On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electronmicroscopic, X-ray diffraction and chemical study.

The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 mum long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of beta-(1 leads to 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was found de novo by the protoplasts. A fine net structure of microfibrils about 7-5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.     

Cell Wall Composition of the Yeastlike and Mycelial Forms of Blastomyces dermatitidis

The thermally induced changes in the cell wall polysaccharides of Blastomyces dermatitidis strain BD64, which produces a yeastlike form (Y form) at 37 C and a mycelial form (M form) at 20 C, were examined. The cell walls of the Y and M forms contained 36 and 51% of hexoses, respectively. The M-form cell wall contained glucose, galactose, and mannose in a molar ratio of 1:0.1:0.2. The Y-form cell wall contained mainly glucose and a very small amount of galactose and mannose. The glucans of the cell wall of the Y form consisted of about 95% alpha-glucan and 5% beta-glucan, whereas those of the M-form cell wall consisted of about 60% alpha-glucan and 40% beta-glucan.     

Fine Structure of Zygotes and Oocysts of Eimeria nieschulzi*

SYNOPSIS. Mature macrogamonts were present in the small intestine of rats 5.5 to 7.5 days postinoculation with Eimeria nieschulzi oocysts; oocysts were present at 6 to 7.5 days. Types I and II wall-forming bodies in macrogamonts began to undergo ultrastructural changes within zygotes to form the outer and inner layers of the oocyst wall. Before and during oocyst wall formation a total of 5 membranes (M1–5) were formed at or near the surface of the zygote. The outer and inner oocyst wall layers formed between M2 and M3, and M4 and M5, respectively. The mature oocyst was loosely surrounded by M1 and M2, had an electron-dense outer layer, 100–275 nm thick, and an electron-lucent inner layer, 160–180 nm thick. It also contained an electron-lucent line consisting of M3 and M4 interposed between the outer and inner layers of the oocyst wall. The micropyle, measuring 935 × 47 nm, was located in the outer layer of the oocyst wall and consisted of 10–14 alternating layers of electron-dense and lucent material. The sporont of mature oocysts was covered by M5, immediately beneath which were M6 and M7. The sporont contained a nucleus and nucleolus, lipid and amylopectin bodies, mitochondria, ribosomes, as well as smooth and rough endoplasmic reticulum. Canaliculi, Golgi complexes, and types I and II wall-forming bodies were absent.     

Isolation and characterization of the rodlet layer of Trichophyton mentagrophytes microconidial wall.

The rodlet layer of the microconidial wall of Trichophyton mentagrophytes was isolated and partially characterized. The purified microconidial walls were first extracted with urea (8M), mercaptoethanol (1%), and sodium dodecyl sulfate (1%) followed by enzymatic digestion with glusulase (snail intestinal enzymes) and purified (1 leads to 3)-beta-D-glucanase and chitinase. The purified rodlet layer was 15 to 30 nm thick and accounted for approximately 10% of the original wall weight. The pattern of rodlet patches, as revealed by electron microscopy of freeze-etched preparations of the isolated layer, was essentially the same as that observed on the intact microconidial wall. The rodlet layer was found to be resistant to most of the common organic solvents, cell wall lytic enzymes, mild acid treatments, and surface-active agents, but was solubilized in boiling 1 N NaOH with concomitant disorientation of the rodlet patterns. A melanin or melanin-like pigment appeared to be intimately associated with this rodlet layer and was solubilized during a hot-alkali treatment. Protein (80 to 85%) and glucomannan (7 to 10%) were the major components of the rodlet layer. The rodlet layer did not contain any appreciable amounts of lipid or phosphorus.     

Chemical analysis of the hyphal wall of Schizophyllum commune.

1. Purified hyphal wall fragments of Schizophyllum commune are analysed and shown to consist of glucose (67.6%), mannose (3.4%), xylose (0.2%), (N-acetyl)glucosamine (12.5%), amino acids (6.4%) and some lipid material (3.0%). 2. The previously proposed structures of two glucans located at the hyphal wall surface (Wessels et al. (1972) Biochim. Biophys. Acta 273, 346-358) were essentially confirmed using methylation analysis. The mucilaginous glucan consists of 1,3-linked beta-glucan chains with branches of single glucose units attached by beta-1,6 linkages on every third unit, on average, along the chain. The alkali soluble S-glucan is an exclusively 1,3-linked alpha-glucan. 3. The alkali-insoluble R-glucan, occurring in close association with chitin, in the inner wall layer, has been characterised by methylation analysis, X-ray diffraction, enzymatic hydrolysis with purified exo-beta-1,3-glucanase and Smith degradation. It appears to be a highly branched beta-1,3,beta-1,6-glucan and a model of this glucan is proposed. Certain parts of this highly insoluble R-glucan bear a close structural similarity to the mucilaginous glucan present at the outer wall surface and in the medium.     

OCCURRENCE OF TRI-LAMELLAR BODIES IN ISOLATES OF NOSTOC (CYANOPHYCEAE)1

Tri-lamellar bodies were observed in eight of 29 isolates of Nostoc examined. They appeared identical to the previously described bodies in various species of Anabaena. The bodies consist of three discoid lamellae each ca. 0.3 μm diam and 8 nm thick. The outer lamella (closest to the plasma membrane) is separated from the middle lamella by a 12 nm space whereas the middle and inner lamellae are ca. 8 nm apart. Osmiophilic striations 3 nm wide were generally observed running between the lamellae. Osmiophilic β granules were usually associated with the inner lamella. The bodies were most always located close to the plasma membrane along the longitudinal wall near the junction of the cross and longitudinal walls. In three isolates the bodies located near the cross walls were associated with gas vesicles and possessed a slightly different morphology. These tri-lamellar bodies consisted of three discoid lamellae, each ca. 2 nm thick, ca. 25 nm apart with electron dense material between the inner and middle lamellae. Pores 20 nm diam and ca. 60 nm apart were observed in layer 2 of the cell wall adjacent to the tri-lamellar bodies. These wall pores were also observed in isolates lacking tri-lamellar bodies.     

Scanning and transmission electron microscopy of the oocyst wall of Isospora lacazei

The oocyst wall of Isospora lacazei from sparrows was studied with scanning (SEM) and transmission (TEM) electron microscopy. In TEM, the oocyst wall consisted of four distinct layers (L1-4). The innermost layer, L1, was moderately electron-lucent and 240--285 nm thick; L2 was electron-dense and 210--240 nm thick; L3 was moderately electron-lucent and 15--150 nm thick; L4, the outer most layer, was discontinuous and consisted of electron-dense discoid bodies which measured 180--220 nm x 320--840 nm. The discoid bodies of L4 as seen by TEM appeared spheroid in shape when observed by SEM. One or two membranes were situated on or between various layers of the oocyst wall. One such membrane occurred on the inner margin of L1, two closely applied membranes were interposed between L1 and L2, one membrane occurred between L2 and L3, and one membrane on the outer margin of L3.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma farciminosum

The cell wall of the yeast form of Histoplasma farciminosum contains 13.2% beta-1,3-glucan, 1.0% galactomannan, and 25.8% chitin, whereas the cell wall of mycelial form has 21.8, 4.5, and 40%, respectively, for the same polymers. Also, the cell wall of the yeast form contains alpha-1,3-glucan (13.5%) and an unidentified polymer (21.5%). Chitin, one of the structural polymers of both yeast and mycelial cell walls, is identified as thin isolated fibers (4 nm wide) or in thick bundles (50 nm wide) of fibers. beta-(1-3)-Glucan is also found as thin isolated fibers indistinguishable from isolated fibers of chitin. Fibers 14 nm wide and resembling alpha-(1-3)-glucan fibers of other fungi are found in the yeast form. The results reported here do not give support to the proposal for a different taxonomic classification.     

Cell wall composition of the yeast and mycelial forms of Paracoccidioides brasiliensis

Isolation and chemical analyses of the cell walls of the yeast (Y form) and mycelial forms (M form) of Paracoccidioides brasiliensis and Blastomyces dermatitidis revealed that their chemical composition is similar and depends on the form. Lipids, chitin, glucans, and proteins are the main constituents of the cell walls of both forms of these fungi. There is no significant difference in the amount of lipids (5 to 10%) and glucans (36 to 47%) contained by the two forms. In both fungi, the Y form has a larger amount of chitin (37 to 48%) than the M form (7 to 18%), whereas the M form has a larger amount of proteins (24 to 41%) than the Y form (7 to 14%). Several properties of the glucan of P. brasiliensis were studied. Almost all of the glucan in the Y form was soluble in 1 n NaOH, was weakly positive in the periodic acid-Schiff reaction, was not hydrolyzed by snail digestive juice, and had alpha-glycosidic linkage. Glucans of the M form were divided into alkali-soluble (60 to 65%) and alkali-insoluble (35 to 40%) types. The alkali-soluble glucan was similar to that of the Y form; the alkali-insoluble glucan was positive in the periodic acid-Schiff reaction and was hydrolyzed by snail digestive juice.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

The cell wall of Fusarium oxysporum.

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

The fine structure of secretion in Hyalophysa chattoni: formation of the attachment peduncle and the chitinous phoretic cyst wall.

The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electrondense dense secretory bodies from the cell's ventral surface.     

The fine structure of the endogenous stages of Eimeria labbeana: 2. Mature macrogamonts and young oocysts.

The fine structure of the mature macrogamonts and intracellular oocysts of Eimeria labbeana from the ileal mucosa of experimentally infected Pigeons (Columbia livia) was investigated and described. The macrogamont reached a maximum size of 12.0 x 9.5 mum (average equals 10.8 x 8.8 mum), and was located within a narrow parasitophorus vacuole. Most of the macrogamonts were limited by two membranes. Intravacuolar tubules, 1.2 mum long and 58 nm in diameter, established direct connections between the parasite and the host cell. Each tabule was composed of 9 subunits arranged around the central lumen. Cytoplasmic canaliculi were composed of bundles of microtubule-like structures (8-10 nm wide). Type 1 wall-forming bodies reached a maximum size of 1.8 x 1.5 mum, and many had centric or eccentric electron transparent portions within them. They were frequently seen lodged within peripherally-located mitochondria. Type 2 wall-forming bodies averaged 1.5 mum in diameter. The role of the two types of wall-forming bodies in forming the outer and inner layers of the wall of the oocyst was similar to that in other species of Eimeria. The oocyst wall was 0.2 mum thick and composed of a limiting membrane (20 nm thick), an outer layer (75 nm thick), and an inner layer (100 nm thick).     

The Fine Structure of Secretion in Hyalophysa chattoni: Formation of the Attachment Peduncle and the Chitinous Phoretic Cyst Wall

ABSTRACT. The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electron-dense secretory bodies from the cell's ventral surface.     

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.     

Ultrastructural studies on the cell walls in Fusarium sulphureum.

The cell walls of Fusarium sulphureum have a microfibrillar component that is randomly arranged. X-ray-diffraction diagrams of the microfibrils are consistent with a high degree of crystallinity and show that they are chitin. The chitin microfibrils of the peripheral walls envelop the hyphal apex and extend across the septae. During the first 8h in culture, the conversion of conidial cells to chlamydospores is evidenced by a swelling of the cells and the original microfibrils remain randomly arranged. Within 24h new wall material is deposited as the cells expand and the wall thickens. The new microfibrils are indistinguishable from those of the original conidial cells. After 3 days in culture, the chlamydospores are fully developed and have the characteristic thick wall which is a continuous layer of randomly arranged microfibrils. Chlamydospores maintained in a conversion medium for 8 days have microfibrils identical with those in 3-day-old cultures; thus a further change in the microfibril orientation did not occur during that period. Alkaline hydrolysis of the walls removes most of the electron-dense staining constituents from the inner wall layer and leaves the outer wall layer intact. This treatment also reveals some of the wall microfibrils. An additional treatment of the walls with HAc/H2O2 completely removes the wall components that react positively to heavy metal stains. The results are discussed in relation to the structure of other fungal cell walls.     

The fine structure of the phoront of Gymnodinioides pacifica, a ciliated protozoan (Ciliophora, Apostomatida) from euphausiids of the Northeastern Pacific

Phoretic stages of the exuviotrophic apostome Gymnodinioides pacifica were examined using transmission and scanning electron microscopy (TEM and SEM). TEM revealed that the mature cyst wall possesses 2 or 3 layers differing by the presence or absence of the third inner layer. This inner layer may represent a different form of the middle wall material. The inner cyst layer is approximately 0.15 microm thick and has striations with a periodicity of approximately 19 nm. The middle cyst layer has a variable thickness and the outer dense layer is approximately 0.1 microm thick. The 3 layered cyst wall had a thickness of 0.3-0.7 microm and averaged 0.5 microm. Advanced phoront stages were enclosed by fully formed cyst walls or by cyst walls thinned to approximately 0.1 microm, as the phoronts prepared to excyst prior to host ecdysis. Additionally, we report the fine structure of the rosette, trichocysts, nuclei, food plaquettes, oral fiber, and other cytoplasmic inclusions. SEM revealed an outer cyst wall layer connected to the secreted peduncle material, which was observed to extend over a wide (15 microm) area on the host setae. Cysts were usually attached at their posterior ends or, less frequently, along their side.     

Microsporidian Spore Wall: Ultrastructural Findings on Encephalitozoon hellem Exospore

A study of the spore wall of Encephalitozoon hellem was performed on thin sections, freeze-fracture, and deep-etched samples to obtain information on spore wall organization and composition. Our observations demonstrate that the spore wall is formed by an inner 30–35 nm electron-lucent endospore and an outer 25–30 nm electron-dense exospore. The exospore is a complex of three layers: an outer spiny layer, an electron-lucent intermediate lamina and an inner fibrous layer. Freeze-fracture and deep-etching techniques reveal that the intermediate lamina and the inner fibrous layer result from the different spatial disposition of the same 4-nm thick fibrils. In thin sections the endospore reveals a scattered electron-dense material that appears in the form of trabecular structures when analyzed in deep-etched samples. The presence of chitin in the exospore is discussed.     

"Nonfibrillar" chitin associated with walls and septa of Trichophyton mentagrophytes arthrospores

Two morphologically distinct forms of chitin were found in the arthrospore walls and septa of Trichophyton mentagrophytes. Two-thirds of the total wall chitin was the microfibrillar and chitinase-sensitive form. The remaining chitin existed in a previously uncharacterized "nonfibrillar" form and was insensitive to the action of Streptomyces chitinase. Exhaustive digestion of the arthrospore walls and septa with beta (1 leads to 3)-glucanase and chitinase followed by extraction with NaOH (1 N, 100 degrees C, 3 h) resulted in a fraction which retained the original wall shape. This fraction consisted of 85% N-acetylglucosamine, 2.0% galactosamine, 2.5% glucose, and 0.4% amino acids, 74% of which were lysine. Both its infrared spectrum and its X-ray diffraction pattern were almost identical to those of authentic chitin. There was no evidence of the presence of muramic acid, hexuronic acid, phosphate, or sulfate in this fraction. Its resistance to chitinase was due neither to the presence of protective wall layers or melanin nor to its close or covalent association with beta-glucan. Aside from its nonfibrillarity, this hexosamine polymer differed from authentic chitin in that it was soluble in 6 N HCl and 7.5 N NaOH. The development of this nonfibrillar chitin layer in the cell wall during arthrosporogenesis of T. mentagrophytes may be related to the arthrospores being resistant to a variety of antifungal agents.     

The glucan components of the cell wall of baker''s yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure

1. Commercial pressed baker's yeast, and cell walls prepared from it, were extracted in various ways and the products examined by a number of techniques, including infrared spectroscopy and electron microscopy. 2. The glucan components of the walls cannot be extracted from intact yeast cells by 3% (w/v) sodium hydroxide at 75 degrees , but at least one-third of the glucan of cell wall preparations is dissolved under these conditions, and more will dissolve after ultrasonic treatment. 3. If intact cells are given a preliminary treatment with acid the wall glucans dissolve in dilute aqueous alkali. 4. Acid conditions as mild as sodium acetate buffer, pH5.0, for 3hr. at 75 degrees are sufficient for this preliminary treatment; the glucan then dissolves in 3% sodium hydroxide at 75 degrees leaving a very small residue, which contains chitin and about 1% of the initial glucan of the wall. Dissolution is hindered by exclusion of air, or by a preliminary reduction with sodium borohydride, suggesting that some degradation of the glucan by alkali is taking place. 5. After treatment with 0.5m-acetic acid for 24hr. at 90 degrees the glucan dissolves slowly at room temperature in 3% sodium hydroxide, or in dimethyl sulphoxide. The extraction with acetic acid removes glycogen and a predominantly beta-(1-->6)-linked glucan (not hitherto recognized as a component of baker's yeast), but none of the beta-(1-->3)-glucan, which remains water-insoluble. 6. Without treatment with acid, the glucan is not significantly soluble in dimethyl sulphoxide, but can be induced to dissolve by ultrasonic treatment. 7. These results are interpreted by postulating the presence of an enclosing membrane, composed of chitin and glucan, that when intact acts as a semipermeable membrane preventing the escape of the alkali- and dimethyl sulphoxide-soluble fraction of the glucan. Mild acid treatments damage this membrane, and ultrasonic and ballistic disintegration disrupt it. 8. Some support for this hypothesis is given by the effects of certain enzyme preparations, which have been found to render a substantial part of the glucan extractable by dimethyl sulphoxide.