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     

Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

Subunit exchange between lectins from different cereal species

Lectins from Triticum monococcum, Secale cereale (rye), and Hordeum vulgare (barley) can exchange their subunits in vitro and thereby form (intergeneric) heteromeric lectins. An analysis of the isolectin pattern of a Triticale variety revealed that intergeneric heterodimers of wheat and rye lectin subunits are normal constituents of the embryo cells. It appears, therefore, that these different cereal lectins are structurally so closely related that their subunits can not distinguish between identical and nonidentical partners when they associate into dimers.Abbreviations CL cereal lectin - SP Sephadex sulfopropyl Sephadex - WGA wheat germ agglutinin     

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     

An ultrastructural search for lectin-binding sites on surfaces of spinach leaf organelles

Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin     

A genetic basis for the origin of six different isolectins in hexaploid wheat

Wheat (Triticum aestivum) germ agglutinin represents a complex mixture of multiple isolectin forms. Upon ion exchange chromatography at pH 3.8, three isolectins can be separated, each of which is composed of two identical subunits. At pH 5.0, however, three additional isolectins can be distinguished, which are built up of two different subunits (heteromeric lectins). Evidence is presented that these heterodimers are normal constituents of the wheat embryo cells. Analyses of the isolectin patterns in extracts from Triticum monococcum, Triticum turgidum dicoccum and Triticum aestivum, provide evidence that each genome, either in simple or complex (polyploid) genomes, directs the synthesis of a single lectin subunit species. In addition, a comparison of the isolectin pattern in these wheat species of increasing ploidy level, made it possible to determine unequivocally the genome by which the individual lectin subunits in polyploid species are coded for. The possible use of lectins in studies on the origin of individual genoms in polyploid species is discussed.Abbreviations CL cereal lectin - PBS phosphate buffered saline - SP Sephadex sulfopropyl Sephadex - WGA wheat germ agglutinin     

Use of labeled lectins to investigate cell wall surfaces of the entomogenous hyphomycete Nomuraea rileyi

Nomuraea rileyi is an entomogenous fungus infecting lepidopterous defoliators; host range of the pathogen varies according to strain. Identifying surface components of infectious stages of different strains of the fungus may be an important step in understanding host-parasite interactions. A variety of fluorescein and ferritin-conjugated lectins, including concanavalin A, peanut, soybean, winged pea and wheat germ were used to investigate surface components on germ tube and hyphal body walls from two strains (FL 74, FL 78) of N. rileyi. Binding was observed on outer wall layers when both Con A and wheat germ agglutinin conjugates were tested. There was no apparent difference in binding between the two strains or between germ tubes and hyphal bodies. These results indicate the presence of mannose (and/or glucose) residues (Con A specifity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin specifity) on outer wall surfaces. Extracellular sheath material especially noticeable on germ tubes from the FL 74 strain was not labeled by any of the lectins tested, but was well stained with ruthenium red, indicating the presence of polysaccharides.Florida Agricultural Experiment Station Journal Series No. 4764.     

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     

Localization of chitin on ultrathin sections of cysts of two ciliated protozoa,Blepharisma undulans andPseudomicrothorax dubius,using colloidal gold conjugated wheat germ agglutinin

Summary The cyst walls of the ciliatesBlepharisma undulans andPseudomicrothorax dubius were examined ultrastructurally and by postembedding labeling with wheat germ agglutinin (WGA)-gold conjugate. Different methods of fixation and embedding were performed. In all procedures, WGA-gold binds selectively to material of the cyst wall. Pretreatment of the sections with chitinase inhibits labeling. The cyst walls of both species contain 3 nm fibrils, which are supposed to be of chitinous nature. In the cyst wall ofB. undulans, several thin layers of WGA-binding fibrils are interspaced with thick layers of other material. InP. dubius, WGA-binding sites are mainly concentrated in the mesocyst, where the microfibrils appear to represent the major component. These results obtained from two phylogenetically distant species confirm that chitin synthesis is an ancestral feature of ciliated protozoa. The amount and distribution of the chitin fibrils may play an important role in the properties and functions of the wall of the resting cyst.     

Lectin binding sites during Drosophila embryogenesis

The spectrum of lectin binding sites as it emerges during embryonic development of Drosophila was analysed by means of fluorescein-labelled lectins. As development and morphogenesis proceed, the reaction pattern becomes more and more complex. Mannose/glucose-, mannose-, N-acetylglucosamine- and poly-N-ace-tylglucosamine-specific lectins bind ubiquitously. Nuclear envelopes only have binding sites for wheat germ agglutinin. N-acetylgalactosamine-binding lectins are specific for ectodermal derivatives. Ga-3-N-acetylgalac-tosamine-binding lectins are highly selective markers for neural structures, haemocytes and Garland cells. It is also shown that Drosophila laminin is differentially glycosylated. The possible implications of differential and germ layer-specific glycosylation are discussed.Dedicated to the memory of Jan Callaerts     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Lectin-receptor interactions in liposomes

The major sialoglycoprotein of mammalian erythrocytes has been incorporated into phosphatidylcholine membranes to generate a model system, glycoprotein-liposomes. Electron microscopic examination revealed these structures to be vesicles, approximately 300 Å in diameter. An aqueous compartment inside the glycoprotein-liposomes has been identified by trapped volume studies with [¹⁴C]sucrose. These glycoprotein-liposomes were found to interact with the lectins, wheat germ agglutinin, and phytohemagglutinin, to form aggregates of mainly unfused vesicles. The aggregation process has been studied by electron microscopy, 90° light scattering, and differential ultracentrifugation analysis. Hapten inhibitors of the lectins were found to inhibit the lectin-induced aggregation of the glycoprotein-liposomes. Binding of ¹²⁵I-labeled wheat germ agglutinin to glycoprotein-liposomes was studied by differential ultracentrifugation. Hapten inhibitors of wheat germ agglutinin were also found to inhibit the binding of ¹²⁵I-labeled wheat germ agglutinin to the glycoprotein-liposomes. The characteristics of the lectin interactions with glycoprotein-liposomes appeared to be phenomenologically similar to lectin-cell interactions.     

The use of iodinated lectins for determining the degree of deglycosylation of high-mannose glycoproteins by endo-beta-N-acetylglucosaminidase H

A method which demonstrates that the removal of polymannosyl chains from glycoproteins by endo-β-N-acetylglucosaminidase H can be monitored reliably using only submicrogram quantities of glycoprotein is described. Glycoproteins and their endoglycosidase-treated forms are subjected to electrophoresis on SDS-polyacrylamide gels, which are then overlaid with [¹²⁵I]concanavalin A or [¹²⁵I]wheat germ agglutinin. The degree to which these lectins bind is measured by autoradiography. The complete loss of [¹²⁵I]concanavalin A binding by glycoproteins such as deoxyribonuclease I, ovalbumin, carboxypeptidase Y, and invertase is associated with the removal of their oligosaccharide chains. Invertase, unlike the above mannose-containing glycoproteins, acquires the capacity to bind [¹²⁵I]wheat germ agglutinin only upon partial or complete deglycosylation, a finding substantiated by wheat germ agglutinin-Sepharose column chromatography. In addition to providing a procedure for monitoring the enzymatic deglycosylation of mannose-containing glycoproteins, the lectin-gel binding technique is shown to provide an estimate of the mannose content of neutral glycoproteins at levels which cannot be detected by conventional methods. In some cases, this method may be useful in distinguishing between N- and O-glycosidic linkages where the oligosaccharide is predominantly mannosyl.     

Enhancement of glycosylation of cellular glycoconjugates in the squamous carcinoma cell line MDA886Ln by β-all-trans retinoic acid

Retinoids have been shown to inhibit the growth and modulate the glycosylation of head and neck squamous cell carcinoma (HNSCC) cells including the MDA886Ln cells. To examine the effects of -all-trans retinoic acid (RA) on glycoconjugates in HNSCC MDA886Ln cells, the cells were grown in the absence or presence of 1 µM RA and then labeled with tritiated monosaccharides, extracted and analysed by polyacrylamide gel electrophoresis and fluorography. RA increased markedly the incorporation of [³H]-glucosamine, [³H]-galactose, and [³H]-mannose into numerous cellular glycoconjugates, however, the incorporation of [³H]-fucose and [³H]-leucine was almost unaffected by RA. RA increased the incorporation of glucosamine and galactose but not mannose into high molecular weight (HMW) glycoconjugates of about 220 and 500–600 kDa. To analyse the steady state level of glycoconjugates by lectin blotting, extracts of unlabeled cells were separated by gel electrophoresis and the gels were probed with¹²⁵I-labeled wheat germ agglutinin (WGA) andMaackia amurensis (MA) agglutinin. Both lectins were found to bind to numerous glycoconjugates including the HMW glycoconjugates, whereas¹²⁵I-peanut agglutinin bound only to the HMW glycoconjugates. RA treatment increased the binding of all three lectins to the HMW glycoconjugates. These findings demonstrate that RA enhanced the incorporation of specific monosaccharides into a variety of glycoconjugates and in particular into HMW mucin-like glycoconjugates. This effect of RA may be the result of induction of a more normal differentiation state of the HNSCC cells.     

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5′-diphospho-N-[²H]-acetyl-glucosamine and the chemical preparation of N-[²H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.     

Mating receptor complex in the flagellar membrane of Chlamydomonas eugametos gametes

Summary The protein composition of the flagellar membrane of C. eugametos mt ^–gametes was analyzed using SDS-polyacrylamide gel electrophoresis. The association of the proteins with the membrane was assessed by differential extraction and an assay for glycosylation. Particular attention was paid to integral membrane proteins that could be associated with the mt ^– agglutinin, the membrane-bound sexual receptor by which the mt ^– gamete binds to its mt ⁺ partner. This agglutinin is a peripheral membrane glycoprotein and must be bound to the flagellar surface by an integral membrane anchor protein that connects the agglutinin with the cell's interior. Immunoaffinity chromatography was performed using Mab 66.3, a monoclonal antibody specific for the mt ^– agglutinin, in order to isolate protein complexes consisting of agglutinin molecules and associated components. Only one integral membrane glycoprotein (M_r = 125 kDa) was isolated that has an association with the agglutinin. It did not bind Mab 66.3, but did bind the lectin wheat germ agglutinin. This was an expected property of the membrane anchor protein because previous research (Kooijman et al. 1989) has shown that cross-linking a WGA-binding glycoprotein by this lectin induces sexual responses that are similar to those induced by agglutinin-agglutinin interactions during mating. We conclude that the 125-kDa glycoprotein is the membrane anchor for the agglutinin.Abbreviations BSA Bovine serum albumin - CBB Coomassie Brilliant Blue - CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate - GTC guanidine thiocyanate - mt ^–/mt ⁺ mating type minus/plus - PAS periodic acid Schiff - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TBS TRIS-buffered saline - WGA wheat germ agglutinin     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests,Nilaparvata lugens andNephotettix cinciteps

Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.