Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

A Comparison of Soybean Agglutinin in Cultivars Resistant and Susceptible to Phytophthora megasperma var. sojae (Race 1)

The amount of soybean agglutinin (SBA) detectable by radioimmunoassay in seeds of resistant cultivars to Phytophthora megasperma var. sojae was approximately twice that of susceptible cultivars. SBA was preferentially released at earlier times (6-9 hours) and in higher amounts in the imbibate from resistant cultivars as compared to susceptible cultivars. The lectin in the imbibate was immunologically identical to the seed lectin, indicating little or no proteolysis had occurred, and was active in hemagglutination. Binding of fluorescein isothiocyanate-labeled SBA to mycelial cell walls could be abolished by adding N-acetyl galactosamine or galactose. Purified SBA at concentrations of 150 to 300 micrograms inhibited mycelial growth by 50%, and the imbibate from Govan (resistant) cultivar was more inhibitory than the imbibate from Shore (susceptible) cultivar. Removal of SBA from the imbibate by affinity chromatography abolished the inhibition of mycelial growth, but the inhibition could be recovered from the eluant containing lectin.     

Pregnancy-related changes in the human endometrium revealed by lectin histochemistry

The binding of 22 fluorescein isothiocyanate (FITC) conjugated lectins to human proliferative phase and pregnant endometrium was studied histochemically. Only the lectin from Bauhinia purpurea (BPA) reacted exclusively with the epithelial cells. All the others reacted to a certain extent with glandular and/or stromal cells. Lectins from soybean (SBA), and Vicia villosa seeds (VVA) reacted with endometrial glands of pregnancy but not with the glands of the proliferative endometrium. In the proliferative endometrium SBA reacted only with cells of the surface endometrium. Lectin from peanuts (PNA) reacted only with some glands in the proliferative endometrium but was unreactive with others. In pregnant endometrium PNA reacted with all glands. Lectins from lentils (LCA) and red kidney beans (PHA-E and PHA-L) reacted with endometrial glands of the proliferative phase but not with the glands from pregnant endometrium. We thus show that FITC labeled lectins define specific carbohydrate moieties selectively expressed on either proliferative phase or pregnant endometrial glands.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

Detection of acrosome-reacted toad sperm based on specific lectin binding to the inner acrosomal membrane

When the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin-conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC-PNA-labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA-labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC-PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.     

Possible surface carbohydrates involved in signaling during conjugation process in Zygnema cruciatum monitored with fluorescein isothiocyanate-lectins (Zygnemataceae, Chlorophyta)

The changes of cell surface carbohydrates were examined with FITC (fluorescein isothiocyanate)‐labeled lectins during the conjugation process of the green alga Zygnema cruciatum. The Ulex europaeus agglutinin (UEA)‐specific materials were detected consistently on the surface of vegetative cells, but were absent on the surface of protruding papillae or conjugation tube. The tips of male and female papillae were labeled with soybean agglutinin (SBA) and peanut agglutinin (PNA) during conjugation. The SBA‐ and PNA‐specific materials appeared first at the tip of male papillae and began to accumulate on the surface of female papillae. No labeling of these lectins was detected on the surface of vegetative filaments throughout the conjugation process. FITC‐ConA (Concanavalin A) and FITC‐RCA (Ricinus communis agglutinin) did not label the vegetative filaments of Z. cruciatum, but a trace labeling of these lectins was observed on the surface of some swollen papillae occasionally. Blocking experiments with various lectins showed that these SBA‐ and PNA‐specific glycoconjugates might be involved in the signaling between male and female papillae.     

Lectin activity in pollen from plants representative of thirty orders of spermatophyta

Summary Pollen extracts from a variety of species representative of thirty orders of spermatophyta, including gymnosperms, dicotyledons and monocotyledons, were examined for the presence of lectin activity by means of a hemagglutination assay. Hemagglutinating activity (HA) was detected in the pollen extracts of all the species examined, indicating that lectins are generally present in the pollen of spermatophyta. The response of this pollen hemagglutinating activity to the sugars and glycoproteins tested as potential inhibitors was identical in all species examined. Moreover, the hemagglutinating activity of pollen extracts from eight species which had been selected as representative of the gymnosperms and both subclasses of angiosperms exhibited similar properties (e.g. distribution by differential centrifugation, stability to heat, response to bivalent ions). The bulk of the hemagglutinating activity was always recovered in the pellet after centrifugation at 1000 g for 5 min. Although sequential treatments with 1% Triton X-100 and 1 M KCl were ineffective, subsequent incubation of the pellet with saline phosphate buffer released hemagglutinating activity. The solubilized hemagglutinating activity was destroyed by protease treatment, indicating that the substance(s) responsible for the activity is (are) protein in nature and, consequently, might be considered to be a lectin. The sugar specifity of the pollen lectin activity from wheat, potato and bean was compared with that of wheat germ agglutinin (WGA), potato agglutinin and bean agglutinin — the lectins present in sporophytic tissues of these plants. For all three plants, the response of the pollen lectin activity to sugars and glycoproteins was different from that shown by the lectin from sporophytic tissues.Abbreviations HA Hemagglutinating activity - PBS 150 mM Na-phosphate buffer (pH 7.2) containing 0.9% NaCl - PHA Phaseolus vulgaris agglutinin - STA Solanum tuberosum agglutinin - WGA wheat germ agglutinin     

An Ouchterlony double diffusion study on the interaction between legume lectins and rhizobial cell surface antigens

Acidic exopolysaccharides and O-antigen containing lipopolysaccharides were isolated from Rhizobium japonicum, R. leguminosarum, R. lupini, R. meliloti, R. phaseoli, cowpea Rhizobium sp. and a non-nodulating soil bacterium. Lectins from seeds of soybean (Glycine max), garden pea (Pisum sativum), lentil (Lens culinaris), alfalfa (Medicago sativa), field bean (Phaseolus vulgaris), jackbean (Canavalia ensiformis) and from wheat germ were tested for their capacity to precipitate rhizobial exopolysaccharides and lipopolysaccharides in the Ouchterlony double diffusion test. Soybean lectin precipitated exclusively with the exopolysaccharide of R. japonicum, whereas the lectins from pea and lentil precipitated exopolysaccharides from all the fast growing strains of Rhizobium. Host range specific interactions between lipopolysaccharides and lectins were observed in the pea/lentil-R. leguminosarum and in the alfalfa-R. meliloti systems. Concanavalin A precipitated the exopolysaccharides of all fast growing strains of Rhizobium, the exopolysaccharide of the cowpea strain and several lipopolysaccharides of different Rhizobium species and thus did not show any correlation between polysaccharide binding and symbiotic specificity. Non-leguminous wheat germ agglutinin did not precipitate any of the rhizobial polysaccharides tested and the lipopolysaccharide of the soil bacterium did not precipitate with any of the lectins examined.Abbreviations Con A Concanavalin A - CPC cetylpyridinium chioride - EPS exopolysaccharide - FITC fluorescein isothiocyanate - KDO 2-keto-3-deoxyoctonic acid - LPS lipopolysaccharide - PBS phosphate-buffered saline - PS polysaccharide     

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.     

Binding and penetration ofRhizobium japonicum to cultured soybean cells

A cultured soybean cell line, SB-1 was used to evaluate the initial interaction between the soybean cells andRhizobium japonicum. Co-culturing ofR. japonicum with SB-1 cells in suspension resulted in strain-specific polar attachment. This attachment can be inhibited by galactose and antibodies raised against seed soybean agglutinin (SBA). A lectin was purified from SB-1 cells which shares properties with SBA in terms of immunological reactivity, sugar binding activity, polypeptide molecular weight and peptide maps. When the SB-1 cells were co-cultured withR. japonicum for three weeks in solid agar medium, histological staining revealed bacterial penetration into certain SB-1 cells. Furthermore, there were focal regions of cells with prominent nuclei representing actively proliferating regions. These observations are analogous to that ofin vivo nodule initiation in soybean roots.     

烟草与蓝猪耳胚胎发生中细胞表面三种凝集素受体的动态分布

以异硫氰酸甲酯(FITC)标记的三种凝集素(伴刀豆凝集素, 麦芽凝集素和大豆凝集素)为荧光探针,对烟草及蓝猪耳各发育时期胚细胞表面的凝集素受体进行了定位.结果显示胚柄基部荧光信号最强,沿胚柄单列细胞向胚体方向渐次减弱.以后随着胚柄功能的逐渐丧失而改变.同时,三种凝集素受体集中分布于胚柄细胞间的分裂面;凝集素受体在原胚中分布的另一个特点是聚集于新形成的细胞壁上.随着胚胎发育至分化阶段,凝集素受体则主要分布在胚体细胞的外切向壁上;三种凝集素受体的动态分布显示了凝集素受体的分布与细胞分裂之间的密切关系及其调控胚胎发育的作用.     

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Summary Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl -D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.Abbreviations ConA Concanavalin A - LCA Lens culinaris agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - DAPI 4,6-diamidinophenylindol×2 HCl     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D.Hyphal tips and septae were strongly labelled by short pulses of [³H] N-acetyl-D-glucosamine ([³H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA).The dynamics of wall formation was followed by pulse and chase as well as by pulse and wash treatments in which the colony was shortly exposed to [³H]GlcNAc and then freed from the radioactive chitin precursor.Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis.FITC-WGA binding, [³H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - GlcNAc N-acetyl-D-glucosamine     

137 Gamete Recognition and Sexual Isolation in a Red Alga,Aglaothamnion Byssoides (Rhdophyta)

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.     

Cellular heterogeneity in human transitional cell carcinoma: an anlysis of optical properties and lectin binding

Summary Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Wardet al., 1987). This difference was most marked withMaclura pomifera agglutinin (MPA),Ricinus communis agglutinin (RCA) andUlex europus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.     

Agglutination ofRhizobium japonicum 3I1b110 by soybean lectin

Summary Conditions leading to agglutination ofRhizobium japonicum 3I1b110 with soybean seed lectin were examined. Ability of cells to be agglutinated was transient and was optimal for cultures grown for 4–5 days on yeast extract mannitol plates. Similar lectin-binding results were obtained with cells from the same cultures using fluorescence microscopy with fluorescein isothiocyanate-labelled lectin. These results revise the previous model for soybean lectin-R. japonicum interactions, since it was based on the inability of soybean lectin to agglutinate these bacteria.     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria

Lectins were extracted from whole fern grindings ofAzolla pinnata (AP) andAzolla filiculoides (AF) by precipitation with ammonium sulfate to 20% of saturation. At high pH both lectins dissociate into inactive subunits (5000 mol wt) which reassociate into active aggregates (>500,000 mol wt) following concentration by ammonium sulfate precipitation or freezing and thawing. Although amino sugars inhibited hemagglutinating activity of both AP and AF lectins,d-fructose was inhibitory only to the AP lectin hemagglutinating activity, andd-galactose was slightly inhibitory to the AP lectin but not to the AF lectin. Both lectins exhibited specificity for freshly extracted cyanobionts from homologous fern species: AP lectin agglutinated cyanobiont filaments from AP, but not from AF; AF lectin agglutinated cyanobiont filaments from AF, but not AP. Neither lectin reacted with cultured cyanobionts from either fern species. Hemagglutinating titers were likewise reduced by adsorption of these lectins to homologous cyanobiont cells. This report provides strong suggestive evidence for specificity in this N-fixing symbiosis between aquatic fern and cyanobacterium.