Formation of a new cell wall by protoplasts of Candida albicans: effect of papulacandin B, tunicamycin and Nikkomycin

Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.     

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

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

Localization of α-galactomannan and of wheat germ agglutinin receptors inSchizosaccharomyces pombe

The location of galactomannan on the surface ofSchizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized onS. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections ofS. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed thatS. pombe cell wall is devoid of chitin.Non-Standard Abbreviations Au gold colloid - RCA_I Ricinus communis lectin - SEM scanning electron microscopy - TEM transmission electron microscopy - WGA wheat germ agglutinin     

Cytochemical demonstration of chitin incorporation in the cuticle of Trialeurodes vaporariorum (Westwood) (Homoptera : Aleyrodidae) after buprofezin treatment

Buprofezin is an insect growth regulator acting specifically against sucking insects. Nymphs of the greenhouse whitefly Trialeurodes vaporariorum (Westwood) (Homoptera : Aleyrodidae) were treated with different concentrations of this compound and the deposition of the chitin nn the integument examined. The lectin, wheat germ agglutinin (WGA), which binds specifically to N-acetylglucosamine-containing polymers, conjugated with colloidal gold, was used as an electron-dense marker for localizing chitin on ultrathin sections. In untreated insects, the epicuticle and epidermis cells were devoid of label, while the procuticle was heavily labelled. In buprofezin-treated nymphs, only the pre-apolysial cuticle remained heavily labelled, independent of the concentration used. The post-apolysial cuticle, however, was hardly labelled, the intensity of labelling dependn: g on the applied concentration, and its thickness severely reduced. These results suggest that buprofezin affects only post-apolysial cuticle in a concentration-dependent way.     

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.     

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     

Retrograde tracing of neural pathways with a protein-gold complex

Summary In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

Retrograde tracing of neural pathways with a protein-gold complex. I. Light microscopic detection after silver intensification

In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.     

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.     

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

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

The effect of monensin on chitin synthesis in Candida albicans blastospores

Monensin, a monovalent cation ionophore, was used to investigate some steps of the wall synthesis and morphogenesis in Candida albicans blastospores. In the presence of the drug, the pathogenic yeast developed enormous wall and septum thickenings that reacted intensely and specifically with wheat germ agglutinin and chitinase coupled to colloidal gold and fluorescein isothiocyanate. Therefore, the aberrant zones are interpreted as sites of chitin accumulation. The increased production of this homopolymer, also demonstrated by the chemical analysis of cell wall preparations, implies that monensin interferes in some way with the regulatory factors that normally control, in space and time, chitin synthetase activity.     

Localization of mannan at the surface of yeast protoplasts by scanning electron microscopy

The (13)glucanase of Basidiomycete QM 806 was used to prepare Saccharomyces cerevisiae and Candida utilis protoplasts. Plasma membranes isolated from S. cerevisiae contained a small amount of mannose and traces of glucose and ribose. Randomly distributed -mannan was detected by scanning electron microscopy at the surface of prefixed protoplasts using colloidal gold labelled with Concanavalin A as a marker. C. utilis protoplasts were also marked with anti-mannan antibodies. Again the distribution of mannan was random. This experiment indicated also that plasma membrane mannan has the same immunochemical determinants as cell wall mannan. It is hypothesized that mannan is mainly located in the outer layer of plasma membranes.     

Cell wall architectures in a mycorrhizal association as revealed by cryoultramicrotomy

Summary In a vesicular-arbuscular mycorrhizal association a different texture in the cell wall of the host and fungus is revealed by using cryoultramicrotomy, that is a powerful tool in studying macromolecular arrangements. Using this technique the host wall displays fibrillar texture, whilst the fungal wall appears amorphous. The latter is not affected by the tested enzymatic digestions. On the contrary, it is labelled by ferritin linked to the wheat germ agglutinin, displaying the presence of N-acetylglucosamine. It is suggested that this sugar is present in single units or in short chains, whilst true chitin fibrils are not formed.     

Polyoxin D inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans

Yeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4.5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.     

Location on chitin in the cyst wall of Entamoeba invadens with colloidal gold tracers

Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.     

A proposed neural pathway for vocalization in South African clawed frogs,Xenopus laevis

Vocalizations of South African clawed frogs are produced by contractions of laryngeal muscles innervated by motor neurons of the caudal medulla (within cranial nerve nucleus IX-X). We have traced afferents to laryngeal motor neurons in male and female frogs using retrograde axonal transport of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA). After iontophoretic injection of HRP-WGA into n. IX-X, retrogradely labelled neurons were seen in the contralateral n. IX-X, in rhombencephalic reticular nuclei, and in the pre-trigeminal nucleus of the dorsal tegmental area (DTAM) of both males and females.     

Characterization by lectin binding of the sugar moiety of glycocompounds stored in inherited diseases

Summary Kidney and liver samples from two cases of Fabry's disease and spleen and liver samples from Gaucher and Niemann—Pick diseases were tested for binding to lectins such as peanut agglutinin (PNA),Bandeiraea simplicifolia, (BSA),canavalia ensiformis (Con A), soybean agglutinin (SBA) and wheat germ agglutinin (WGA) labelled with horseradish peroxidase using histochemical techniques. These techniques allowed the localization of compounds with -galactosyl residues in tissues from Fabry's disease. In tissues from the Gaucher and Niemann—Pick cases, the storage material was found to be more complex than expected, and some problems regarding the significance of lectin binding are discussed.     

Cytochemical localization of carbohydrates in intercalated duct and acinar cells of mouse parotid gland

Summary The glycoconjugate composition of mouse intercalated duct and acinar cells of parotid gland has been compared. Mucins containing 1,2-glycols were demonstrated by the tannic acid-uranyl acetate technique. Hexose residues of glycoconjugates were identified using ferritin conjugated withCanavalia ensiformis agglutinin (Con A),Triticum vulgare or wheat germ agglutinin (WGA),Ricinus communis I agglutinin (RCA-I),Phaseolus vulgaris agglutinin (PHA-E) andArachis hypogaea agglutinin (PNA). Whereas qualitative and quantitative differences were observed in sugar residues of secretory granules in intercalated duct and acinar cells, apical plasmalemmae were labelled sparsely and similarly. This indicates that the glycocalyx composition of apical plasma minae in the parotid acinar and intercalated duct cells is little influenced by secretory granule composition.     

Candida albicans mycelial wall structure: supramolecular complexes released by Zymolyase,chitinase and β-mercaptoethanol

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118–1125]. Zymolyase, chitinase and -mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (>180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.     

Double label freeze-etch study of the relative topography of concanavalin A and wheat germ agglutinin receptors at the myoblast surface

This report records for the first time double-label freeze-etch electron microscopy of cells in culture. On L6 rat myoblasts, receptors for wheat germ agglutinin and concanavalin A were found distributed together in irregular granular microclusters of cell surface material up to 60 nm in diameter. Simultaneous localization of two different receptor families to such small regions using colloidal gold and ferritin to differentiate between lectin markers proved difficult in our hands. We were able to achieve the desired result using native concanavalin A and ferritin-conjugated wheat germ agglutinin, whose shadowed diameters are measurably different.