Structure of an α-D-galactosaminoglycan from Physarum polycephalum Spherule walls

The spherule walls Physarum polucephalum have been reexamined and found to contain 88% of galactosamine (as anhydrogalactosamine), 6.80% of protein, 4.7% of phosphate groups, and a small proportion of acetyl groups (0.5%). Methylation studies indicated that the spherule-wall polysaccharide is a long-chain galactosamino- glycan linked exclusively (1→4) and without phosphate linkages. The specific optical rotation of this unique glycan. [x]_D, + 118° (6_M HCI), indicated that it is α-D-linked.     

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H₂O₂-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.     

Hyphal polysaccharides as potential phylogenetic markers forEupenicillium species

The hyphal wall polysaccharide fractions of certain species ofEupenicillium have been utilized to study their phylogenetic relationships.E. abidjanum andE. shearii (group 1) cell walls have an alkali-soluble polysaccharide fraction (20%) containing an α-glucan.E. alutaceum, E. baarnense, E. catenatum, andE. erubescens (group 2) contain β-linked polysaccharides in all their polysaccharide fractions. These results suggest that some of the previously assigned taxonomic groups ofEupenicillium may not be natural.     

Cytochemical detection of polysaccharides on the surface of the cell membrane complex in fungi

Cytochemical staining in toto (periodic acid, thiosemicarbazide, O_SO₄) revealed the presence of polysaccharide lamellae on the surface of the cell membrane complex of fungi. The membraneous clusters in the vacuolar bodies of Claviceps purpurea were covered with these lamellae at both surfaces, as it was also the case with the endoplasmic reticulum membranes, the tonoplast and the cytoplasmic membrane. In Saccharomyces cerevisiae, the polysaccharide lamellae were visible on the surface of the endoplasmic reticulum membranes and the plasmalemma; the strain revealed polysaccharide deposits also on the tonoplasts of small vacuoles and in glucanase vesicles. We assume that these observations give precision to the localization of the enzymes synthetizing the glycoprotein components of the fungal cell wall.     

Flow cytometric determination of nuclear DNA content during differentiation (spherulation and germination) of the myxomycete Physarum polycephalum

Summary Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G₂ phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of ³H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G₂ phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

Fine-structural Correlates of Growth in Hyphae of Ascodesmis sphaerospora

Mycelial mats of Ascodesmis sphaerospora were fixed and embedded for electron microscopy, and thin sections of 1-mm blocks, taken from the 1st to the 7th mm behind the hyphal tips, were cut parallel to the long axis of the hyphae. The hyphal tip region is characterized by an outer zone of electron-transparent vesicles, 500 to 1,000 A in diameter, and is apparently associated with wall elaboration. Immediately behind this region, dense granules become evident along convoluted membrane systems and along the plasma membrane; in the same region are numerous small lomasomes in the lateral wall. As the hypha grows, septa are laid down at 3- to 7-min intervals at a distance of 200 to 250 μ behind the hyphal tip. A cylinder of endoplasmic reticulum is intimately involved in cross-wall deposition from its earliest stages; as the wall grows in, it becomes increasingly constricted in the pore region, finally assuming a torus-like configuration. Woronin bodies are shown to have a crystalline substructure and to originate in pouch-like membrane systems. Cross-walls from a 7- to 13-hr-old mycelium frequently show highly ordered structures in the vicinity of the pore. These structures may appear either as laminar stacks of discs to one side of the pore or as series of stubby concentric rings within the pore area itself. In the latter case, a mass of granular material is frequently seen plugging the pore. Other unusual organelles and inclusions in 7- to 13-hr hyphae are vesicles containing swirls of beaded or dilated membrane, membrane-enclosed rods, and stacks of unit membranes associated with spherical, electron-transparent vesicles.     

Electron microscopy of Bacillus subtilis protoplast membrane after treatment with phospholipase A2 and phospholipase C

The effects of phospholipase A₂ and phospholipase C on Bacillus subtilis protoplast membrane have been studied by electron microscopy and by chemical methods. Phospholipase A₂ (from porcine pancreas) almost quantitatively converted cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol to fatty acids and lysoderivatives. The fatty acids like the lysophospholipids remained in the membrane. Phospholipase C (from Bacillus cereus) hydrolyzed about 80% of the phosphatidylethanolamine and about 40% of the cardiolipin. Electron microscopy has been carried out with respect to general morphology of the affected protoplasts, the occurrence of a triple-layered membrane structure in thin sections, and the ultrastructure of membrane fracture faces upon freeze fracturing. Phospholipase A₂ treatment resulted in fragmentation of the protoplasts. In all cases the triple-layered membrane profile was preserved in thin sections. The membrane fracture faces appeared normal, i.e. they showed a convex face with many particles and a concave face with few particles. This indicated that the hydrophobic interior of the membrane was not too much damaged after incubation with phospholipases, presumably because of the stabilizing action of membrane proteins.     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Ultrastructural localization of β-galactan in the nuclei of the myxomycete Physarum polycephalum

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated β-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei ofP. polycephalum by electron microscopy using a specific “sandwich” technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactin was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasma and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.     

THE STRUCTURE OF SOME CYTOPLASMIC COMPONENTS OF PLANT CELLS IN RELATION TO THE BIOCHEMICAL PROPERTIES OF ISOLATED PARTICLES

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

Scanning electron microscope studies on the parasitic cycle of Coccidioides immitis

The process involved in the in vivo conversion of the arthroconidia of Coccidioides immitis into endosporulating spherules was studied with the aid of a scanning electron microscope. By the fifth and sixth day after inoculation of laboratory mice, complete conversion had occurred in their kidneys, lungs, and spleens. The progressive stages of cleavage that occurred in the enlarged arthroconidia were initiated by invagination at several points of the cytoplasmic membrane that covers the inner surface of the developing spherule's cell wall. Through repeated branching of the cleavage cell walls, the spherule's cytoplasm was divided into progressive smaller segments. These segments were aggregated in packets and enclosed in a membranous sac. At maturity the membrane dissolved, and the endospores were freed within the spherules. Finally, the spherule wall ruptured, and the endospores were released.     

STRUCTURE AND DEVELOPMENT OF HYPHAL SEPTA IN ALLOMYCES

Development of hyphal septa (pseudosepta) in Allomyces macrogynus begins with the formation of five or more discontinuous pieces of wall material that project inward from the hyphal wall. Lateral fusion of these projections leaves a central pore in the septum that is later filled in by centripetal deposition of wall material. However, lateral fusion of the projections is not complete; peripheral pores remain in the rim of the mature septum. The position of cytoplasmic microtubules corresponds to the position of actively moving cellular particles and organelles. Allomyces reticulatus and A. arbuscula have similar septa.     

ULTRASTRUCTURE OF THE CORALLINACEAE (RHODOPHYTA) II. VEGETATIVE CELLS OF LITHOTHRIX ASPERGILLUM1

The ultrastructure of the calcareous red coralline alga Lithothrix aspergillum Gray and the development of the various tissue types has been studied. The sub-apical meristematic tissue alternately produces genicular or intergenicular cells. The genicular cells rapidly elongate and their cell walls thicken and become denser as more fibrillar wall material is laid down within the cell wall. These cells contain little cytoplasm and few organelles. The inter genicular cells which elongate only slightly during development have a small vacuole and many free starch grains in the cytoplasm. The peripheral cells in each inter genicular layer remain meristematic and form a cortical cell layer over the genicular cells. These cortical cells and the apical meristematic cells are covered by small epidermal cells which have extensive cell wall ingrowths between the chloroplasts. The inter genicular cells are calcified. Although the CaCO₃ is laid down within the cell walls, there is always a thin layer of CaCO₃-free organic cell wall material between the plasmalemma and the CaCO₃ impregnated wall. Only the distal tips of the genicular cells are calcified. In old genicular tissues of Lithothrix, secondary deposits of CaCO₃ of unknown crystallography are also found in the spaces between the cell walls. Thus there appear to be at least two mechanisms of calcification in this alga.     

Morphology ofEntomophthora muscae protoplasts grownin vitro

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

感染病毒的小麦全蚀菌的超微结构

将感染病毒的小麦全蚀菌山东烟台株培养 20天的菌体细胞,进行超微结构的研究。于电镜下观察到球状病毒颗粒,平均直径23—30nm,多是无规则松散的分布于胞质中;或紧密聚集于液泡、线粒体周围;或排列成线状;或7—8个颗粒排列成环状。病毒仅分布于细胞质中,细胞核、脂肪体内均未见病毒颗粒。病毒浓度在较老的菌体内有增加的趋势。全蚀菌的菌丝细胞壁有三层,外层电子致密内含纤维状物,内层电子较为透明,中层为一电子致密度很深的狭窄夹层。壁的厚度不均,外缘不规则;在菌丝体产生隔膜的早期阶段,于隔膜附近有1—3个外被膜结构的沃罗宁体 Woronin body,隔膜形成的后期,见电子致密物质沉积在核膜孔上,形成中的隔膜顶端为尖状突起向基部逐渐增宽略成金字塔形。     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : II. Electrophysiological Observations

The electrical membrane potential (E_m) and electrical membrane resistance (R_m) were measured continuously during the first cleavage of Xenopus eggs, using intracellular microelectrodes. A sharp hyperpolarization of E_m and decrease in R_m can be observed from 6 to 8 min after the onset of cleavage. This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the interblastomeric membrane during normal cleavage. Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the membrane newly formed during cleavage. These conditions allow for a direct measurement of the permeability properties of the new membrane. It was found that under these conditions E_m reaches values about 3 times more negative and R_m reaches values about 1.5–3 times smaller than during normal cleavage. The extent of reduction of R_m can be correlated with the surface area of the newly formed membrane. We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K⁺ ions). Its mean specific resistance is 1–2 kΩ·cm², as against 74 kΩ·cm² for the pre-existing membrane. No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected.     

The Fine Structure of the Rod-Bipolar Cell Synapse in the Retina of the Albino Rat

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mµ in thickness and 400 mµ in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mµ in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.