarly observed in fibers of dog and pig lenses (Figures 2, 3). These results strongly support the possibility that the expression of several GSLs bearing Lex, sialyl-Lex, and [alpha]-galactosyl epitopes might be associated with the differentiation of epithelial cells to fibers in mammalian lens.
Our previous immunohistochemical study revealed distribution profiles of lens GSLs in non-cataractous Wistar rat (Ogiso et al., 1995c). Several ganglio-series gangliosides including GM3, GD2, GD3, GM1, GD1a, and GD1b were expressed in anterior epithelial cells and entered the cortex, accompanying the differentiation of epithelial cells to elongating fibers. Although neolacto-series GSLs (nLc4 and IV3Gal[alpha]nLc4) were restricted to fibers of rat lens, the localization profile was slightly different from those in dog, pig, and Japanese monkey lenses.
The current immunohistochemical study also suggested that several GSLs play important roles in the multiple layering of fibers, which become progressively more internalized from the equatorial region toward the central region (Bloemendal, 1977). Most neolacto-series GSLs bearing Lex, sialyl-Lex, and [alpha]-galactosyl epitopes were localized in the inner cortical and nuclear fibers of dog, pig and monkey lenses, but not in the superficial cortical fibers beneath the epithelia (Figures 5, 7, 8). Interestingly, no Lex or sialyl-Lex epitopes were identified on lens glycoproteins from several mammals (Ogiso et al., 1994a). MAb Gal-13 specifically binds to GSLs with the [alpha]-galactosyl epitope, but not glycoproteins (Galili et al., 1987b). In addition, the distribution patterns strongly suggest that the synthesis of sialyl-Lex ganglioside from Lex GSL may occur in the inner cortical fibers of monkey lens (Ogiso et al., 1996).
Immunoelectron microscopic analyses confirmed the involvement of cell surface GSLs containing Lex and sialyl-Lex epitopes in the cell adhesion between mature elongated fibers. In primate lens, ball-and-socket joints first appear in the bow region and in the elongated fibers of the cortical regions. There is a decrease in the number of ball-and-socket joints from the cortex to the nucleus, coincident with an increase in tongue-and-groove junctions (Kuwabara, 1975). Although the inner cortical fibers possessed many ball-and-socket joints along their length, Lex and sialyl-Lex epitopes were detected in the intercellular space of both ball-and-socket joints and long flat surfaces (Figure 10).
The [alpha]-galactosyl epitope was similarly detected in the inner cortical and nuclear fibers of dog and pig lenses (Figures 7, 8). In pig lens, sialyl-Lex gangliosides were, however, restricted to the lens nucleus, which originates from posterior lens epithelial cells during the fetal stage (Dickson and Crock, 1975; Bloemendal, 1977). No Lex epitope was detectable in lens GSLs from nonprimate mammals (Ogiso et al., 1994a). Thus, the expression of the [alpha]-galactosyl epitope appeared to be crucial in the progression of terminal differentiation and maturation of lens fibers, even if the sialyl-Lex epitope coexisted in nonprimate lens. The involvement of the [alpha]-galactosyl epitope in GSLs and glycoproteins in lens fiber development during the embryonic stages in rat lens is reported (Ogiso et al., 1997).
Sialyl-Lex tetrasaccharide has been demonstrated to be a natural ligand for selectins (Polley et al., 1991; Asa et al., 1992; Laskey, 1992; Erbe et al., 1993). However, we could not detected selectins in lens proteins by immunoblotting analysis using some MAbs. Recently, we successfully identified the lectin domain of putative selectin(s) by the sequencing of mRNA from monkey lens tissues (unpublished observations). Since the [alpha]-galactosyl epitope has not been reported to be involved in cell-to-cell interactions, the cell adhesion mechanism, which is mediated through interaction of the sialyl-Lex epitope and selectin, may partly account for the evolutionary alteration in the shape of primate lens, coupled with dynamic accommodation.
On the other hand, a globo-series GSL, Gb3, is well known as Pk antigen (CD 77), one of the blood P-related antigens of human red blood cells, and also as a tumor-associated antigen of Burkitt's lymphoma (Nudelman et al., 1983). Although Gb3 was only detected in dog and primate lenses among several mammalian lenses (Ogiso et al., 1994a), Gb3 was widely distributed in lens epithelia and the cortical and nuclear fibers of dog and monkey lenses (Figures 6B, 9). In monkey lens, intense immunoreaction was seen in posterior subcapsular region, where vitreous humor communicates through posterior lens capsule. Thus, although the expression of Gb3 may be partly associated with the entry of epithelial cells into multiple layers of fibers, the physiological significance of Gb3 in lens tissues remains unknown.
Among several mammalian lenses, primate lenses contained a-series gangliosides, including GM1 and GD1a, but not complex b-series gangliosides (Ogiso et al., 1994a). In contrast, only GM3 and GD3 could be detected in dog, pig, and cow lenses. In monkey lens, GM3 and GM1 were immunostained more intensely in the lens epithelial cells than in fibers. GD3, which is considered a minor ganglioside (Figure 1), was not detected through immunohistochemical study.
In conclusion, neolacto-series GSLs are considered to be differentiation-associated antigens of lens fibers in mammals. The physiological roles of the [alpha]-galactosyl epitope in the differentiation of lens fibers is replaced by the Lex and sialyl-Lex epitopes in Old World monkeys, apes, and humans. The alternative expression of the Lex and sialyl-Lex epitopes is of special interest in lens tissues in terms of exploring the development of primate lens morphology, as mentioned in Introduction. Immunohistochemical findings of neolacto-series GSLs indicate that the differentiation and maturation of epithelial cells to lens fibers is regulated by at least three steps, as the cortical fibers pass from the equator (bow region) and elongate to the anterior and posterior poles: entry to lens fibers; expression of Lex, sialyl-Lex, and [alpha]-galactosyl epitopes; and final maturation accompanying denucleation and the breakdown of cell organelles.
Accommodation, the capacity of the optical system to adjust to near and distant vision, is not operative in all vertebrates, since a small lens with a short focal length has a greater depth of focus than a large lens (Duke-Elder, 1958; de Jong, 1981). Dynamic accommodation is acquired through movement of the lens as a whole or deformation of the lens. In the lower mammals, accommodative activity is lacking because the ciliary muscle is vestigial if present, except in squirrels. Accommodation by deformation of the lens and the consequent change in refractivity is found in some mammals. Primates have the most effective range of accommodation among mammals, but the range of accommodation decreases with age. Although it remains an open question whether soft, pliable lenses are characterized by particular protein properties, such as crystallins (de Jong, 1981), glycobiological studies on the arrangement and interaction of membrane GSLs may throw light on the evolutionary development of the structure and accommodation of mammalian lenses.
Materials and methods
Partial purification of neutral GSLs and gangliosides from lenses
Dog lenses were obtained from the Experimental Animal Center of Toho University School of Medicine, and pig lenses from a local slaughterhouse. Noncataractous lenses from Japanese monkey (Macaca fuscata) were supplied under a cooperative program of the Primate Research Institute, Kyoto University, Inuyama. Lenses from dog, pig, and Japanese monkey were separated under a binocular microscope into the anterior capsule with lens epithelial cells, and cortical and nuclear regions.
Neutral GSLs and gangliosides were partially purified from lens portions as described previously (Ogiso et al., 1994a). Briefly, neutral and acidic GSLs were separated from the total lipid fraction by a column of DEAE-Sephadex A-25 (acetate form, LKB-Pharmacia Biotechnology, Uppsala, Sweden). Neutral GSLs were partially purified by column chromatography on an Iatrobeads 6RS-8060 (Iatron, Tokyo, Japan), and Folch's partition (Ogiso et al., 1992). Gangliosides were desalted by being applied to a Sep-Pak C18 cartridge (Waters Associates, Milford, MA). Neutral GSLs were separated on precoated silica gel HP-TLC plates (5556, Merck, Darmstadt, Germany) or plastic TLC sheets (Polygram Sil-G, Macherey-Nagel, Düren, Germany) in the developing solvent of chloroform/methanol/water (65:25:4, v/v/v), and detected with orcinol spray. Gangliosides were developed in chloroform/methanol/0.25% CaCl2 (55:45:10, v/v/v), and detected with resorcinol spray. Neutral GSL and ganglioside standards were obtained from Iatron, Tokyo.
TLC-immunostaining of lens GSLs
TLC-immunostaining by an indirect method using a horseradish peroxidase-linked second antibody was performed as described previously (Ogiso et al., 1992). The primary antibodies were monoclonal anti-GM3 (M2590) antibody (1:50, IgM, Meiji, Tokyo) (Hirabayashi et al., 1985), polyclonal anti-GM1 antiserum (1:200, Iatron), monoclonal anti-GD3 (R24) antibody (1:20, IgM, Wako Pure Chemicals, Osaka) (Pukel et al., 1982), monoclonal anti-Gb3 (1A4) antibody (1:20, IgM, a kind gift of Dr. Reiji Kannagi, Aichi Cancer Center), monoclonal anti-nLc4 ([alpha]FGF-H11) antibody (1:100, IgM, a kind gift of Dr. Takao Taki, Tokyo Medical and Dental University) (Myoga et al., 1988), and monoclonal anti-sialyl-Lex (CSLEX-1) antibody (1:200, IgM, UCLA Tissue Typing Laboratory, Los Angeles) (Fukushima et al., 1984). The second peroxidase-conjugated antibodies (1:500, affinity-purified, Cappel, West Chester, PA) to the primary antibodies were overlaid, and peroxidase activity was visualized using a Konica immunostaining HRP kit IS-50B (Konica, Tokyo).
For the detection of [alpha]-galactosyl GSLs on TLC sheets, biotinylated GS-I lectin (25 µg/ml, Vector Lab. Inc., Burlingame, CA) and the Vector ABC reagent were used according to Galili et al. (1987).
Immunohistochemical study of GSLs in lens tissues
The localization of lens GSLs was immunohistochemically examined using frozen sections as described previously (Ogiso et al., 1995c). Lenses from dog, pig, and 4-month-old Japanese monkey were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C overnight, and washed several times in cold phosphate-buffered saline (PBS). Samples were placed in 10, 15, and 20% sucrose in PBS and embedded in Tissue-Tek O.C.T. Compound (Miles Inc., Elkhart, IN). Six-micrometer sections were blocked with 10% rabbit serum and incubated with monoclonal anti-ganglioside antibodies to GM3 (M2590, 1:50, IgM), GM1 (MAB 306, 1:20, IgG, Chemicon, Temecula, CA), GD3 (R24, 1:20, IgM), and sialyl-Lex (CSLEX-1, 1:100), and monoclonal anti-neutral GSLs to nLc4 ([alpha]FGF-H11, 1:100, IgM), Lex (Y12, 1:20, IgM, a kind gift of Dr. Reiji Kannagi, Aichi Cancer Center) and IV3Gal[alpha]nLc4 (Gal-13, 1:1, IgG1, a kind gift of Dr. Uri Galili, the Medical College of Pennsylvania) (Galili et al., 1987b) at 4°C overnight. Immunoreaction was detected using the Nichirei alkaline phosphatase-conjugated SAB kit (Nichirei, Tokyo) and Vector substrate kit IV (Vector Lab., CA). Experiments with control ascites fluid showed no immunoreaction. Parallel sections were stained with hematoxylin solution to locate the nuclei of epithelial cells and the elongating fibers.
Immunoelectron microscopy of lens GSLs
Lenses from 2-year-old Japanese monkey were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) at 4°C overnight and separated into several pieces in the fixative. Small segments were washed several times with cacodylate buffer and rapidly frozen by immersing them into liquid propane cooled by liquid nitrogen. Freeze-substitution was performed at -85°C in ethanol for 72 h, followed by raising the temperature to -20°C. Infiltration of LR-White (The London Resin Co., London) to the segments was carried out in the graded series of LR-White/ethanol mixtures (1:1 for 1 h and 2:1 for 1 h) and finally in LR-White at -20°C overnight. The samples were polymerized in gelatin capsules at 50°C for 24 h.
Ultrathin sections were cut with a LKB Ultrotome V ultramicrotome and placed on Formvar-coated nickel grids. The sections were blocked with 1% bovine serum albumin in PBS and incubated with anti-sialyl-Lex antibody (CSLEX-1, 1:50) or anti-Lex antibody (Y12, 1:10) at 4°C overnight. After washing with PBS, the sections were incubated with 15 nm-diameter colloidal gold-conjugated antimouse IgG + IgM antibody (1:30, E-Y Laboratories, San Mateo, CA) for 4 h at room temperature. The samples were postfixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) for 10 min and poststained with uranyl acetate and lead citrate. The specimens were examined and photographed using a JOEL 1200 EX electron microscope at 80 kV.
Acknowledgments
We thank Dr. Uri Galili of the Medical College of Pennsylvania, Dr. Takao Taki of Tokyo Medical and Dental University, and Dr. Reiji Kannagi of Aichi Cancer Center for their generous gifts of monoclonal antibodies. We also thank Dr. Takashi Kageyama of the Primate Research Institute, Kyoto University, for the generous supply of Japanese monkey lenses under the cooperative research program, and Dr. Nobuyuki Saito of Okura National Hospital for his technical assistance.
Abbreviations
GS-I, Griffonia (Bandeiraea) simplicifolia-IB4; GSL, glycosphingolipid; HP-TLC, high-performance thin-layer chromatography; Lex, Lewisx; MAb, monoclonal antibody; PBS, phosphate-buffered saline. Neutral GSLs are abbreviated according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1977), but the suffix OseCer is omitted. Ganglio-series gangliosides are abbreviated according to Svennerholm (1964).
References
2To whom correspondence should be addressed at: 8-9-303 Higashimaita-machi, Minami-ku, Yokohama 232, Japan
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