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1.
Svend Kirkeby Thorkild C. B?g-Hansen Dennis Moe Charly Garbarsch 《The Histochemical journal》1991,23(8):345-354
Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections. 相似文献
2.
Jarig Darbès Florian Colbatzky Günter Minkus Walter Hermanns 《Histochemistry and cell biology》1993,100(1):83-91
Canine and feline platelet cytocentrifuge preparations (CCPs), cryostat and paraffin-embedded bone marrow sections were used in this study. We evaluated whether platelets, megakaryocytes and megakaryocyte precursor cells could be labelled by monoclonal antibodies (Y2/51, CLB-thromb/1, HPL1) against human platelet membrane glycoprotein GP IIIa and the GP IIb/IIIa complex or by the following 10 biotinylated lectins: concanavalin A (Con A), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PsA), wheat germ agglutinin (WGA), peanut agglutinin (PNA), Phaseolus vulgaris lectin (PHA-L), Ricinus communis agglutinin 120 (RCA120), Ulex europaeus agglutinin — I(UEA-1), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). Monoclonal antibodies Y2/51 and HPL1 cross reacted with platelets and megakaryocytic cells from both species, whereas CLB-thromb/1 was unreactive with canine preparations. Only Y2/51 labelled megakaryocytic cells in paraffin-embedded samples. LCA, PSA, WGA and PHA-L labelled feline and canine platelets and different numbers of morphologically identifiable megakaryocytes and numerous other, mostly myeloid, cells. Immunoblots of dog and cat platelet lysates using Y2/51 visualized a single protein of 95 kDa (unreduced), a mol·wt value within the range of those reported for GP IIIa. Some of the platelet (but not necessarily megakaryocyte) glycoproteins reacting with LCA, PSA and WGA could be identified in lectin blots following one- or two (nonreduced/reduced)-dimensional sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Thus in dogs and cats, the immunohistochemical detection of GP IIIa (and eventually GP IIb/IIIa) rather than lectin binding patterns could be important for the diagnosis of megakaryoblastic leukaemias. 相似文献
3.
T. G Honegger 《Experimental cell research》1982,138(2)
The effect of concanavalin A (conA), fucose-binding protein (FBP), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) on fertilization of the ascidian Phallusia mammillata was investigated. ConA, FBP, and RCA had no influence on fertilization and did not bind to the chorion or sperm, as determined with FITC-conjugated conA and by electron microscopy with gold-labelled FBP. WGA (100 μg/ml) prevented fertilization of eggs by sperms in concentrations which gave 100% fertilization in controls (2 × 107 sperm/ml). N-Acetyl-glucosamine (50 mM) abolished the effect of WGA, whereas an excess (100 mM) of this competitive sugar alone did not affect fertilization. FITC-conjugated and gold-labelled WGA revealed binding sites on the chorion, but not on follicle cells nor sperms. Electron microscopy showed that WGA gold-markers are bound to the fibrillar network forming the outer layer of the chorion and indicate that WGA inhibits fertilization by interfering with sperm binding to the chorion. Binding of WGA to the chorion may either mask sperm binding receptors or cause chorion resistance to sperm enzymes. 相似文献
4.
This study determined the effects of lectin binding to theronts of Ichthyophthirius multifiliis on theront immobilization, invasion, trophont development and survival in channel catfish Ictalurus punctatus excised fins in vitro. Soybean agglutinin (SBA), lentil agglutinin (LCA), gorse agglutinin (UEA-I) and wheat germ agglutinin (WGA) were used to treat theronts. Percentages of theronts immobilized by 4 lectins ranged from 12.0 to 19.4% at a concentration of 1000 microg ml(-1). These lectins bound more than half of the theronts at a concentration of 50 microg ml(-1). More theronts were labeled by SBA and WGA than by lectin LCA at concentrations of 50 and 100 microg ml(-1), respectively. The binding of these lectins to theronts indicated that monosaccharides (D-galactose, L-fucose, D-mannose and D-glucose) and amino sugar derivatives (N-acetylgalactosamine and N-acetylglucosamine) were present on the surface of theronts. Invasion was reduced significantly for theronts treated with LCA, UEA-I and WGA. No difference in invasion was found between control and SBA bound theronts (p > 0.05). The binding of lectin LCA, UEA-I and WGA to theronts significantly reduced the development of trophonts (p < 0.05). The mean volumes of trophonts labeled with these 3 lectins were smaller than volumes in control trophonts from 8 to 48 h after exposure. Survival was lower in trophonts labeled with lectins than in control trophonts at 48 h after exposure. 相似文献
5.
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. 相似文献
6.
Enrique J. del Pino Marcelo O. Cabada María C. Fonio 《Molecular reproduction and development》1981,4(2):97-104
Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination. 相似文献
7.
SYNOPSIS. Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing α-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and α-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with α-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with α-1,4 or repetitive α-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera. 相似文献
8.
Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa. 相似文献
9.
Dissociated epidermal cells derived from the backskin of scaleless chick embryos (stage 34 or 35) form larger agglutinates with wheat germ agglutinin (WGA) than epidermal cells from normal embryonic skin. [3H]Acetyl WGA binding to the scaleless cells is twice as great as to normal epidermal cells. Treatment of these cells with concanavalin A (conA) results in equivalent agglutination of both mutant and normal epidermal cells, whereas neither scaleless nor normal epidermal cells are agglutinated by Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) or Ulex europeus agglutinin (UEA). This alteration in cell surface carbohydrates may be related to the failure of the scaleless mutant embryonic epidermis to undergo normal morphogenesis. 相似文献
10.
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 相似文献
11.
Pamela Stanley Louis Siminovitch 《In vitro cellular & developmental biology. Plant》1976,12(3):208-215
Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney
beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (PhaR) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis;
and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine
(GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased
ability to bind125I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase
activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each
of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive
cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation
data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.
Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture
Association, Montreal, Quebec, June 2–5, 1975. 相似文献
12.
CELSO VATARU NAKAMURA MANUEL JOSE GIL ESTEVES ARNALDO FEITOSA BRAGA ANDRADE CELUTA SALES ALVIANO WANDERLEY DE SOUZA JAYME ANGLUSTER 《The Journal of eukaryotic microbiology》1992,39(2):303-309
ABSTRACT. Cell surface carbohydrates of three phytoflagellates, Phytomonas francai. Phytomonas serpens and Phytomonas sp. from different hosts including cassava, coreid insect Phthia picta and the milkweed plant Euphorbia hyssopifolia, respectively, were analysed by agglutination assays employing a battery of highly purified lectins with affinity for receptor molecules containing N-acetylglucosamine (d-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose, mannose-like (D-Man-like) residues and fucose, and by binding assay using radiolabeled [125I]-wheat germ agglutinin (WGA) and fluorescent WGA lectin, as well as glycosidases of known sugar specificity, Escherichia coli K with mannose-affinity fimbrial lectin was also used as an agglutination probe. In general, the presence of D-GlcNAc. D-GalNAc and D-Man-like residues was detected in the phytomonads' plasma membrane. These sugar moieties were confirmed in whole cell hydrolysates as assessed by gas-liquid chromatography (GLC) which in addition, also showed the presence of galactose and xylose. However, marked differences in cell surface carbohydrate structures were observed. Wheat germ agglutinin, which binds to sialic acid and/or d-GlcNAc-containing residues, shows selective agglutinin activities for P. francai and Phytomonas sp., while Bandeiraea simplicifolia II agglutinin (which recognizes d-GlcNAc units) specifically bound to Phytomonas sp. Helix pomatia agglutinin which binds to D-GalNAc-containing residues reacted preferentially with Phytomonas sp. and P. serpens. Con A, which recognizes D-Man-like receptors, agglutinates all the phytomonads; however, the higher interaction was observed with Phytomonas sp. P. francai was selectively agglutinated in the presence of E. coli fimbrial lectin. Fluorescence WGA binding was significantly decreased by N-acetylglucosaminidase activities and the cell agglutination was not altered by neuraminidase treatment, suggesting the presence of an exposed D-GlcNAc moiety on the P. francai and Phytomonas sp. surfaces. Binding studies with [125I]-WGA essentially confirmed the fluorescence WGA binding and agglutination assays. 相似文献
13.
L. D. Truong V. T. Phung Y. Yoshikawa C. A. Mattioli 《Histochemistry and cell biology》1988,90(1):51-60
Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies. 相似文献
14.
Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens. 总被引:1,自引:0,他引:1
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. 相似文献
15.
In the present work we have compared the binding of fluorescein-conjugated lectins (concanavalin A (ConA), wheat germ agglutinin (WGA), fucose binding protein (FBP) and soybean agglutinin (SBA)) to the sperm surface and to the egg and its envelopes of Ciona intestinalis. Only WGA is bound to the follicle cells: yet this lectin has no binding sites on the sperm surface. Both ConA and FBP are bound by the chorion, the oolemma and the sperm surface. However, while ConA reacts only with the sperm head, FBP is bound both to the head and to the flagellum. Experiments on the effect of ConA and FBP on the fertilization reaction have been carried out. The role of the lectin-binding sites that are shared by the surfaces of both gametes is discussed in connection with the nature of the sperm-binding sites. 相似文献
16.
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. 相似文献
17.
PETER R. JACKSON B. M. HONIGBERG STANLEY C. HOLT 《The Journal of eukaryotic microbiology》1978,25(4):471-481
SYNOPSIS. Living, intact bloodstream trypomastigotes and culture procyclic forms of Trypanosoma congolense were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose binding protein (FBP). Similar experiments were conducted with living bloodstream and culture forms treated with trypsin or dextranase. Parasites were incubated for 30 min at 25 C in various concentrations of each lectin, then examined for agglutination by dark-field microscopy. Control preparations consisted of parasites incubated alone or with 0.5 M of the specific competing sugar, with or without the corresponding lectin. Electron-microscopic localization of lectin binding sites on the surface of intact and dextranase-treated bloodstream and intact culture forms was accomplished with Con A, reacted with horseradish peroxidase (HRP) and then diaminobenzidine (DAB). In addition, FBP and SBA were coupled to HRP, then utilized for the localization of binding saccharides on the surface of blood-stream forms by the DAB technic. Similar studies were conducted with culture procyclics incubated with WGA-, SBA, PP- or FBP-HRP conjugates and then reacted with DAB. Controls were utilized to confirm the sugar specificity of all positive reactions. Intact living bloodstream forms were agglutinated in a concentration-dependent manner with all the lectins tested. Agglutination levels were scored as Con A > FBP > WGA = PP = SBA. Sugars resembling α-D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and α-L-fucose are evidently present on the surface of the parasites. No agglutination was noted in any control preparations. Identical lectin-induced agglutinations were obtained with trypsin- or dextranase-treated bloodstream forms. Trypsin disrupted but did not entirely remove the surface coat of bloodstream forms, while dextranase did not alter the ultrastructure of the parasites. Con A-, SBA- and FBP-binding saccharides were distributed uniformly on the surface coat of intact bloodstream forms; a similar distribution of Con A receptors was noted also on the surface of dextranase-treated cells. No lectin-binding saccharides were visualized by electron microscopy on any control preparations. Intact, trypsin- or dextranasetreated, procyclics were agglutinated in a concentration-dependent fashion by Con A and WGA, but not by the other lectins tested. Control preparations did not agglutinate and the enzymes did not affect the ultrastructure of the parasites. Con A- and WGA-specifically binding saccharides were uniformly distributed on intact procyclics and control preparations were lectin-negative. Thus, T. congolense procyclics retained surface saccharides resembling α-D-mannose and N-acetyl-D-glucosamine but lost sugars resembling N-acetyl-D-galactosamine (or D-galactose) and α-L-fucose. The failure of dextranase to remove the lectin-binding saccharides from the surface of bloodstream and procyclic forms suggests that α-1,6-glucan bonds do not link these carbohydrates. The results are contrasted with lectin research on other trypanosome species and discussed with relation to the biology of T. congolense. 相似文献
18.
SYNOPSIS. Surface saccharides in 2 Trichomonas vaginalis strains, the moderately pathogenic, JH34A, and the mild, JH162A, were analyzed with the aid of plant lectins. Concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and lectin from the garden pea (GPA) were employed in agglutination tests and in treatment of ultrathin sections for electron microscopy according to the horseradish peroxidase-3,3′-diaminobenzidine method. With Con A and WGA, small quantitative differences were noted between the 2 strains in the results of agglutination and in the reaction-product deposits observed by electron microscopy. Distribution of the binding sites for the 2 lectins was also somewhat different in the JH34A and JH162A trichomonads. In general, the reactions with the more pathogenic strain were slightly stronger. Although the reactions with SBA and CBA lectins were weaker than those with Con A or WGA, they provided the means for qualitative differentiation between the 2 trichomonad strains. SBA alone agglutinated the JH34A strain and formed demonstrable deposits on the cell surfaces. On the other hand, only CBA reacted with JH162A flagellates. The garden pea lectin failed to bind to the surface of either strain. On the basis of results obtained with the control preparations incubated in the presence of specific inhibitors, it was concluded that both strains had α-methyl-D-mannoside and/or α-methyl-D-mannoside-like as well as N-acetyl-D-glucosamine residues on their surfaces. In addition, JH34A strain had D-lactose-containing residues while JH162A trichomonads had residues with D-galactose. Neither strain appeared to possess residues containing N-acetyl-D-galactosamine. 相似文献
19.
The effects of cytochalasin B (CB) and colchicine on the lectin-mediated agglutination of dissociated cells from chick embryos at the early primitive streak stage were studied. Cells incubated in the absence of the above-mentioned drugs were agglutinable with concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA). A pre-incubation with neuraminidase was required to render the cells agglutinable with soybean agglutinin (SBA). This treatment had no appreciable effect on the agglutinability of the cells with the other three lectins. Treatment with the drug colchicine had no appreciable effect on the extent of agglutination with any of the above-mentioned lectins. Cells treated with CB dissolved in dimethylsulfoxide (DMSO) in saline, exhibited a reduced lectin-mediated agglutinability. However, a similar decline in agglutinability was observed in controls incubated in saline containing DMSO alone. It is suggested that structures sensitive to colchicine and CB do not play a major role in the control of surface lectin receptors in early embryonic cells. 相似文献
20.
Summary Lectin-histochemical studies were performed on formalin-fixed, paraffin-embedded tissues from ten mammalian species to demonstrate the pattern of carbohydrate residues in vascular endothelium. Ten different biotinylated lectins were used as probes and avidin-biotin-peroxidase complex (ABC) was used as visualant. Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA) stained vascular endothelium in all species. Peanut agglutinin (PNA) stained vascular endothelium in all species only after preincubation with neuraminidase. Bandeirea simplicifolia agglutinin-I (BS-I) stained vascular endothelium in all species but human, while, Ulex europeus agglutinin-I (UEA-I) stained only human endothelium. Individual differences in staining of human vascular endothelium were noted with BS-I and succinylated-WGA (SWGA). Similarly, individual differences in staining of animal vascular endothelium were noted with soybean agglutinin (SBA) after preincubation with neuraminidase. Finally, Concanavalia ensiformis agglutinin (Con A), Dolichos biflorus agglutinin (DBA) and Lens culinaris agglutinin (LCA) did not stain vascular endothehuman in any of the species studied. 相似文献