Lectin histochemistry of glycolipid storage diseases on frozen and paraffin-embedded tissue sections

Lectin histochemical studies were performed on frozen and paraffin-embedded brain tissue sections from six cases of galactosylceramide lipidosis (i.e., globoid cell leukodystrophy, or Krabbe's disease) in Twitcher mice and one case of canine infantile GM1-gangliosidosis. The globoid cells in Krabbe's disease stained with Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), and Bandeirea simplicifolia agglutinin-I (BS-I) in frozen sections. However, paraffin sections and frozen sections pretreated with chloroform-methanol or xylene, from the same animals, stained with Concanavlia ensiformis agglutinin (ConA), wheat germ agglutinin (WGA), and succinylated-WGA (S-WGA), in addition to staining with RCA-I, PNA, and BS-I. The affected neurons of canine infantile GM1-gangliosidosis stained only with RCA-I in frozen sections. In paraffin sections, however, these cells were negative with RCA-I but positive with BS-I, ConA, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Ulex europaeus agglutinin (UEA-I) in paraffin sections. These results indicate that in paraffin processing of glycolipid storage disease tissue, some lectin receptors are lost and others are unmasked. The retained receptors can be stained with specific lectins and could serve as markers to characterize and differentiate among the various glycolipid storage diseases.     

Eosinophilic globule cells in mouse MFH-like sarcomas: lectin histochemistry.

Lectin binding patterns in ten mouse malignant fibrous histiocytoma (MFH)-like sarcomas containing eosinophilic globule (EG) cells and in granular metrial gland (GMG) cells of mouse placenta were stained with nine lectins (Con A, LCA, WGA, DBA, SBA, e-PHA, PNA, RCA-I and UEA-I) by an avidin-biotin-peroxidase-complex method. EG cells stained strongly with DBA, SBA and PNA which are specific for N-acetyl-D-galactosamine and/or D-galactose. DBA and SBA bound throughout the cytoplasm including the globules; PNA reacted preferentially at the cell surface. There was no evidence that these three lectins were reactive for immature EG cells. WGA, RCA-I and e-PHA also gave a slightly to moderately positive reaction to globules of EG cells. The results indicate that the globules contain abundant O-linked sequences of sugars, but also a few N-linked residues. MFH tumor cells showed a variable degree of binding with Con A, RCA-I, and WGA, but did not react with DBA, SBA and PNA. On the other hand, GMG cells exhibited specific affinities for DBA, SBA and PNA with staining patterns similar to those of EG cells. These findings suggest that EG and GMG cells may be of the same cellular lineage.     

The glycoprotein-bound large carbohydrates from embryonal carcinoma cells carry receptors for several lectins recognizing N-acetylgalactosamine and galactose

When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.     

Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase

Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.     

Genetic Differences in Androgen Receptors and in Autoregulation of Testicular Human Chorionic Gonadotropin Binding Sites in the Mouse

Abstract

The autoregulation of testicular human chorionic gonadotropin (hCG) binding sites was studied in two strains of mice known to differ in their endocrine and reproductive characteristics (C57BL/10J and DBA/2J), and in their F₁ progeny (B10D2F₁). Basal hCG binding levels were higher in C57BL/10J than in DBA/2J mice, while B10D2F₁ mice had intermediate levels. Twenty-four h after injection, hCG produced dose-related changes in hCG binding in C57BL/10J and B10D2F₁ mice not observed in DBA/2J mice. However, 72 h after treatment with hCG there was a decrease in hCG binding in all the strains studied. These results suggest the participation of genetic factors in determining basal levels, dose-related changes and temporal response of testicular hCG binding sites to hCG administration. Androgen receptor levels were measured in the same strains of mice. DBA/2J mice had higher receptor levels in the kidney and coagulating gland, and lower levels in the hypothalamus and seminal vesicle when compred to C57BL/10J mice. B10D2F₁ mice had androgen receptor levels similar to those measured in C57BL/10J mice in all tissues studied, with the exception of the coagulating gland, where levels were similar to those observed in DBA/ZJ mice. These observations may indicate the existence of several loci coding for androgen receptors, with only one being expressed per tissue     

Cell-surface changes during in vitro differentiation of pluripotent embryonal carcinoma cells

When aggregates of HM-1 embryonal carcinoma (EC) cells were exposed to 10(-6) M retinoic acid for 2 days and cultured in medium lacking retinoic acid, they differentiated to nerve cells, endoderm cells, and myoblasts. Cells 2 days after initial exposure to retinoic acid were not significantly different from the parental EC cells, as judged by cell-surface architecture and by reactivity to lectins. On the fourth day, the surface of the aggregates was covered with two kinds of cells distinguishable from the parental cells. The round cells with short villi seemed to be precursors to endoderm cells. Receptors for Dolichos biflorus agglutinin (DBA) newly appeared and receptors for peanut agglutinin (PNA) were still expressed on their surfaces. The other cells, which were round cells with a few processes, might be precursors to nerve cells. PNA receptors had disappeared from their surfaces, and DBA receptors were not expressed. On the sixth day of differentiation, possible precursors to myoblasts were detected; they were flat cells with smooth surfaces. These cells lacked cell-surface receptors for the two lectins, while the precursor cells and the myoblasts excreted intercellular fibers reacting with PNA. HM-1 cells synthesized much embryoglycan, the structure of which was similar to that of the glycan isolated from quasinullipotent F9 cells. The only difference was that the glycan from HM-1 cells lacked DBA binding sites. Synthesis of fucosylated embryoglycan mainly decreased between the second and fourth day of differentiation. As above, cell-surface changes occurred mainly between the second and fourth day. The period seems to be important in determining the fate of the cells, since endoderm cells were scarcely seen among differentiated cells which had been continuously exposed to 10(-6) M retinoic acid during the period.     

Interaction of lectins with membrane receptors on erythrocyte surfaces

The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.     

Lectin binding pattern in normal human labial mucosa

Summary The pattern of lectin binding in normal human labial mucosa was examined by light and electron microscopy using eight different lectins (ConA, LCA, WGA, UEA-1, RCA-1, SBA, DBA and PNA) and compared with the patterns in normal human skin and oesophageal mucosa. As seen by light microscopy, ConA, LCA, and WGA stained cell membranes in all layers of the mucosae. RCA-1 stained the plasma membrane of cells in the basal and middle layers, whereas cells in the superficial layers showed little positive staining. UEA-1, SBA, and PNA stained the cells in the middle layers weakly in some cases. No positive staining for DBA was seen. By electron microscopy, reaction product indicating ConA-binding sites was observed in the plasma membrane, cisternae of the endoplasmic reticulum, nuclear envelope and the Golgi apparatus. Binding of LCA, WGA, and RCA-1 was observed in the plasma membrane. These results show that the binding pattern of PNA, SBA, and RCA-1 in labial mucosa is different from that in the normal skin or oesophageal mucosa, although the labial mucosal epithelium, epidermis, and oesophageal epithelium are all stratified squamous epithelia. These differences in the cell-surface sugar residues are likely to be related to the possible functional differences in these tissues.     

Histochemical characteristics of glycoproteins in the bile duct system of mice immunized with swine serum

The bile duct system of BALB/c and DDY mice, which were immunized with swine serum (SS) or not, was examined histochemically. Biliary epithelial cells of the SS-treated BALB/c mice, which were positively stained with periodic acid-Schiff (PAS) and had binding sites of Dolichos biflorus (DBA), were thought to secrete neutral glycoproteins with terminal N-acetyl-D-galactosamine residues. Those of the SS-treated DDY mice were however negatively or weakly stained with any histochemical stainings. On the other hand, glandular epithelial cells of the SS-treated mice of both strains, which were positively stained with high iron diamine-alcian blue (HID-AB) and had binding sites of DBA, Griffonia simplicifolia-II (GS-II), Ulex europaeus-I (UEA-I), and Triticum vulgaris (WGA), were thought to secrete glycoproteins with terminal sialic acid residues. Biliary and glandular epithelial cells of the normal mice contained only a small amount of glycoproteins showing similar histochemical characteristics to those in the SS-treated BALB/c mice. BALB/c mice immunized with SS were thought to be very useful for the investigation of production and secretion of glycoproteins in the bile duct system as well as being good model of bile duct disease.     

Metabotropic glutamate receptors are differentially regulated under elevated intraocular pressure

Glaucoma is a leading cause of blindness, ultimatively resulting in the apoptotic death of retinal ganglion cells. However, molecular mechanisms involved in ganglion cell death are poorly understood. While the involvement of ionotropic glutamate receptors has been extensively studied, virtually nothing is known about its metabotropic counterparts. Here, we compared the retinal gene expression of metabotropic glutamate receptors (mGluR) in eyes with normal and elevated intraocular pressure (IOP) of DBA/2J mice, a model for secondary angle-closure glaucoma using RT-PCR and immunohistochemistry. Elevated IOP in DBA/2J mice significantly increased retinal gene expression of mGluR1a, mGluR2, mGluR4a, mGluR4b, mGluR6 and mGluR7a when compared to C57BL/6 control animals, while mGluR5a/b and mGluR8a were decreased and no difference was observed for mGluR3 and mGluR8b. Specific antibodies detected an increase of mGluR1a and mGluR5a/b in both synaptic layers and in the ganglion cell layer of the retina under elevated IOP. Because ganglion cell death in DBA/2J mice occurs most likely by apoptotic mechanisms, we demonstrated up-regulation of mGluRs in neurons undergoing apoptosis. In summary, we support the idea that the specific gene regulation of mGluRs is a part of the glaucoma-like pathological process that develops in the eyes of DBA/2J mice.     

A locus for circadian period of locomotor activity on mouse proximal chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (~1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.     

Opisthorchis viverrini: ultrastructure and cytochemistry of the glycocalyx of the tegument

The ultrastructure and cytochemistry of the glycocalyx of the tegument of Opisthorchis viverrini during maturation from newly excysted juvenile to adult stages were investigated using colloidal iron, ruthenium red and lectin stainings. The results showed that the glycocalyx was intensely stained by the first two dyes, thus indicating the presence of relatively high amounts of negative charges. However, the thickness and intensity of the staining decreased during the fluke's maturation. Binding studies using lectin probes on the surface of adult parasites showed that binding sites for Canavalia ensiformis (Con A), Triticum vulgaris (WGA) and Ricinus communis I (RCA I) were present in relative large amounts on the glycocalyx of the adult tegument, whereas those for Dolichos biflorus (DBA) were relatively fewer in number, and those for Ulex europaeus I (UEA I) were absent. The binding patterns of Con A, WGA, RCA I and DBA were generally similar, and the reaction product was uniformly distributed over the dorsal and ventral surfaces of the parasite's body. These bindings, therefore, indicate the presence of D-mannose/D-glucose, N-acetyl-D-glucosamine/sialic acid, D-galactose and N-acetyl-D-galactosamine residues on the glycocalyx of the adult tegument.     

A Locus for Circadian Period of Locomotor Activity on Mouse Proximal Chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (?1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.     

Spontaneous calcified tongue lesions in DBA mice

Large numbers of spontaneously occurring polypoid or slightly elevated lesions were observed in the tongue, mainly the dorsum linguae near the margo linguae, of DBA mice. Histologically the lesion consisted of granulation tissue with focal calcification, and involved superficially the tongue muscle. Often the calcareous deposits were encircled by multinuclear giant cells. The frequency of the calcified tongue lesion was high in lines of DBA/2 (DBA/2NCrj, DBA/2NJcl and DBA/2J), and DBA/1 (DBA/1Jcl and DBA/1J); the SM/J, BALB/c, C57BL/6 and C3H/He strains did not have the lesion. Among hybrid mice, CDF1, a hybrid of DBA/2 and BALB/c, a few had the lesions but no BDF1 mice, a hybrid of DBA/2 and C57BL/6, had any. The frequency was high in the hybrids of DBA/1 and SM/J. These results seem to indicate that the occurrence of the tongue calcified lesions was controlled by polygene.     

Histochemical characteristics of glycoproteins in the bile duct system of mice immunized with swine serum

Summary The bile duct system of BALB/c and DDY mice, which were immunized with swine serum (SS) or not, was examined histochemically. Biliary epithelial cells of the SS-treated BALB/c mice, which were positively stained with periodic acid-Schiff (PAS) and had binding sites of Dolichos biflorus (DBA), were thought to secrete neutral glycoproteins with terminal N-acetyl-d-galactosamine residues. Those of the SS-treated DDY mice were however negatively or weakly stained with any histochemical stainings. On the other hand, glandular epithelial cells of the SS-treated mice of both strains, which were positively stained with high iron diamine-alcian blue (HID-AB) and had binding sites of DBA, Griffonia simplicifolia-II (GS-II), Ulex europaeus-I (UEA-I), and Triticum vulgaris (WGA), were thought to secrete glycoproteins with terminal sialic acid residues. Biliary and glandular epithelial cells of the normal mice contained only a small amount of glycoproteins showing similar histochemical characteristics to those in the SS-treated BALB/c mice. BALB/c mice immunized with SS were thought to be very useful for the investigation of production and secretion of glycoproteins in the bile duct system as well as being good model of bile duct disease.     

Receptors to agglutinin fromDolichus biflorus (DBA) at the synaptic basal lamina of rat neuromuscular junction

Summary The binding of agglutinin fromDolichus biflorus (DBA) and other lectins (Concanavalin A, agglutinin from wheat germ and lectin fromBandeiraea simplicifolid) to synaptic and extrasynaptic portions of the basal lamina of muscle fibers, was studied with histochemical methods. In rat muscle, DBA-binding is specifically detected at the basal lamina of neuromuscular junction. However, long-term (6 months) denervated end-plate in adult rat muscle failed to bind DBA. During normal development, synaptic DBA receptors appear later than acetylcholine receptors or acetylcholinesterase at the rat neuromuscular junction. Generalized DBA-binding to motor end-plates is first visualized in 3-day-old rats, but section of sciatic nerve in 1-day-old rats prevents the appearence of synaptic DBA-binding on the leg end-plates. It is suggested, therefore, that the synaptic DBA receptors could be related to the postnatal stabilization of rat neuromuscular synapses.     

Genetic background determines the extent of islet amyloid formation in human islet amyloid polypeptide transgenic mice

Genetic background is important in determining susceptibility to metabolic abnormalities such as insulin resistance and beta-cell dysfunction. Islet amyloid is associated with reduced beta-cell mass and function and develops in the majority of our C57BL/6J x DBA/2J (F(1)) male human islet amyloid polypeptide (hIAPP) transgenic mice after 1 yr of increased fat feeding. To determine the relative contribution of each parental strain, C57BL/6J (BL6) and DBA/2J (DBA2), to islet amyloid formation, we studied male hIAPP mice on each background strain (BL6, n = 13; and DBA2 n = 11) and C57BL/6J x DBA/2J F(1) mice (n = 17) on a 9% (wt/wt) fat diet for 1 yr. At the end of 12 mo, islet amyloid deposition was quantified from thioflavin S-stained pancreas sections. The majority of mice in all groups developed islet amyloid (BL6: 91%, F(1): 76%, DBA2: 100%). However, the prevalence (%amyloid-positive islets; BL6: 14 +/- 3%, F(1): 44 +/- 8%, DBA2: 49 +/- 9%, P < 0.05) and severity (%islet area occupied by amyloid; BL6: 0.03 +/- 0.01%, F(1): 9.2 +/- 2.9%, DBA2: 5.7 +/- 2.3%, p < or = 0.01) were significantly lower in BL6 than F(1) and DBA2 mice. Increased islet amyloid severity was negatively correlated with insulin-positive area per islet, in F(1) (r(2) = 0.75, P < 0.001) and DBA2 (r(2) = 0.87, P < 0.001) mice but not BL6 mice (r(2) = 0.07). In summary, the extent of islet amyloid formation in hIAPP transgenic mice is determined by background strain, with mice expressing DBA/2J genes (F(1) and DBA2 mice) being more susceptible to amyloid deposition that replaces beta-cell mass. These findings underscore the importance of genetic and environmental factors in studying metabolic disease.     

Strain‐specific proportion of the two isoforms of the dopamine D2 receptor in the mouse striatum: associated neural and behavioral phenotypes

Genetic variability in the proportion of the two alternative dopamine D2 receptor (D2R) mRNA splice variants, D2R‐long (D2L) and D2R‐short (D2S), influence corticostriatal functioning and could be implicated in liability to psychopathology. This study compared mesostriatal D2L/D2S ratios and associated neural and behavioral phenotypes in mice of the DBA/2J and C57BL/6J‐inbred strains, which differ for schizophrenia‐ and addiction‐like phenotypes. Results showed that DBA/2J mice lack the striatal predominance of D2L that has been reported in the rat and in C57BL/6J mice and confirmed in the latter strain by this study. Only C57BL/6J mice showed enhanced striatal c‐Fos expression under D1R and D2/3R co‐stimulation, indicating synergistic interaction between the subtypes of DA receptors. Instead, DBA/2J mice were characterized by opposing effects of D2/3R and D1R stimulation on striatal c‐Fos expression, in line with a more pronounced influence of D2S isoform, and did not express stereotyped climbing under D1R and D2/3R co‐stimulation, as reported for D2L?/? mice. Finally, strain‐specific modulation of c‐Fos expression by D1R and D2/3R co‐stimulation was selectively observed in striatal compartments receiving inputs from the prefrontal cortex and involved in the control of motivated behaviors. These results show differences in tissue‐specific D2R splicing in mice with intact genotypes and support a role for this phenotype in individual variability of corticostriatal functioning and in liability to psychopathology.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.     

Treatment with digestive agents reveals several glycoconjugates specifically associated with rat neuromuscular junction

Summary The lectins DBA, WGA, SBA, Con A, GS-1, LFA and PNA were used to characterize the carbohydrate domains of the rat neuromuscular junction. DBA stained only the synaptic domains of muscle surface. All other lectins stained the whole muscle surface but the intensity of staining was stronger at the synaptic regions. However, when sections were treated with several digestive agents prior to lectin application, the lectin staining pattern changed dramatically. Collagenase-sensitive GlcNac, Mannose, Sialic acid, and GalNac-containing glycoconjugates associated with synaptic regions but not present extrasynaptically were revealed after chemical treatment. On the basis of these modifications it is proposed that, apart from the synapse-specific Gal-Nac-containing glycoconjugate already described elsewhere, new carbohydrate-containing compounds are evidenced. These results provide a new insight into regional specialization of the extracellular matrix associated with the neuromuscular junctions and indicates that pretreatment with various agents, not necessary digestive substances, may alter molecular properties of muscle membrane and uncover previously unknown binding sites.