Influence of mucosal cell origin on the in vitro adherence of Candida albicans: Are mucosal cells from different sources equivalent?

The influence of collecting mucosal cells from various anatomical sites, and varying the date of collection and cell donor on adhesion of Candida albicans to human epithelial cells was examined by using an in vitro adherence assay. Examination of buccal mucosal cells from twenty-four donors showed statistically significant differences in the number of attached yeasts between individuals. Sex did not exert a significant influence on adhesion. Examination of buccal mucosal cells from ten donors collected on five different dates revealed that yeast attachment to mucosal epithelial cells varied significantly within subjects across time. Epithelial cells from some donors manifested greater date-to-date variations in yeast adhesion than others. Adherence of Candida to mucosal cells from three anatomical sites (mouth, vagina and urinary tract) collected from ten different donors was also tested. Yeast adherence to buccal cells was highest, lowest using urinary tract cells, while vaginal epithelium was intermediate. Adherence to mucosal cells from three sites was significantly different both within and between individuals although some subjects manifested larger variations than others. These data suggest that the in vitro adherence of Candida albicans is influenced by mucosal cell donor, date of collection and body site of origin. Mucosal cells from different sources do not appear to be equivalent in receptiveness to C. albicans and this might explain some of the discrepancies observed when adhesion studies performed by different investigators are compared. The existing need for a more uniform methodology with which to pursue studies on fungal attachment to mucosal surfaces is emphasized.     

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons     

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     

Chemical characterization of the lectin from Amaranthus leucocarpus syn. hypocondriacus by 2-D proteome analysis

In this work, we characterized chemically the N-acetyl-D-galactosamine specific lectin from Amaranthus leucocarpus syn hypocondriacus lectin (ALL). It is a dimeric glycoprotein composed by three isoforms with pl at 4.8, 4.9, and 5.2. Circular dichroism analysis indicated that the secondary structure of ALL contains 45% of -sheet and 5% of -helix. Amino acid sequence of the purified lectin and its isoforms was determined from peptides obtained after trypsin digestion by MALDI-TOF (Matrix assisted laser desorption ionization-time of flight). The tryptic peptides prepared from the purified lectin and the three isoforms showed different degrees (80 to 83%) of identity with the amino acid sequence belonging to a previously described high nutritional value protein from A. hypocondriacus not shown at the time to be a lectin. Furthermore, analysis of tryptic peptides obtained from ALL previously treated with peptide N-glycosidase, revealed a 93% identity with the aforementioned protein. Presence of N-glycosidically linked glycans of the oligomannosidic type and, in minor proportion, of the N-acetyllactosaminic type glycans was determined by affinity chromatography on immobilized Con A.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

Protection of catheter surfaces from adhesion of Pseudomonas aeruginosa by a combination of silver ions and lectins

A catheter surface was modified by coating a cellulose acetate polymer. Adhesion of Pseudomonas aeruginosa ATCC 27853 to the surface was investigated by exposing bacterial cultures to three treatments: polymer impregnated with silver ions (Ag⁺), polymer surfaces coated with lectins and a combination of Ag⁺ and a lectin coating. The effective concentration of Ag⁺ providing protection against bacterial biofilm development was 100g/ml and higher. Lectins alone at 10% also showed inhibition of bacterial attachment. However, the best result was achieved against bacterial adhesion and growth on surfaces using a combination of 100 g Ag⁺/ml and a lectin coating as a surface treatment. This surface treatment was also effective against both fresh culture and a two-week-old culture containing P. aeruginosa producing exopolymers. Our results suggest that Ag⁺impregnation combined with a lectin coating warrants further investigation as a potential means of protecting catheters.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

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.     

Adhesion of sea-urchin embryonic cells to substrata coated with cell adhesion molecules

Summary— A cell-to-substratum adhesion assay is developed to study the adhesion of sea-urchin embryonic cells to coated substrata. The involvement in this process of both carbohydrate and protein molecules is reported. Concanavalin A (Con A) increases the attachment of cells to the substratum in a dose-dependent manner and this effect is completely abolished when the incubation is carried out in the presence of the specific monocarbohydrate Con A-inhibitor, α-methyl-d -mannoside. A Con A-mediated enhancement of cell-to-substratum adhesion was also detected on cells deprived of toposome, a glycoprotein complex responsible for cell-to-cell adhesion. The involvement of other molecules as well as toposome in the process of cell-to-substratum adhesion is also investigated. Results of these in vitro experiments indicate that all the molecules tested contribute to the process of cell-to-substratum adhesion.     

Modification of Drosophila cell surfaces by concanavalin A

Summary The effect of Con A on the surface morphology of cultured cells of Drosophilia melanogaster growing on coverglasses was examined by scanning electron microscopy. With low lectin concentrations (5–10g/ml) surface filaments disappeared and the cells flattened and spread against the glass surface. Cytoplasmic fusion bridges were observed in areas where cells made contact. Concentrations of Con A ranging between 50–500 g/ml caused cell shrinkage and surface distortions without cell flattening and filament loss. These morphologic effects were not apparent if Con A binding sites were blocked by preincubation with -methyl-D-mannopyranoside before application to the cell cultures. However, once the Con A-mediated changes were in effect, the cells failed to show recovery when they were returned to growth medium and a majority of the cells on the coverglasses degenerated. Presumably the cells whose morphology appears unaffected by Con A treatment are the survivors that repopulate cultures returned to growth medium.Supported by Grants CA-12600 and CA 16619 awarded by the National Cancer Institute, DREW and in part by NIH Biomedical Sciences Grant No. RR-07050. CAA's participation in this project was supported by Training Grant No. 5T01-GM-71-17We wish to thank Dr. Imogene Schneider for providing the cell lines     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Microscopical evidences for the presence of a nucleus inCandida albicans chlamydoconidia

By using the fluorescent, DNA specific stain DAPI (4,6-diamidino-2-phenylindole) some microscopic observations ofCandida albicans pseudomycelium and chlamydoconidia were performed. In this manner blue fluorescent dots were noted both in yeasts, psudomycelium and chlamydoconidia, so evidencing the presence of a nucleus inC. albicans chlamydoconidia.     

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

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

Fine sugar specificity of theButea frondosa seed lectin

Various monosaccharides and oligosaccharides were used to define the specificity of theButea frondosa lectin using the hapten inhibition technique of human erythrocyte agglutination. AlthoughB. frondosa lectin exhibited higher affinity forN-acetylgalactosamine, lactose andN-acetyllactosamine appeared to be relatively good inhibitors of haemagglutination. The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column ofB. frondosa lectin immobilized on Sepharose 4B showed that the sugar-binding specificity of the lectin is directed towards unmaskedN-acetyllactosamine sequences. Substitution of theseN-acetyllactosamine sequences by sialic acid residues completely abolished the affinity of the lectin for the saccharides. The presence of one or several Fuc(1-3)GlcNAc groups completely inhibited the interaction between the glycopeptides and the lectin. Substitution of the core -mannose residue by an additional bisecting (1-4)GlcNAc residue decreases the affinity of the lectin for these structures as compared with the unsubstituted ones.     

Lectin Analysis of Trypanosoma congolense Bloodstream Trypomastigote and Culture Procyclic Surface Saccharides by Agglutination and Electron Microscopic Technics*

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.     

Endogenous β-d-galactoside binding lectin during the expansion of the yolk sac in the developing chick embryo

Summary A lectin with an affinity for -d-galactoside-containing saccharides is present in the developing yolk sac from the chick embryo at stages from 2 to 7 days of incubation. This activity is present in the area vitellina (less differentiated) and the area vasculosa (more differentiated). In both areas, lectin activity increases significantly during the spreading of the yolk sac up to 5 days of incubation. At all of the stages studied lectin activity was significantly higher in the area vasculosa, as compared to the area vitellina.Lectins were purified by affinity chromatography and examined by SDS-PAGE. Under reducing conditions two components are evident. A more prominent band of subunit molecular weight of 14,200±100 for the area vitellina and 13,700±300 for the area vasculosa and a second band with molecular weight of about 68,000±700 and 68,000±1,200 for the area vitellina and area vasculosa respectively, were observed. The -d-galactoside-binding lectin appears to be similar if not identical to that of the early chick blastoderm.     

Localized accumulation of cell wall mannoproteins and endomembranes induced by brefeldin A in hyphal cells of the dimorphic yeastCandida albicans

Summary Candida albicans, a dimorphic yeast, has the abililty to switch its growth form between budding growth and hyphal growth. Since fungal growth involves secretory processes, spatial control of secretion should play a crucial role in such a morphogenetic transition. Brefeldin A (BFA), an inhibitor of the membrane trafficking system of eukaryotes, increases the occurrence of Golgi-like cisternae in the yeast. In the present study, BFA was used to obtain further insights into the spatial organization of secretory processes in hyphal growth ofC. albicans. BFA completely inhibited the formation and growth of germ tubes at a concentration of 35 M or higher. Electron microscopy of BFA-untreated germinated cells revealed many vesicles in the apical region and Golgi-like cisternae in the cytoplasm. In cells treated with 35 M BFA, the vesicles disappeared from the apical region, and, instead, stacked membrane cisternae and membrane-enclosed spherical dense bodies accumulated in the subapical region. These accumulated structures were positive for both polysaccharide staining and immunocytochemical staining with antibodies raised against cell surface antigens ofC. albicans, as were Golgi cisternae in BFA-untreated cells. In cells treated with a higher concentration of BFA (140 M), the structures that appeared in cells treated with 35 M BFA were no longer observed and the endoplasmic reticulum was extended and positive for polysaccharide staining. These results suggested that BFA affects different steps of membrane trafficking in a concentration-dependent manner. The accumulated structures induced by 35 M BFA seemed to be the altered forms of Golgi cisternae. Their accumulation in the subapical region of the germ tube might indicate that the step(s) in membrane trafficking that are associated with the Golgi pathway are vectorially organized in hyphal growth ofC. albicans.Abbrevations BFA brefeldin A - BSA bovine serum albumin - CBB Coomassie brilliant blue - Con A concanavalin A - HRP horseradish peroxidase     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

Changes in sexual agglutination ability during the formation of vegetative cells from spores in Saccharomyces cerevisiae

Summary Spores of heterothallic diploid cells of Saccharomyces cerevisiae had neither a nor agglutination substance in either cell wall or cytoplasmic fraction; they, however, showed selfagglutination not caused by sex-specific agglutination substances. Meanwhile, practically no sexual agglutination was detected during germination and outgrowth of the spores; it arose after emergence of the first buds and progressed with incubation time. Its ability increased gradualy until the first bud emergence and rapidly thereafter. a and agglutination substances were detected in both cell wall and cytoplasmic fractions of cells from an 8h-old spore culture. Only germinated spores with buds had the ability to produce and to respond to the a pheromone.