Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Lectin binding to neural crest cells. Changes of the cell surface during differentiation in vitro

To examine possible changes in cell surface carbohydrates, fluorescent lectins were applied at various times during differentiation of neural crest cells in vitro. The pattern and intensity of binding of several lectins changed as the crest cells developed into melanocytes and adrenergic cells. Considerable amounts of concanavalin A (Con A) and wheat germ agglutinin (WGA) bound to all unpigmented cells throughout the culture period. Melanocytes, however, bound much less of these lectins. Soy bean agglutinin (SBA), unlike Con A and WGA, only bound later in development to unpigmented cells at about the time when catecholamines were detected histochemically. Binding of SBA could be induced in younger cultures by pretreating the cells with neuraminidase. Melanocytes, however, did not bind detectable amounts of SBA even if treated with neuraminidase. The SBA-binding sites were often concentrated on cytoplasmic extensions and on contact points between neighboring cells, even when receptor mobility was restricted by prefixation of the cells or adsorption of lectin at 0 degrees C. All three lectins bound to cell processes resembling nerve fibers in particularly high amounts.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of glycolipid binding sites for soybean agglutinin and differences in the surface glycolipids of cultured adrenergic and cholinergic sympathetic neurons

Cultured adrenergic neurons from newborn rat superior cervical ganglia bind the lectin soybean agglutinin (SBA) at a fivefold higher density than the same neurons which have been induced to become cholinergic (M. Schwab and S. L. Landis, 1981, Dev. Biol.84, 67–78). In the present experiments, the binding sites for this lectin on the surfaces of living neurons were identified by labeling the surfaces with the galactose oxidase-[³H]sodium borohydride reduction technique, with and without prior incubation with the lectin. SBA binds to and inhibits the labeling of two neutral glycolipids, a glycolipid comigrating with globoside on thin-layer chromatograms and an unidentified glycolipid. When neuronal proteins are extracted and separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, SBA shows only very faint labeling of this fraction. Thus the SBA binding sites on these neurons appear to be two neutral glycolipids. Further support for this conclusion comes from the finding that the two neutral glycolipids detected by SBA are present in smaller amounts or are less accessible on the cholinergic than on the adrenergic neurons as measured by surface labeling. In addition to the difference in neutral glycolipids, external labeling revealed quantitative differences in the major gangliosides of the two types of cultured neurons. Thus, by using pure cultures of sympathetic neurons which can be induced to become either adrenergic or cholinergic, specific glycolipid profiles were correlated with the two neurotransmitter phenotypes.     

Search for the presence of lectin-binding sites on Toxoplasma gondii

Evidence for the presence of carbohydrate on the surface membrane of Toxoplasma gondii trophozoites and on the cell wall of toxoplasma brain cysts was sought by fluorescent lectin staining. Using FITC-conjugated preparations of Concanavalin A (Con A), wheat germ agglutinin (WGA), or soy bean agglutinin (SBA), we have failed to obtain evidence for the binding of these lectins on the surface of T. gondii trophozoites. In contrast, the three test lectins bound effectively and specifically to the wall of toxoplasma brain cysts. Prefixation of cysts with glutaraldehyde or brief trypsinization of cysts did not affect the intensity of cyst wall fluorescence when stained with FITC-conjugated Con A, SBA, or WGA. The results are interpreted to indicate that whereas exposed Con A, SBA, and WGA binding sites are associated with the wall of toxoplasma brain cysts, such lectin-binding saccharide residues are not present on the surface of trophozoites in exposed or reactive form.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Summary Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair. The 16-day fetal tissue is mitotically active, does not exhibit a well defined monolayer, and demonstrates weak fluorescence binding for WGA, Con A and RCA. Conversely, SBA binding is readily detected on many cell surfaces. By 19 days in utero, the endothelial monolayers becomes organized and cell proliferation greatly diminishes. WGA, Con A and RCA now exhibit binding similar to that seen in the adult tissue. SBA binding is not detected at this time. Thus, changes in lectin binding during wound repair of the adult rat corneal endothelium mimic changes in lectin binding seen during the development of the tissue.Supported by grant EY-06435 from The National Institutes of Health     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Ultrastructural localization of WGA, RCA I, LFA and SBA binding sites in the seven-day-old mouse embryo

In the present investigation we localized binding sites for the lectins WGA (wheat germ agglutinin), RCA I (Ricinus communis agglutinin), LFA (Limax flavus agglutinin) and SBA (soya bean agglutinin) in the 7-day-old mouse embryo at the ultrastructural level. Lectin binding sites were localized on formaldehyde fixed embryos, embedded in LR-Gold, using gold-labelled lectins. Binding sites for WGA and RCA I were observed at the surface of the embryonic ectoderm oriented towards the proamnion cavity and the outer surface of the extraembryonic and the embryonic endoderm. Staining for SBA and LFA binding sites was seen in the basement membrane of the ectoderm. Moreover, binding sites for LFA were observed in the nucleoli of cells of the ectodermal, the mesodermal and the endodermal layer and in free ribosomes located in the cytoplasm of these cells.     

Modulation of endothelial cell surface glycoconjugate expression by organ-derived biomatrices

Cell surface molecules play an important role in cellular communication, migration, and adherence. Here, we show the effect of organ-derived biomatrices on endothelial cell surface glycosylation. Five different lectins (with and without neuraminidase treatment) have been used as probes in an enzyme-linked lectin assay to quantitatively detect glycoconjugates on endothelial cells (BAEC) grown on tissue culture plastic or biomatrices isolated from bovine lung, liver, and kidney. BAEC generally exhibit strong binding of concanavalin A (Con A), Ricinus communis agglutinin I (RCA-I), wheat germ agglutinin (WGA), and soybean agglutinin, and peanut agglutinin after neuraminidase pretreatment of cells (Neu-SBA and Neu-PNA), while SBA and PNA consistently bind weakly to BAEC. BAEC grown on organ-derived biomatrices exhibit significantly altered binding intensities of Con A, RCA-I, WGA, and Neu-PNA: BAEC cultured on lung- or kidney-derived biomatrices express significantly stronger binding affinities for Con A and RCA-I than BAEC grown on liver-derived biomatrix or tissue culture plastic. In contrast, BAEC binding of WGA and PNA (after treatment of cells with neuraminidase) is significantly reduced when BAEC are grown on liver- or kidney-derived biomatrix. Quantitative lectin immunogold electron microscopy reveals consistently stronger lectin binding over nuclear regions compared to junctional regions between neighboring cells. These results indicate that extracellular matrix components regulate endothelial cell surface glycoconjugate expression, which determines cellular functions, e.g., preferential adhesion of lymphocytes or metastatic tumor cells.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Retrograde axonal transport of specific macromolecules as a tool for characterizing nerve terminal membranes

The uptake of macromolecules by nerve terminals which is followed by retrograde axonal transport seems to occur by two different mechanisms, a specific and a nonspecific one. The nonspecific uptake depends on the presence of macromolecules (e.g., horseradish peroxidase) in the vicinity of the nerve terminals at very high concentrations and is enhanced by neuronal activity. In contrast, the specific uptake and subsequent retrograde axonal transport becomes apparent at much lower concentrations of the appropriate macromolecules, depends on the affinity of these ligands for specific binding sites on the surface of the neuronal membrane, and is independent of neuronal activity. The fact that lectins and some bacterial toxins bind to specific membrane glycoproteins or glycolipids allows conclusions to be drawn regarding qualitative and even quantitative aspects of the composition of the plasma membrane of the nerve terminals. ¹²⁵I-labelled nerve growth factor (NGF), tetanus toxin, cholera toxin, wheat germ agglutinin (WGA), ricin II, phytohemagglutinin (PHA), and concanavalin A (ConA) were injected into the anterior eye chamber of rats where they were taken up by adrenergic nerve terminals and transported retrogradely to the superior cervical ganglion. The saturation of the uptake-transport found for NGF, WGA, choleragenoid and an atoxic binding-fragment of tetanus toxin indicates that limited numbers of binding sites, which showed also different affinites, are present for each ligand on the membrane of the nerve terminals. Competition experiments showed that the binding sites for the ligands investigated are largely independent. Two different classes of binding sites (high affinity-low capacity and intermediate affinity-intermediate capacity) seem to be involved in the saturable retrograde axonal transport of NGF. In contrast, WGA seems to have only a single class of binding-uptake sites with high capacity and relatively low affinity. Strong evidence for positive cooperativity was obtained for the uptake and subsequent transport of the tetanus toxin fragment.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

138 Molecular Evolution of Glutamine Synthetase In Protists

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N-acetyl-D-galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus-specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi-step process and gamete binding is the initial step.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.