Wheat Germ Agglutinin Binding Research Articles

Fusarium sp. growth inhibition by wheat germ agglutinin

The antifungal role of wheat germ agglutinin (WGA) isolated from a Romanian dihaploid variety of wheat against two pathogenic fungal species of Fusarium, F. graminearum and F. oxysporum, is demonstrated. WGA was prepared from unprocessed wheat germs by a new purification procedure using chitin and fetuin-Sepharose as affinity chromatography supports. SDS-PAGE and chitinase assay showed that the WGA preparation migrated as a single protein band and was devoid of any contaminating enzyme chitinase, well known for its antifungal effects. Based on its affinity for N-acetylglucosamine residues, WGA binding to the chitin-containing walls of the fungi was detected by fluorescence microscopy using WGA coupled with fluorescein isothiocyanate (FITC). In vitro testing of WGA action on early developmental stages of both fungal strains resulted in various modifications of the germ tubes, visualised by light microscopy: swelling, vacuolation of the cellular content and lysis of cell walls. Viability tests performed on potato tuber slices showed that the microbial infection was prevented from spreading by pretreatment of the fungal suspension with WGA.

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells.

Lectin binding specificities for carbohydrate allow phenotypic and functional characterization of membrane-associated glycoproteins expressed on cancer cells. This analysis examined wheatgerm agglutinin binding to pancreatic cancer cells in vitro and the resulting toxicity. Membrane preparations of nine human pancreatic carcinoma cell lines were studied for lectin binding using wheatgerm agglutinin (WGA), concanavalin A (ConA) and phytohaemagglutinin-L (PHA-L) in a lectin blot analysis. Cell proliferation in vitro was measured by thymidine incorporation in the absence or presence of lectins at various concentrations. Sialic acid binding lectins or succinyl-WGA (succWGA) served as controls. WGA toxicity was tested after swainsonine or neuraminidase pretreatment. Binding and uptake of fluorescein-labelled lectins was studied under fluorescence microscopy. All pancreatic cell lines displayed high WGA membrane binding, primarily to sialic acid residues. Other lectins were bound with weak to moderate intensity only. Lectin toxicity corresponded to membrane binding intensity, and was profound in case of WGA (ID50 at 2.5–5 μg ml−1). WGA exposure induced chromatin condensation, nuclear fragmentation and DNA release consistent with apoptosis. Important steps for WGA toxicity included binding to sialic acid on swainsonine-sensitive carbohydrate and lectin internalization. There was rapid cellular uptake and subsequent nuclear relocalization of WGA. In contradistinction to the other lectins studied, WGA proved highly toxic to human pancreatic carcinoma cells in vitro. WGA binding to sialic acid residues of N-linked carbohydrate, cellular uptake and subsequent affinity to N-acetyl glucosamine appear to be necessary steps. Further analysis of this mechanism of profound toxicity may provide insight relevant to the treatment of pancreatic cancer. © 1999 Cancer Research Campaign

The pycnidiospore of Phyllosticta ampelicida: surface properties involved in substratum attachment and germination

Pycnidiospores of Phyllosticta ampelicida germinate only after they become attached to a hydrophobic substratum. They do not readily attach or germinate on hydrophilic surfaces or on nutrient-rich media. The properties of the spore surface that function in attachment and signal mediation for germination were investigated in this study. Pycnidiospores were surrounded by a sheath that bound Concanavalin A (Con A)-TRITC and only weakly bound wheat germ agglutinin (WGA)-FITC. Galanthus nivalis lectin-FITC selectively bound primarily to the apical region of the sheath associated with the appendage. Con A, but not WGA or GNL, disrupted spore attachment. The Con A effect on spore attachment was significantly reduced when the lectin was pre-mixed with methyl-D-α-mannoside. The sheath as well as the associated appendage was removed with either Novozym 234 or crude β-glucuronidase. Such sheathless spores were initially adhesion incompetent but, by 40 min, the spores regained their ability to adhere to hydrophobic surfaces through the secretion of material that had an affinity toward colloidal gold. Non-treated pycnidiospores surrounded by sheath remained spatially separate; however, sheathless spores readily agglutinated. Addition of cations enhanced attachment to both hydrophobic and hydrophilic surfaces presumably through the masking of electronegative groups on the sheath or by reduction of the ordered water-shell surrounding the sheath. In particular, Ca 2+ induced both attachment and germination on normally non-inductive hydrophilic surfaces, suggesting an additional role for Ca 2+ in signal mediation for germination.

Copper induced immunomodulation in the marine mussel, Mytilus edulis

The effects on the immune response of the mussel, Mytilus edulis, of short-term, in-vivo exposure to copper were investigated under laboratory-controlled conditions. Parameters measured concentrated on the ability of the blood cells to destroy invading pathogens and included changes in the number and character of the circulating haemocytes, peroxidase and phenoloxidase enzyme activity in the blood cells, intra- and extracellular superoxide radical production, phagocytosis and uptake of neutral red. Copper concentrations of 0.02 and 0.05 ppm were found to increase significantly the total number of circulating haemocytes, while 0.2 and 0.5 ppm decreased the proportion of eosinophilic to basophilic cells. Intracellular superoxide production significantly decreased on exposure to 0.5 ppm copper, whereas phagocytic activity was stimulated at 0.2 ppm but not at 0.5 ppm. Copper exposures of 0.2 and 0.5 ppm reduced the percentage of haemocytes showing binding of lectins from Galanthus nivalis and Helix pomatia compared with haemocytes from mussels not dosed with copper. No significant alterations were found in peroxidase and phenoloxidase activity, binding of wheat germ agglutinin or uptake of neutral red. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in aquatic organisms. The benefits of using a multi-assay approach to monitoring environmental pollution using such methods are highlighted.

Wheat germ agglutinin binding is a useful prognostic indicator in stomach cancer

Background. Recent studies suggest that lectin binding activity is correlated with the metastasis and prognosis of several human carcinomas. Wheat germ agglutinin (WGA) is a lectin that recognizes mainly N-acetyl-glucosamin (GalNAc) and acetyl-neuramic acid. However, little is known about WGA expression in gastric carcinoma. The purpose of this investigation was to clarify the significance of WGA expression in human gastric carcinoma.

Quantitative studies of the binding of wheat germ agglutinin (WGA) to chitin-oligosaccharides and partially N-acetylated chitosans suggest inequivalence of binding sites

We have investigated the interactions between wheat germ agglutinin (WGA) and highly de- N-acetylated chitosans of low molecular weight (prepared by nitrous acid degradation and subsequent reduction) by 1H and 19F NMR spectroscopy. Three isolectins were separated from an unfractionated WGA preparation. No significant differences among isolectins were found with respect to binding properties towards GlcNAc or towards chitosans. We observed a specific interaction between WGA and N-acetylated sugar residues in highly de- N-acetylated chitosans of low molecular weight, as observed by line-broadening of the N-acetyl 1H NMR resonance. The interaction was not inhibited by fully de- N-acetylated chitosan of low molecular weight, although evidence of weak interaction between WGA and de- N-acetylated residues was found. We estimated a dissociation constant, K D, of 0.15–0.20 mM for the complex between WGA and a single N-acetylated residue in the chitosan chain (i.e. an N-acetylated residue surrounded primarily by de- N-acetylated residues). Competitive binding experiments with tri- N-fluoroacetylglucosamine (F 3-GlcNAc) were performed by 19F NMR spectroscopy. Surprisingly, the N-acetylated residues of highly de- N-acetylated chitosans were much less effective competitors for binding to WGA than expected from K D-values, while GlcNAc and (GlcNAc) 3 showed the expected competition with F 3-GlcNAc for binding to WGA. These data suggest that internal N-acetylated sugar residues in chitosans bind primarily to other sites of WGA than chitin-oligosaccharides. Support of this suggestion is found in recent reports on the structure of WGA.

Quantitative measurements of the interaction between monosialoganglioside monolayers and wheat germ agglutinin (WGA) by a quartz-crystal microbalance

Monosialogangliosides (GM1, GM2, GM3 and GM4) were reconstituted in lipid monolayers at the air–water interface. The binding amounts and the initial binding rates of wheat germ agglutinin (WGA) to the monosialoganglioside monolayers were quantitatively studied by use of a quartz-crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase, and the binding behavior (mass increase) was followed by the frequency decrease of the QCM. WGA binding affinities for the ganglioside monolayers were influenced by hydrophilic head groups of lipid matrices, densities of gangliosides, and sequences of oligosaccharide in gangliosides. Binding of WGA to the gangliosides reconstituted in a phosphatidylcholine (sphingomyelin and distearoylphosphatidylcholine) matrix was strongly suppressed, but not in a neutral glycolipids (GlcCer, GalCer, and LacCer), dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine matrix. WGA showed high affinity for monolayers containing 20 mol% gangliosides, but only low affinity for 100% ganglioside monolayers. WGA preferably binds to gangliosides in the following sequence: GM3>GM4>>GM2=GM1. No affinities of WGA for GM2 and GM1 were observed. The combined techniques of monolayer and QCM have the advantages of investigating recognition properties of gangliosides.

Lectin-Mediated Drug Targeting: Quantification of Binding and Internalization of Wheat germ Agglutinin and Solatium tuberosum Lectin Using Caco-2 and HT-29 Cells

For the potential use of Wheat germ agglutinin (WGA) and Solarium tuberosum lectin (STL) as auxiliary excipients for targeting drugs to colonocytes, the number of Caco-2 and HT-29-bound lectins was determined by fluorimetry using fluorescein-labelled derivatives of the N-acetylglucosamine-specific lectins. After 1 h of incubation, the WGA-binding capacity of 5 × 104 Caco-2 cells was 26.9 ± 0.5 pmol at 4°C and 27.2 ± 1.0 pmol at 37°C respectively. In comparison, 19.5 ±2.9 pmol (37°C) and 16.7 ±0.9 pmol (4°C) WGA were bound within 1 h to 5 × 104 HT-29 cells referring to about 80% of the total amount of WGA bound within 4 h of incubation. In contrast, binding of STL to the colon carcinoma cell lines was independent of incubation times and temperatures tested exhibiting a binding rate of 8.4 ± 0.6 pmol (HT-29) and 9.9 ± 0.8 pmol (Caco-2) STL/ 5 × 104 cells. As determined by flow cytometry, non-specific binding is lower than 1.0% (WGA) and 3.4% (STL). Uptake and intracellular accumulation of the lectins were investigated by confocal laser scanning microscopy at 4°C and 37°C respectively. A decrease of initially membrane-bound lectins concurrent with increasing cytoplasmic enrichment by time was observed by digital cell image analysis. Due to specific and numerically sufficient adhesion as well as internalization, WGA and STL are anticipated as targeting tools in lectin-mediated drug delivery systems.

The wheat germ agglutinin binding sites and development of the mitochondria-rich cells in gills of tilapia ( Oreochromis mossambicus)

By labelling with gold-colloid-conjugated wheat germ agglutinin (WGA), the WGA-binding sites were identified in the apical region of certain mitochondria-rich (MR) cells in gills of freshwater-adapted tilapia (Oreochromis mossambicus). Bromo-deoxyuridine (BrdU) injection was used to discern the differentiation of the WGA binding sites during development of MR cells. Double labeling of gill sections of fish 2, 4, 6, and 8 days after a single BrdU injection showed that the BrdU labeled MR cells 2 and 4 days after injection were mostly WGA negative, whereas 6 and 8 days after injection, WGA and BrdU-labeled MR cell increased gradually in number. Furthermore, the ratio of the WGA-positive to total MR-cells was higher in gills of tilapia adapted to low calcium ([Ca] = 0.015 mm) freshwater than in hard freshwater ([Ca] = 1.898 mm). The results indicate that WGA binding site may be a marker expressed late during differentiation of MR cells, and physiologically functional in assisting uptake of calcium when environmental calcium is low. According to their WGA binding and also characteristics their likely function in calcium absorption, the WGA positive MR cells shown in this study are considered to be similar to the β cells described by Pisam et al. (1995).

Effect of cholecystokinin blockade on the recovery of alterations induced by acute pancreatitis in glycoconjugates of rat zymogen granules.

Lectin-binding studies have been performed on rat zymogen granules to investigate alterations in the carbohydrate membrane composition that occur in acute pancreatitis induced by caerulein. The influence of treatment with hydrocortisone for seven days before inducing pancreatitis was also studied. Lectin labeling on zymogen granules was also analyzed seven days after inducing pancreatitis in rats that had previously received a hydrocortisone treatment. During this period L 364,718 (0.1 mg/kg)--specific cholecystokinin (CCK) receptor antagonist--was administered daily to some of the rats, and no treatment was applied to others. Using fluorescein-labelled T. purpureus (TP)lectin, a significant decrease in the amount of L-fucose in the granule membrane was observed in rats with caerulein-induced pancreatitis. This effect was directly caused by the pancreatitis and was not influenced by previous hydrocortisone treatment. Seven days later, the density of TP receptors in the granule membrane was similar to the controls both in L-364,718-treated and untreated rats. Therefore, we suggest that endogenous CCK is not an essential factor in the recovery of L-fucose containing glycoconjugates the granule membrane after pancreatitis. Acute pancreatitis did not alter the expression of wheat germ agglutinin (WGA) receptors in the zymogen granule membrane. WGA specifically binds N-acetyl glucosamine and sialic acids. L 364,718 administered for seven days after inducing pancreatitis significantly reduced WGA binding, untreated rats showed a normal zymogen granule membrane. Therefore, the blockade of CCK-induced alterations in membrane glycoconjugates enriched in N-acetyl glucosamine and sialic acid of newly formed granules after pancreatitis, a finding that could explain the delay in the regression of the disease.

Glucocorticoids regulate l-fucose glycoconjugates in rat pancreatic zymogen granules

Lectin-binding studies were performed on rat pancreatic zymogen granules to investigate the influence of glucocorticoid levels on saccharide membrane composition. The following animal groups were used: (1) control rats; (2) rats treated with hydrocortisone (1, 10 and 25 mg/kg/day) for 1, 3 and 8 days; (3) postadrenalectomized rats at days +1, +3 and +8; and (4) adrenalectomized rats receiving hydrocortisone therapy (10 mg/kg/day) for 8 days. By flow cytometry, fluoresceinated (FITC) lectins were used to measure the amount of Concanavalin A (Con A) (specific for d-mannose), wheat germ agglutinin (WGA) (specific for N-acetyl- d-glucosamine) and sialic acids and Tetragonolobus purpureus (TP) (specific for l-fucose) bound to individual zymogen granules from two subpopulations, Z1 and Z2, identified on the basis of their forward and side scatter properties. The molar ratio of the different FITC-lectins revealed significant differences in the glycoconjugate composition of Z1 and Z2 granules, the Z1 granules showing higher ratios of N-acetyl- d-glucosamine: l-fucose and N-acetyl- d-glucosamine: d-mannose, both in control, adrenalectomized and hydrocortisone-treated rats. It was also observed that N-acetyl- d-glucosamine and/or sialic acids were more abundant than l-fucose and d-mannose in the zymogen granule membrane. Z1 and Z2 granules had different glycosylation patterns. Neither adrenalectomy nor hydrocortisone treatments varied the Con A binding to zymogen granules. An increase in WGA binding was only induced by administration of very high doses of hydrocortisone (25 mg/kg/day) for 8 days, an effect not directly related to glucocorticoids. In contrast, a correlation between the FITC-TP labelling and glucocorticoid levels can be established, so that, in a time-dose dependent way, an increase was observed in zymogen granules of rats treated with hydrocortisone while a decreased TP binding was found in adrenalectomized rats—an effect which was reversed with hydrocortisone therapy. Therefore, glucocorticoids exert a direct influence on the saccharide composition of rat pancreatic zymogen granules, regulating the amount of l-fucose glycoconjugates, with Z2 granules more sensitive than Z1 ones.

Ultrastructure of the Cuticle of Loricifera and Demonstration of Chitin using Gold‐Labelled Wheat Germ Agglutinin

AbstractThe pharyngeal and lorical cuticles of adult and larval Loricifera were investigated by transmission electron microscopy. LR White sections of larval and adult Loricifera were labelled with the lectin wheat germ agglutinin (WGA) conjugated to colloidal gold. The pharyngeal cuticle of Nanaloricus mysticusexhibits a multilaminate epicuticle and an amorphous basal layer with osmiophilic fibres. The lorical cuticle consists of an osmiophilic or trilaminate epicuticle, one to three amorphous layer(s), and a basal fibrous layer which is strongly labelled by the lectin‐gold conjugate. Chitinase treatment or competitive inhibition with N‐, N′‐, N″‐triacetylchitotriose exclude labelling almost completely, whereas competitive inhibition with N‐acetyl‐D‐glucosamine does not affect labelling intensity. The binding of WGA in connection with competition experiments indicates the presence of chitin in the fibrous layer. In most areas of a section, three amorphous layers extend below the epicuticle of the Nanaloricidae. Only in favourably orientated sections can all three “amorphous” layers be seen to be formed by stacks of lamellae. Modified articulation sites with bundles of osmiophilic longitudinal fibres and an osmiophilic plate (Nanaloricidae only) occur in adult Loricifera, but not in the larval stages. The ultrastructure of the lorical cuticle of the Loricifera resembles that of other Nemathelminthes (= Aschelminthes). The morphology of the articulation sites and the number of lorical plates seem to differ between the Loricifera and Priapulida. Therefore, it is currently not possible to conclude whether the lorica of the Loricifera and Priapulida are homologous structures. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.

Mosquito midgut glycoproteins and recognition sites for malaria parasites

Midgut glycoproteins of the malaria vector Anopheles tessellatus were partially characterised by gel electrophoresis and lectin binding. Specific binding to wheat germ agglutinin (WGA) and Concanavalin A (Con A) indicated the presence of N-linked core oligosaccharides in many proteins. Rabbit antibodies were produced against wheat germ agglutinin binding proteins (WGABP). These antibodies also recognised distinct proteins in the peritrophic membrane which is secreted into the midgut to enclose a bloodmeal. Rabbit anti-WGABP antibodies ingested in a bloodmeal containing infective gametocytes of the human malaria parasites Plasmodium falciparum and P. vivax tended to reduce infectivity of the parasites to vector mosquitoes. Chitotriose added to a bloodmeal also inhibited parasite development in the mosquito. The results are consistent with a hypothesis that N-acetyl glucosamine residues in mosquito midgut glycoproteins and/or midgut chitin and proteoglycan function as recognition sites for malaria parasites.

Identification of O-Linked N-Acetylglucosamine Modification of AnkyrinG Isoforms Targeted to Nodes of Ranvier

AnkyrinGs of 270 and 480 kDa are localized at nodes of Ranvier and are candidates to couple the voltage-dependent sodium channel and neurofascin to the spectrin/actin network. This study presents evidence that these ankyrins contain O-linked GlcNAc residues and identifies as the site of glycosylation a serine-rich domain that distinguishes them from other ankyrin isoforms. The 480-kDa ankyrinG, extracted from brain membranes associated with wheat germ agglutinin-affinity columns, was [3H]galactose-labeled with UDP-[3H] galactose and galactosyltransferase, and cross-reacted with an antibody against O-GlcNAc monosaccharides. AnkyrinG-associated sugars are O-linked monosaccharides based on resistance to peptide-N-glycosidase F and analysis of saccharides released by beta-elimination. The serine-rich domain is the site of glycosylation based on wheat germ agglutinin binding activity of polypeptides produced by in vitro translation in reticulocyte lysates. Immunofluorescence revealed co-localization of ankyrinG and O-GlcNAc immunoreactivity at nodes of Ranvier. These observations suggest that ankyrin at the node of Ranvier is O-GlcNAc-glycosylated and are the first demonstration of a post-translational modification that is concentrated at the node of Ranvier and not in adjacent areas of myelinated axons.

Changes in lectin binding to bovine sperm during heparin-induced capacitation.

Changes in the plasma membrane of bovine sperm during heparin-induced capcitation were detected by the binding of fluorescent labeled lectins to unfixed sperm. Of the seven lectins evaluated, only binding of wheat germ agglutinin (WGA) changed with capacitation. Sperm were classified into one of 5 patterns (p1-p5) based on staining with WGA, presence or absence of propidium iodide (PI) staining (dead or alive), and acrosomal integrity (acrosome intact or reacted). The major changes associated with capacitation occurred in p1 and p2. Sperm in p1 exhibited diffuse WGA binding over the anterior sperm head, were alive, and had intact acrosomes. In p2, sperm were also acrosome intact and alive, but lacked WGA binding. When sperm were incubated under capacitating conditions with heparin, there was a decrease over time in the percentage of sperm classified in p1 (p < 0.05) and an increase in the percentage of sperm in p2 (p < 0.05). When capacitation by heparin was delayed by the inclusion of glucose in the culture medium, the same heparin-dependent changes in the percentage of sperm in p1 and p2 were delayed (p < 0.05). When capacitation by heparin was inhibited by including protamine in the culture medium, the percentage of sperm in p1 or p2 was not different from sperm incubated without heparin. Heparin-induced capacitation was associated with a loss of WGA binding to the bovine sperm head.

Neuropeptide Y Y2 receptors in hypothalamic neuroendocrine areas are up-regulated by estradiol and decreased by progesterone cotreatment in the ovariectomized rat.

Central neuropeptide Y (NPY) systems are known to stimulate, via the Y1 subtype of NPY receptor, the release of LHRH that leads to surges of LH. Levels of NPY receptors in relation to the above modulation have not been assessed. This study, therefore, examined profiles of NPY receptors in ovariectomized (Ovx) rats and in Ovx rat treated with estradiol (E2) with or without cotreatment with progesterone (P4). [125I]Human PYY containing proline in position 34 [(Pro 34 )hPYY] was used as the Y1 receptor ligand, and [125 I]human peptide YY-(3-36) [hPYY-(3-36)] was used as the Y2 site ligand. Treatment with E2 over 3 days increased Y2 binding in the preoptic hypothalamus and the medial basal hypothalamus, whereas no changes were found in the lateral anterior hypothalamus or the piriform cortex. Administration of P4 (1.5 mg/animal) on the third day of E2 treatment reduced Y2 binding in both the preoptic hypothalamus and the medial basal hypothalamus to or below the density found in Ovx controls. The Y1 receptor levels and the affinity of either Y1 or Y2 binding did not change appreciably with any of the treatments. No significant changes in the binding of wheat germ agglutinin were found at the time of the largest reduction in Y2 receptor numbers by P4, indicating the absence of a major membrane receptor reduction in response to the progestin. The down-regulation of Y2 sites by P4 preceded and accompanied the surge of serum LH induced by the progestin in E2-treated animals. A short term P4 treatment thus appears to reduce the Y2 tone in hypothalamic areas involved in LHRH secretion. This reduction might reinforce Y1 drives known to stimulate the output of LHRH, and thus contribute to LH release.

Distribution of concanavalin A and wheat germ agglutinin binding sites in the rat peripheral nerve fibres revealed by lectin/glycoprotein-gold histochemistry.

The affinity of rat peripheral nerve fibres for concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested in semi-thin sections of Epon-embedded material. A two-step post-embedding technique was used. As a first step, Con A and WGA were used in pure form. As a second step, peroxidase-gold (for Con A) and ovomucoid-gold (for WGA) complexes were applied. The lectin-binding sites, visualized by means of signal amplification with the photochemical silver reaction, were associated mainly with the myelin sheaths and the surfaces of Schwann cells.

Molecular Cloning, Expression, and Characterization of Podocalyxin-like Protein 1 from Rabbit as a Transmembrane Protein of Glomerular Podocytes and Vascular Endothelium

Podocytes are responsible in part for maintaining the size and charge filtration characteristics of the glomerular filter. The major sialoprotein of the podocyte foot process glycocalyx is a 140-kDa sialoprotein named podocalyxin. Monoclonal antibodies raised against isolated rabbit glomeruli that recognized a podocalyxin-like protein base upon size, Alcian blue staining, wheat germ agglutinin binding, and distribution in renal cortex were used to expression clone cDNAs from a rabbit glomerular library. On Northern blot the cDNAs hybridized to a 5.5-kilobase pair transcript predominantly present in glomerulus. The overlapping cDNAs spanned 5,313 base pairs that contained an open reading frame of 1,653 base pairs and were not homologous with a previously described sequence. The deduced 551-amino acid protein contained a putative 21-residue N-terminal signal peptide and a 26-amino acid transmembrane region. The mature protein has a calculated molecular mass of 55 kDa, an extracellular domain that contains putative sites for N- and O-linked glycosylation, and a potential glycosaminoglycan attachment sites. The intracellular domain contains potential sites for phosphorylation. Processing of the full-length coding region in COS-7 cells resulted in a 140-kDa band, suggesting that the 55-kDa core protein undergoes extensive post-translational modification. The relationship between the cloned molecule and the monoclonal antibodies used for cloning was confirmed by making a fusion protein that inhibited binding of the monoclonal antibodies to renal cortical tissue sections and then raising polyclonal antibodies against the PCLP1 fusion protein that also recognized glomerular podocytes and endothelial cells in tissue sections in a similar distribution to the monoclonal antibodies. We conclude that we have cloned and sequenced a novel transmembrane core glycoprotein from rabbit glomerulus, which has many of the characteristics of podocalyxin. We have named this protein podocalyxin-like protein 1.

Effect of early dietary deficiency in polyunsaturated fatty acids on two lectin binding sites in the small intestine of postweanling rats.

This study was designed to determine whether dietary lipids influence the development of intestinal cell glycosylation, in relationship to diet-induced changes in phospholipid fatty acid composition. The ability of two different lectins, wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA), to combine specifically with particular carbohydrate residues was used to investigate the surface characteristics of epithelial cells of rats fed different dietary lipids from birth to 6 weeks of age. Diets contained 5% (weight) peanut oil (PO), rich in n-6 fatty acids; salmon oil (SO), rich in n-3 fatty acids; hydrogenated palm oil (HPO), deficient in both n-6 and n-3 fatty acids or a PO and rapeseed oil (RO) mixture (PRO), the control diet. Pieces of jejunal and ileal villi were excised from postweanling rats and prepared for lectin histochemical study. Concurrently, epithelial cells were removed from jejunal and ileal segments for determining their phospholipid fatty acid compositions. Polyunsaturated fatty acid (PUFA) deficiency was evidenced in the HPO group by the appearance of eicosatrienoic acid (20:3n-9) in both jejunal and ileal phospholipids, which paralleled the decrease in arachidonic acid content. Accretion of 18:1n-9 and 20:3n-9 in cell phospholipids of group HPO was not sufficient to match the unsaturation level in rats fed nonhydrogenated vegetable oils (PRO, PO) or fish oil (SO). The lectin histochemical study showed that WGA strongly labelled the brush border membrane microvilli whereas binding of MAA was specific to goblet cells and mucus. Regardless of the type of diet, WGA binding was weaker in the ileum than in the jejunum. In comparison to all other groups, WGA-labelling of villi was less intense in the jejunum and disappeared almost completely in the ileum of HPO-fed rats. Although SO- and PO-fed rats had, respectively, very low and high ratios of n-6 to n-3 in their intestinal phospholipids, binding of WGA in both groups was not markedly different from that in the control (PRO). MAA-labelling was very intense in jejunal and ileal villi of n-3-fed (SO) rats, whereas it was strongly attenuated in the n-3- and n-6 deficient (HPO) group. These results suggest that intestinal glycosyltransferase activities involved in cell differentiation were altered relative to the overall unsaturation index of dietary fatty acids. Alterations of epithelial glycosylation mainly resulted from a drop in total n-6 and n-3 fatty acids, although it may be speculated that there is a specific effect of n-3 fatty acids.

Use of the fluorescence-activated cell sorter (FACS) for in vitro assays of developmental toxicity

Our objective is to predict embryotoxicity with reliable in vitro techniques. In several experimental systems, differentiation is accompanied by changes in the glycosylation pattern of cell-surface glycoconjugates. This is also the case with embryonal carcinoma cells. We have monitored the expression of receptors for wheat germ agglutinin (WGA). Murine embryonal carcinoma cells (P19 and F9) were exposed in vitro to xenobiotics for 1–3 days, then incubated successively with WGA-biotin (15 μg/ml) and streptavidin-phycoerythrin (SA-PE) (20 μg/ml), each for 30 min at room temperature. Cell-surface fluorescence was then analysed using a fluorescence-activated cell sorter (FACS). Exposure to 1 μm retinoic acid, a known inducer of differentiation, altered glycosylation as indicated by changes in WGA binding. Clear-cut effects were also observed after exposure to salts of arsenic (20 μM), or nickel (50 μm), and to methotrexate (1 μg/ml), fluorouracil (1.3 μg/ml) or actinomycin D (0.04 μg/ml). These compounds affected the percentage of positive cells the intensity of labelling, or both. Two non-teratogenic compounds (metronidazole and sulfonilamide) have also been tested and had no effect. Lectin histochemistry of embryonal carcinoma cells exposed to potentially toxic agents holds promise as a method for predicting embryotoxicity. FACS analysis allows rapid quantification.