Immunohistochemical Characterization of the Distribution of Galectin-4 in Porcine Small Intestine
Veterinary Science Department (MAW,MBH,EAN,AKE), Biology/Microbiology Department (MBH), South Dakota State University, Brookings, South Dakota
Correspondence to: Alan K. Erickson, Veterinary Science Department, N. Campus Drive, PO Box 2175, South Dakota State University, Brookings, SD 57007. E-mail: alan.erickson{at}sdstate.edu
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Summary |
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Key Words: galectin-4 porcine intestine monoclonal antibodies epithelial cells
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Introduction |
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The early occurrence of galectins in animal evolution, along with the expansion of the family during evolution, suggests that galectins perform critical functions within cells (Yang and Liu 2003). Numerous important biological functions have been assigned to various galectins including regulation of pre-mRNA splicing, apoptosis, and cell-cycle progression; activation of inflammatory cells; and adhesion of cells to each other and to the extracellular matrix (reviewed in Rabinovich 1999
). Galectins have also been shown to be involved in cancer cell invasion and metastasis (Van Den Brule and Castronovo 2000
; Huflejt and Leffler 2004
). Just as there is variation in the distribution of galectins in cells and tissues, there also seems to be variation in the biological functions of each galectin in specific cells at different times during development and growth (reviewed in Rabinovich 1999
). Consequently, to gain an overall understanding of the diversity of functions performed by galectins, individual galectins need to be evaluated in specific cells at particular times during the development and growth of an animal, organ, or cell.
Galectin-4 is expressed in the epithelium lining the entire length of the alimentary canal in mammals (reviewed in Huflejt and Leffler 2004). The long-term goal of our research is to determine the biological functions of galectin-4 in the small intestine of postnatal animals. Because galectin-4 is a tandem-repeat galectin with two distinct CRDs in a single polypeptide chain, it seems logical that it accomplishes some of its biological functions by crosslinking glycoconjugates. The molecules that are crosslinked by galectin-4 and the subsequent effects of that crosslinking are not known. In the current study, we determined the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its crosslinking functions in the intestine. Using immunohistochemistry (IHC) with an anti-galectin-4 monoclonal antibody (MAb), we found that galectin-4 is primarily expressed in the cytoplasm of the absorptive epithelial cells covering the villi in the three major regions (duodenum, jejunum, and ileum) of porcine small intestine. The highest level of expression of galectin-4 was observed in mature epithelial cells at villous tips where, in addition to cytoplasmic staining, we also observed nuclear staining, indicating that the localization of galectin-4, and quite possibly its function, change during the terminal maturation of intestinal epithelial cells. We also identified a subset of intestinal crypt cells, which may be enteroendocrine cells that express galectin-4 at a high level.
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Materials and Methods |
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Purification of MAbs from Ascites Fluid
To remove lipids, ascites fluid was filtered through glass wool. Filtered ascites fluid was clarified by centrifugation (30 min, 20,000 x g, 4C), diluted 10-fold with 0.1 M sodium acetate, pH 5.0 and loaded onto a HiTrap protein G column (5 ml; Pharmacia, Piscataway, NJ) equilibrated with 0.1 M sodium acetate, pH 5.0. The column was washed with five column volumes of 0.1 M sodium acetate, pH 5.0. Bound MAbs were eluted from the column with 0.1 M glycineHCl, pH 2.8. To minimize exposure of MAbs to low pH conditions, 50 µl of 2 M Tris base was added per milliliter of eluate as fractions from the column were collected. The MAb-containing fractions were pooled, dialyzed against PBS at 4C with two buffer changes, and stored at 20C.
ELISA for Detection of Anti-galectin-4 MAbs
Purified galectin-4 (2 µg/well; diluted in 50 mM sodium carbonate, pH 9.6) was immobilized to 96-well Immulon I polystyrene plates (Dynatech; Alexandria, VA) at 37C for 2 hr. The wells were washed three times with PBS containing 0.05% Tween 20 (PBS-Tween). The wells were blocked with 2% bovine serum albumin (BSA) in PBS for 1 hr at 37C and then washed three times with PBS-Tween. Culture supernatants from hybridomas or dilutions of ascites fluid were added to the wells and incubated for 1 hr at 37C. The plate was washed three times with PBS-Tween. Goat-anti-mouse peroxidase conjugate (100 µl of 0.047 mg/ml conjugate in PBS containing 0.01% BSA; ICN, Costa Mesa, CA) was added to the plate and incubated for 1 hr at 37C. The wells were then washed three times with PBS-Tween and once with PBS. Peroxidase activity was detected using 2,2'-azino-di-3-ethyl-benzthiazoline sulfonic acid (ABTS) as previously described (Erickson et al. 1992).
Immunoblotting
Lactose-binding proteins were prepared from the jejunum of a 5-week-old pig using the procedure of Mandrell et al. (1994) as modified by Wooters et al. (unpublished data). The resulting lactose-binding proteins were dialyzed against Milli-Q water (Millipore; Bedford, MA) and dried in a Speed Vac (Thermo Savant; Holbrook, NY). Lactose-binding proteins were dissolved in XT SDS-PAGE sample buffer containing XT reducing agent (Bio-Rad; Hercules, CA) and heated to 95C for 5 min prior to electrophoresis. Lactose-binding proteins (5 µg/lane) were separated on Criterion X Precast Bis-Tris SDS-PAGE gels (12% resolving gel; Bio-Rad) using XT MES running buffer (Bio-Rad) according to the manufacturer's suggested protocol. Separated proteins were electrotransferred to polyvinylidene fluoride (PVDF) sheets (0.45 µm pore size; Millipore) as previously described by Towbin et al. (1979)
. The proteins on the PVDF membrane were stained with Ponceau S (0.1% in 1% acetic acid) for 5 min. Unbound stain was removed from the membrane by washing the membrane twice with 1% acetic acid for 5 min. A digital image of the stained membrane was acquired using a Hewlett-Packard Scanjet 4400C (Palo Alto, CA) scanner. The Ponceau S stain was removed from the proteins by three 10-min washes in PBS-Tween. The membranes were blocked for 1 hr with PBS-Tween containing 2% BSA and washed three times for 5 min each with PBS-Tween containing 0.1% BSA. Purified MAb A3-166 (0.1 µg/ml in PBS-Tween containing 0.1% BSA) was incubated with the membrane for 1 hr at room temperature (RT). The membrane was washed three times for 5 min with PBS-Tween containing 0.1% BSA and incubated with peroxidase-conjugated rabbit anti-mouse IgG H+L (0.18 µg/ml in PBS-Tween containing 0.1% BSA; Jackson ImmunoResearch, West Grove, PA) for 1 hr at RT. The membrane was washed three times for 5 min each with PBS-Tween containing 0.1% BSA and once for 5 min with Milli-Q water (Millipore). Bound peroxidase activity was detected by incubating the membrane in a stabilized solution of 3,3',5,5'tetramethylbenzidine (TMB) for horseradish peroxidase (Promega; Madison, WI) for 10 min. Color development was stopped by rinsing the membrane three times with Milli-Q water.
Immunoprecipitation with MAb A3-166
MAb A3-166 was immobilized to Protein G-Sepharose beads using a Seize X Protein G Immunoprecipitation kit (Pierce Biotechnology, Inc.; Rockford, IL). Briefly, MAb A3-166 (0.4 ml of 0.24 mg/ml in PBS) was incubated with 0.1 ml of packed Immunopure Immobilized Protein G Plus beads at RT for 1 hr with end-over-end rotation. This solution was centrifuged at 3000 x g for 1 min in a Handee Spin Cup Column (Pierce). The beads were washed three times with PBS by suspension in 0.5 ml of PBS followed by centrifugation at 3000 x g for 1 min in a Handee Spin Cup Column. The Protein G beads were suspended in 0.4 ml of PBS. To immobilize the MAb to the Protein G beads, 25 µl of a solution containing disuccinimidyl suberate (DSS; 25 mg/ml DSS in dimethyl formamide) was added to the beads and incubated with mixing for 1 hr at RT. To remove unbound antibody and quench the DSS reaction, the beads were washed five times with Immunopure IgG elution buffer and three times with IP buffer (50 mM sodium acetate buffer, pH 5.0, 500 mM NaCl, 0.1% SDS, 1% NP-40) by suspending the beads in 0.5 ml of the appropriate buffer followed by centrifugation at 3000 x g for 1 min in a Handee Spin Cup Column. The washed beads were incubated for 16 hr at 4C with lactose-binding proteins (400 µg in IP buffer) from porcine intestine. Unbound antigen was removed by washing the beads five times with 0.4 ml of IP buffer and once with 0.4 ml Milli-Q water (Millipore). Bound antigen was eluted from the beads by incubating the beads for 5 min at 95C with 200 µl of the XT SDS-PAGE sample buffer (Bio-Rad) with no reducing agent. The eluted antigen was collected by centrifugation at 3000 x g for 1 min in a Handee Spin Cup Column and 10 µl of XT reducing agent (Bio-Rad) was added to each sample. The resulting samples were then analyzed using the immunoblotting technique described above with 10 µl of immunoprecipitation sample run in each lane.
Immunohistochemistry
Small intestine was harvested from a 4-week-old pig immediately after euthanasia. The small intestine was rinsed with PBS containing fresh 2 mM PMSF. Tissue segments (75 mm) were cut from the duodenum, jejunum, and ileum. The tissues were then fixed in 10% neutral buffered formalin for 48 hr. The tissues were trimmed, dehydrated through a graded ethanol and propanol series, cleared through three changes of propar, and infiltrated and embedded with paraffin. Five-µm-thick sections were cut using a Leica Microtome (Nussloch, Germany) and placed on Surgipath Snowcoat Xtra slides (Winnipeg, Canada). The slides were air dried overnight and then placed at 60C for 1 hr. The tissues were deparaffinized and treated with 30% hydrogen peroxide. The tissues were washed three times with deionized water and soaked in TBS-Tween (0.05 M Tris-HCl buffer containing 0.3 NaCl and 0.1% Tween 20, pH 7.6) for 5 min. Slides were loaded into a DAKO Autostainer (DakoCytomation; Carpinteria, CA) where they were incubated with primary antibody, MAb A3-166 (0.0048 mg/ml) for 30 min at RT and rinsed with TBSTween. Antibody binding was detected using the DAKO Envision+ SystemHRP (DakoCytomation) according to the manufacturer's protocol. The tissues were counterstained with Meyer's hematoxylin, coverslipped, and examined under the microscope. Negative controls were performed using the same steps as above with the following changes. For the negative control without antibody, no MAb A3-166 was incubated with the tissue. For the control using an antibody for a non-intestinal target, a MAb for NS-1, a mouse tumor cell line, was incubated on the tissue in place of MAb A3-166. For the blocking control, a 10-fold molar excess of purified galectin-4 was incubated with MAb A3-166 for 30 min at RT prior to the MAb being placed on the tissue. A Fujix Digital Camera HC-300Z (Fuji Photo Film Co. Ltd.; Tokyo, Japan) was used for IHC photographs. Exposure time for each picture was selected by the photomicroscopy system.
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Results |
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Discussion |
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Previously, Danielsen and van Deurs (1997) assessed the distribution of galectin-4 in porcine small intestine using IHC and electron microscopy experiments performed with anti-galectin-4 polyclonal antibodies (PAb) made against a partially purified preparation of porcine galectin-4. Many of the results of the current study, which used a highly specific anti-galectin-4 MAb, are consistent with the findings of this previous study (Danielsen and van Deurs 1997
). Both studies found galectin-4 primarily in the cytoplasm of the absorptive intestinal epithelial cells lining intestinal villi with the most intense staining in the villous tip cells and with no staining in the other layers that comprise the wall of the intestinal tract. Also consistent in both studies, galectin-4 was detected at the highest intensity on the apical side of the absorptive intestinal epithelial cells, suggesting that at least some of the functions of galectin-4 are performed at the microvillar (brush border) surface of the cells. In addition to verifying the findings of the study by Danielsen and van Deurs (1997)
, the current study provides the first evidence that galectin-4 is associated with nuclei in villous tip cells and identifies a subset of galectin-4 expressing crypt cells that may be enteroendocrine cells. The current study is also the first to demonstrate that galectin-4 distribution is similar in all three major regions of porcine small intestine.
Our results indicate that virtually all galectin-4 is found inside the absorptive intestinal epithelial cells that line the intestinal villi, indicating that galectin-4 performs at least some intracellular functions for these cells. Because it is possible that extracellular galectin-4 may have been washed away during the rinse step before fixation of the tissues, our studies do not eliminate the possibility that some galectin-4 is secreted and has extracellular functions under certain physiological conditions. Within most absorptive intestinal epithelial cells, galectin-4 staining was distributed throughout the cytoplasm with slightly more intense staining observed on the apical side of the cell. This result is consistent with a previous study in which subcellular fractionation techniques were used to demonstrate that 46% of galectin-4 is bound to microvilli and its associated actin filaments, 29% is bound to tubulovesicular structures, and the other 25% is bound to unrecognizable cytoplasmic components (Danielsen and van Deurs 1997
). One intracellular function proposed for galectin-4 that is consistent with this distribution pattern is that galectin-4 may be involved in the formation and/or stabilization of lipid rafts which are used for intracellular transport of glycosphingolipids (GSL) and membrane proteins to the apical surface of the plasma membrane of intestinal epithelial cells (Danielsen and van Deurs 1997
). In support of this hypothesis, galectin-4 has been found to copurify with lipid rafts isolated from villous intestinal epithelial cells (Hansen et al. 2001
). Lipid rafts form when GSL, cholesterol, glycosylphosphatidylinositol-anchored plasma membrane proteins, and various transmembrane proteins self-associate in the trans-Golgi membrane (Alberts et al. 2002
). Lectins, like galectin-4, are found associated with these rafts likely functioning to stabilize the raft structure by interacting with the glycans on the GSL and transmembrane proteins. After the lipid rafts form in the trans-Golgi, they pinch off into transport vesicles that are targeted for the apical cell surface. When the lipid raft reaches the apical membrane, it is incorporated into the plasma membrane with the GSL and the associated proteins expressed in the outer leaflet of the plasma membrane. This process would lead to externalization of galectin-4 without the necessity for a secretion sequence on galectin-4. Externalization of galectin-4 into the oxidizing extracellular environment would likely inactivate the galectin leading to its release into the intestinal lumen where its crosslinking ability would be rapidly destroyed by proteases found in the intestinal lumen.
Participation in apoptotic pathways is another intracellular function that has been proposed for a number of galectins in a variety of different cells (Perillo et al. 1995; Wada and Kanwar 1997
; Hotta et al. 2001
; Yang and Liu 2003
). In the small intestine, epithelial cells are initially produced from stem cells that are found in the intestinal crypts. In the crypts, these cells undergo differentiation followed by a series of mitotic divisions before migrating up the sides of the villi. When these cells reach the villous tips, they undergo apoptosis and are released into the intestinal lumen (Hall et al. 1994
; Ramachandran et al. 2000
). In the current study, the highest expression of galectin-4 was observed in the most mature (villous tip) cells. In these villous tip cells, galectin-4 appeared to be associated with the nucleus. Both of these observations are consistent with galectin-4 playing a role in apoptosis in villous tip cells, resulting in death and turnover of these cells. Consistent with our observations, a previous study showed that galectin-4 mRNA was strongly downregulated in colorectal tumor tissue, indicating that lack of expression of galectin-4 might permit cell proliferation by preventing normal apoptosis of intestinal cells (Rechreche et al. 1997
).
Most cells in the intestinal crypts do not express galectin-4 to any great extent, except for a set of widely dispersed cells that stained with anti-galectin-4 MAb on their basal or vascular side. Based on this distribution pattern, these cells are likely enteroendocrine cells. Enteroendocrine cells have been shown to be scattered among epithelial cells in the crypt and along the villi and to contain small secretory granules, which contain hormones, located on the basal side of the cells (Cheng and Leblond 1974). The most intense staining of galectin-4 in this subset of crypt cells was observed on the basal side of the cells, indicating that galectin-4 is likely associated with the components (e.g., hormones) of the cells that are secreted out of the vascular side of the cells. A similar observation of galectin-4 expression in secretory cells of the gastrointestinal tract was recently reported for rat gastric secretory cells including endocrine, parietal, and chief cells (Niepceron et al. 2004
). More studies in this area need to be performed to establish that these galectin-4-expressing crypt cells are enteroendocrine cells, and then to determine what role galectin-4 performs in these crypt cells.
The distribution pattern of galectin-4 in porcine small intestine established in the current study indicates that galectin-4 likely serves multiple intracellular functions in small intestine. The distribution of galectin-4 in the cytoplasm of the absorptive epithelial cells lining the villi, with slightly higher expression at the apical surface, is consistent with a role for galectin-4 in lipid raft formation and stabilization. The observations that galectin-4 expression is highest and localized to the nucleus in mature epithelial cells at the villous tips are consistent with the hypothesis that galectin-4 promotes apoptosis of mature intestinal epithelial cells. Also, the presence of galectin-4 in enteroendocrine cells indicates galectin-4 may be involved in packaging hormones in secretory granules in these cells. Further studies to test the role of galectin-4 in all of these putative functions are needed. Also, studies designed to gain a better understanding of the mechanisms by which galectin-4 accomplishes these functions are needed. One method to address these mechanisms is to identify the intracellular glycoconjugate ligands that are recognized and crosslinked by galectin-4. The results of the current study suggest that these glycoconjugate ligands will be found inside absorptive intestinal epithelial cells, especially villous tip cells, and also in enteroendocrine cells in intestinal crypts.
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Acknowledgments |
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We thank Laura Mills, Lori Zobel, Mitzi Trooien, Ying Fang, and Pam Steen for technical advice and assistance.
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Footnotes |
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Received for publication June 10, 2004; accepted November 10, 2004
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular Biology of the Cell. 4th ed. New York, Garland Science, 763764
Cheng H, Leblond CP (1974) Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. III. Entero-endocrine cells. Am J Anat 141:503519[Medline]
Chiu ML, Parry DA, Felman SR, Klapper DG, O'Keefe EJ (1994) An adherens junction protein is a member of the family of lactose-binding lectins. J Biol Chem 269:3177031776
Colnot C, Fowlis D, Ripoche MA, Bouchaert I, Poirier F (1998) Embryonic implantation in galectin-1/galectin-3 double mutant mice. Dev Dynam 211:306313[CrossRef][Medline]
Danielsen EM, van Deurs B (1997) Galectin-4 and small intestinal brush border enzymes form clusters. Mol Biol Cell 8:22412251
Erickson AK, Willgohs JA, McFarland SY, Benfield DA, Francis DH (1992) Identification of two porcine brush border glycoproteins that bind K88ac adhesin of Escherichia coli and correlation of these binding glycoproteins with the adhesive phenotype. Infect Immun 60:983988[Abstract]
Hall PA, Coates PJ, Ansari B, Hopwood D (1994) Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107:35693577
Hansen GH, Immerdal L, Thorsen E, Niels-Christiansen LL, Nystrøm BT, Demant EJF, Danielsen EM (2001) Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes. J Biol Chem 276:3233832344
Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor NY, Cold Spring Harbor Laboratory
Hirabayashi J, Kasai K (1993) The family of metazoan metal-independent ß-galactoside-binding lectins: structure, function and molecular evolution. Glycobiology 3:297304[Abstract]
Hotta K, Funahashi T, Matsukawa Y, Takahashi M, Nishizawa H, Kishida K, Matsuda M, et al. (2001) Galectin-12, an adipose-expressed galectin-like molecule possessing apoptosis-inducing activity. J Biol Chem 276:3408934097
Huflejt ME, Leffler H (2004) Galectin-4 in normal tissues and cancer. Glycoconj J 20:247255[CrossRef][Medline]
Leffler H, Masiarz FR, Barondes S (1989) Soluble lactose-binding vertebrate lectins: a growing family. Biochemistry 28:92229229[Medline]
Levi G, Teichberg VL (1981) Isolation and physicochemical characterization of electrolectin, a ß-D-galactoside binding lectin from the electric organ of Electrophorus electricus. J Biol Chem 256:57355740
Liu FT, Patterson RJ, Wang JL (2002) Intracellular functions of galectins. Biochim Biophys Acta 1572:263273[Medline]
Mandrell RE, Apicella MA, Lindstedt R, Leffler H (1994) Possible interactions between animal lectins and bacterial carbohydrates. Methods Enzymol 236:231254[Medline]
Niepceron E, Simian-Lerme F, Louisot P, Biol-N'garagba MC (2004) Expression and localization of galectin 4 in rat stomach during postnatal development. Int J Biochem Cell Biol 36:909919[CrossRef][Medline]
Oda Y, Herrmann J, Gitt MA, Turck CW, Burlingame AL, Barondes SA, Leffler H (1993) Soluble lactose-binding lectin from rat intestine with two different carbohydrate-binding domains in the same peptide chain. J Biol Chem 268:59295939
Perillo NL, Pace KE, Seihamer JJ, Baum LG (1995) Apoptosis of T-cells mediated by galectin-1. Nature 378:736739[CrossRef][Medline]
Rabinovich GA (1999) Galectins: an evolutionarily conserved family of animal lectins with multifunctional properties; a trip from the gene to clinical therapy. Cell Death Differ 6:711721[CrossRef][Medline]
Ramachandran A, Madesh M, Balasubramanian KB (2000) Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions. J Gastroenterol Hepatol 15:109120[CrossRef][Medline]
Rechreche H, Mallo GV, Mantalto G, Dagorn JC, Iovanna JL (1997) Cloning and expression of the mRNA of human galectin-4, an S-type lectin down-regulated in colorectal cancer. Eur J Biochem 248:225230[Abstract]
Tardy F, Deviller P, Louisot P, Martin A (1995) Purification and characterization of the N-terminal domain of galectin-4 from rat small intestine. FEBS Lett 359:169172[CrossRef][Medline]
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose: procedure and some applications. Proc Natl Acad Sci USA 76:43504354[Abstract]
Van Den Brule FA, Castronovo V (2000) Laminin-binding lectins during cancer invasion and metastasis. In Caron M, Seve AP, eds. Lectins and Pathology. Amsterdam, Harwood Academic Publishers, 79123
Wada J, Kanwar YS (1997) Identification and characterization of galectin-9, a novel ß-galactoside-binding mammalian lectin. J Biol Chem 272:60786086
Wasano K, Hirakawa Y (1995) Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium. Cell Tissue Res 281:7783[CrossRef][Medline]
Wasano K, Hirakawa Y (1997) Recombinant galectin-1 recognizes mucin and epithelial cell surface glycocalyces of gastrointestinal tract. J Histochem Cytochem 45:275283
Wu AM, Wu JH, Tsai MS, Liu JH, Andre S, Wasano K, Kaltner H, et al. (2002) Fine specificity of domain-I of recombinant tandem-repeat galectin-4 form rat intestinal tract (G4-N). Biochem J 367:653664[CrossRef][Medline]
Yang RY, Liu FT (2003) Galectins in cell growth and apoptosis. Cell Mol Life Sci 60:267276[Medline]