Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.     

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.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L．)、鸢尾(Iris tectorium Maxim．)和朱顶红(Hippeastrum vittatum Herb．)的生殖细胞质膜表面的凝集素受体进行标记。结果显示：在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布。这可能是导致部分精子表面不能被同种凝集素标记的重要原因。此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同。在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体。在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体。此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示。     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L.)、鸢尾(Iris tectorium Maxim.)和朱顶红(Hippeastrum vittatum Herb.)的生殖细胞质膜表面的凝集素受体进行标记.结果显示:在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布.这可能是导致部分精子表面不能被同种凝集素标记的重要原因.此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同.在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体.在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体.此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示.     

Purification and Amino-terminal Sequencing of a 68 kD Glycopolypeptide of the Plasma Membrane from Maize Sperm Cells

Based on author's previous work on detection and immunolocalization of glycoproteins of the plasma membrane of maize ( Zea mays L. ) sperm cells, a 68 kD peripheral specific glycopolypeptide of the plasma membrane from maize sperm cells was purified by IEF-SDS two-dimensional electrophoresis. It presents specif- ically positive reaction in Con A-HRP (concanavalin A-horseradish peroxidase) staining, and its pi value is 5.5. The search in protein sequence database reveals that the amino-terminal sequence of this glycopolypeptide is identical with that of Con A. But its difference from Con A in molecular weight and pi value indicates that it could be related to a Con A receptor on the plasma membranes of maize sperm cells instead of being Con A itself. It is fascinating to study further the function of the above glycopolypeptide in gametic recognition, adhesion and fusion of the double fertilization in maize.     

玉米精细胞质膜68kD特异糖蛋白组分的纯化和N—端序列测定

经ＳＤＳ＿ＰＡＧＥ和ＩＥＦ＿ＳＤＳ双向电泳，从玉米（ＺｅａｍａｙｓＬ．）精细胞质膜分离纯化得到等电点（ｐＩ）为５．５的６８ｋＤ糖蛋白的单一组分。伴刀豆球蛋白＿辣根过氧化物酶（ＣｏｎＡ＿ＨＲＰ）染色结果表明，该组分的糖链中含有甘露糖及葡萄糖残基。氨基酸序列分析表明，该组分的Ｎ＿端１５个氨基酸序列与ＣｏｎＡ的Ｎ＿端相应序列相同。根据分子量及ｐＩ值的差异，认为该组分并非ＣｏｎＡ，而可能与玉米精细胞质膜上的ＣｏｎＡ受体有关。它在玉米的精卵识别、粘附及融合中有何作用，无疑是令人感兴趣的问题。     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

Localization of l-fucose in the root cap and slime of Zea mays L. utilizing fluorescent lectin conjugates and colloidal gold-lectin techniques

Ulex europaeus lectin (UEA) labelled with fluorescein isothiocyanate (FITC), rhodamine or colloidal gold, localized l-fucose in maize root cap cells and secreted root cap slime. Free-hand sections of maize root apices stained with FITC-UEA or rhodamine-UEA and examined by fluorescence microscopy yielded satisfactory results as long as the stains were freed of unconjugated dye, the sections treated with osmium tetroxide vapour to quench autofluorescence, and the samples incubated at 37°C. This resulted in successful labelling with a lower concentration of fluorochrome-lectin conjugate than reported by previous workers. Rhodamine-UEA was superior to FITC due to the lower primary fluorescence of the root tip observed under green light.Thin sections from glutaraldehyde fixed and Spurr's resin embedded maize root tips were treated with UEA bound to colloidal gold. Gold particles were found within sloughed cells and root cap cells, particularly concentrated over the Golgi complex, Golgi-derived vesicles and within the secretory slime products.     

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     

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     

The effects of shRNA-mediated gene silencing of transcription factor SNAI1 on the biological phenotypes of breast cancer cell line MCF-7

Developing spermatozoa require a series of posttesticular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation-dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5 kDa wheat germ agglutinin (WGA)-binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA-stained bands, the presence of a 17.5 kDa WGA-binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5 kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5 kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide.     

A phloem-specific,lectin-like protein is located in pine sieve-element plastids by immunocytochemistry

Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from homogenates of Pinus sabiniana Dougl. phloem by differential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucosamine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloemspecific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133–137; A.K. Gietl et al., 1979, Planta 144, 367–371). Fluorescence microscopy and immuno-gold electron microscopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals peculiar to the sieve-element plastids of the Pinaceae.Abbreviations DAB diamino benzidine tetrachloride - FITC fluorescein isothiocyanate - kDa kilodalton - PBS phosphate-buffered saline - PP phloem polypeptide(s) - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Confocal and fluorescence microscopic study using lectins of the distribution of cortical granules during the maturation and fertilization of pig oocytes

A study was carried out to determine whether pig cortical granules (CGs) could be visualized using fluorescein isothiocyanate (FITC)-labelled lec-tins. Following labelling with FITC-labelled peanut agglutinin (FITC-PNA), fluorescent spots were observed that had a distribution during maturation and fertilization entirely consistent with that observed by electron microscopy. For the first 18 h of in vitro maturation, most of the fluorescent spots of FITC-PNA were distributed throughout the cortical cytoplasm. Thereafter, the CGs underwent centrifugal migration to form a monolayer next to the plasma membrane. Following penetration by sperm, fluorescent spots were extruded into the perivitelline space, where they aggregated forming fluorescent clumps, which subsequently formed a reticulate structure surrounding the egg. Fluorescence was gradually lost such that by 18 h after insemination none could be detected in 70% of the eggs. The results indicate that CGs in pig oocytes contain galactosyl-rich glycoconjugates and that FITC-PNA is a useful probe for their rapid visualization and examination. © 1993 Wiley-Liss, Inc.     

Electrophoretic map of boar sperm plasma membrane polypeptides and localization and fractionation of specific polypeptide subclasses

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.     

Concanavalin A binds to the endoplasmic reticulum and the starch grain surface of root statocytes

Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the amyloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of membrane components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the membrane is a glycoprotein tightly inserted in other components of the compartment.Abbreviations Con A Concanavalin A - ER endoplasmic reticulum - FITC fluorescein isothiocyanate - NP 40 nonidet P40     

Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: A flow cytometric study using lectins

Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 μg/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. © 1996 Wiley-Liss, Inc.     

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.     

Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry

Summary Many of the flagellates inhabiting the hindgut of lower termites are associated with ectobiotic, rod-like bacteria or spirochetes. Different types of attachment sites are present. Electron dense material underlies, e.g., the plasma membrane ofJoenia annectens at the contact site, whereas other attachment sites do not show any visible specializations. The host cell's glycocalyx may, however, be reduced at the attachment sites as it is the case inDevescovina glabra. The thick glycocalyx ofStephanonympha nelumbium is not changed at the sites where bacterial rods attach, but spirochetes penetrate to a certain extent. Bacteria which colonize the extracellular surface structures ofMicrorhopalodina multinucleata express their own glycocalyx to mediate a contact. In this study we focussed on the examination of one common mode of interaction between bacteria and their host cells, i.e., adhesion via lectins and sugars. The sugar composition was analysed by light and electron microscopic labelling experiments using the lectins Con A, WGA and SBA. In general, only the posterior body surface ofJoenia which is colonized with bacteria is labelled. The demonstrated sugars are found in fibrous glycocalyx portions surrounding the attachment sites of the bacteria. Such glycocalyx fibres in combination with the electron dense material supporting the attachment sites seem to be the prerequisites for bacterial attachment. InD. glabra, however, a role for sugars in mediating the attachment could not be demonstrated. Removal of the ectobiotes using antibiotics revealed that the specialized contact sites ofJoenia are present in the absence of bacteria and thus possibly serve to attract bacteria. Nothing, however, remains of the former attachment sites in bacteria-freeDevescovina cells. Attachment sites in this case could be induced by bacterial contact. There is not one general mechanism for bacterial attachment to termite flagellates; rather, adhesion seems to follow different strategies.Abbreviations Con A concanavalin A - DAB 3,3-diaminobenzidine tetrahydrochloride - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - FA formaldehyde - FITC fluorescein isothiocyanate - GA glutaraldehyde - PB Soerensen's phosphate buffer - PC phase contrast - pen/strep penicillin and streptomycin - SBA soybean agglutinin - SEM scanning electron microscope - TBS Tris buffer saline - TEM transmission electron microscope - WGA wheat germ agglutinin Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Lectin in five soybean cultivars previously considered to be lectin-negative

Hemagglutinating proteins were isolated by affinity chromatography from seeds of each of five cultivars of soybeans (Clycine max (L.) Merr.) previously reported to lack detectable lectin (S.P. Pull et al., 1978; Science 200, 1277). Quantities were between 1,000 and 10,000 times less than that found in the seeds of the reference cultivar, Chippewa. The sensitivity of the hemagglutinating assay was 0.05 g ml^-1. Hemagglutinating activity was demonstrated in affinity-purified fractions from bulk seeds and seeds from individual plants in two cultivars, 30–70% ammonium-sulfate-precipitable fractions of seeds from individual plants of all five cultivars, and in whole crude extracts of individual seeds from each cultivar. In all instances, hemagglutinating activity was inhibited by galactose, anti-soybean agglutinin (SBA), and lectin-binding polysaccharide produced by Rhizobium japonicum. Affinity-purified lectin from seeds of a single Columbia plant was labeled with fluorescein isothiocyanate (FITC) and observed by fluorescence microscopy to bind to R. japonicum cells with specificity, intensity and localization indistinguishable from FITC-SBA. Lectins from distinguishable from FITC-SBA. Lectins from three cultivars in sufficiently high concentration for study had molecular properties very similar to Chippewa SBA.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - SBA soybean agglutinin