STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE : I. Observations on the Fine Structure of the Yeast Cell

Vegetative cells of Saccharomyces cerevisiae were fixed with potassium permanganate followed by uranyl nitrate, embedded in methacrylate, and studied in electron micrographs of thin sections. Details of the structure of the cell wall, cytoplasmic membrane, nucleus, vacuole, and mitochondria are described. Cell membranes, about 70 to 80 A thick, have been resolved into two dense layers, 20 to 25 A thick, separated by a light layer of the same dimensions, which correspond in thickness and appearance to the components of the "unit membrane" as described by Robertson (15). The cell wall is made up of zones of different electron opacity. Underlying the cell wall is the cytoplasmic membrane, a sinuous structure with numerous invaginations. The nucleoplasm, often of uneven electron opacity, is enclosed in a pair of unit membranes in which nuclear pores are apparent. The vacuole, limited by a single unit membrane, is usually irregular in outline and contains some dense material. Rod-shaped mitochondria, 0.4 to 0.6 µ in length and 0.2 to 0.3 µ in diameter, are smaller in size, but similar in structure to some of those described in plant and animal cells. Attempts to use osmium tetroxide as fixative were unsuccessful, a result similar to that obtained by other workers. It is suggested that yeast cells are impermeable to osmium tetroxide, except when grown under specific conditions.     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS. II. THE STRUCTURE OF THE CELL WALL OF NAVICULA PELLICULOSA (BRÉB.) HILSE

The cell wall of the freshwater diatom Navicula pelliculosa (Bréb.) Hilse is composed of the silica shell and an organic skin which surrounds it. Isolated skins can be prepared by first removing the contents of the cell by mechanical shaking, followed by a posttreatment of these isolated cell walls with HF vapor to remove the silica shell. T h e skins can also be seen in sections, particularly well after the silica shell has been removed B y H. F; vapor. The origin and morphological composition of the shin in N. pelliculosa are not yet completely ascertaincd. As parts of the cell wn11, both the silica shell and the skin are extracellularly located. The growth of the silica shell, however, occurs intracellularly inside a vesicle delimited by a triple-layered membrane, the silicalemma. This membrane or secondary excreted organic material or both in various proportions may compose the skin.     

Fine Structure of Silica Deposition and the Origin of Shell Components in a Testate Amoeba Netzelia tuberculata1

ABSTRACT Netzelia tuberculata secretes a test composed of siliceous particles cemented together by organic plaques forming a single-layered spheroidal shell. The siliceous particles are produced within cytoplasmic vacuoles by three mechanisms: 1) synthesis de novo by deposition of the silica on a matrix; 2) deposition of silica on particles remaining in digestive vacuoles, including starch grains and undigested walls of yeast cells; and 3) secretion of silica as a hollow sphere at the periphery of vacuoles enclosed by the silicasecreting membrane. The silicalemma (silica-secreting membrane) originates as fibril-containing vesicles (GFV) secreted by the Golgi body. Fusion of these vesicles with membranes surrounding digestive vacuoles or with membranes surrounding specialized vacuoles containing a silica-binding matrix apparently converts the vacuole into a silica-depositing organelle. Small spherules of silica occur on the vacuolar side of the membrane surrounding the developing test granules, marking the presence of silicalemma activity. These colloidal spherules become aggregated into larger spherules that condense to form the siliceous surface of the developing test particle. Other Golgi vesicles, designated Golgi plaque vesicles (GPV), produce the organic plaques that are deposited among the siliceous particles at the periphery of the cell during new test construction during cell division. The fine structure of the GFV and GPV and their role in test wall deposition are discussed in relation to other silica-biomineralizing protozoa, including radiolaria.     

A microstructure study on silicified wood from the Permian Petrified Forest of Chemnitz

Three typical plant taxa from the fossil assemblage of the 290-million-year-old Chemnitz Petrified Forest (Zeisigwald Tuff Horizon, Leukersdorf Formation) were studied with regard to the microstructure of the petrifactions: samples of the tree fern Psaronius sp., the seed fern Medullosa stellata, and the gymnosperm Dadoxylon sp. The plant’s tissues are anatomically preserved by silica exhibiting different crystalline order and by other mineralisations. Specimens were studied by means of electron backscatter imaging and electron backscatter diffraction in a scanning electron microscope. The cell walls were largely preserved by quartz crystals, the cell lumina by cryptocrystalline silica. The former organisation and chemical composition of the vascular tissue are mirrored by varying grain formation and grain size. Results are discussed in terms of extant xylem cell wall organisation showing highly hydrophilic cellulose and hemicellulose cross-linked by hydrophobic lignin. The effect of polar and non-polar wood components on the precipitation of silica from aqueous solution and on the formation of crystals is convincing, and the reported results provide a better understanding of how silica replaced organic matter during the petrifaction process.     

Characterization and Localization of Insoluble Organic Matrices Associated with Diatom Cell Walls: Insight into Their Roles during Cell Wall Formation

Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species. Results show that an insoluble organic matrix enriched in mannose, likely the diatotepum, is localized on the proximal surface of the silica cell wall. We did not identify any organic matrix embedded within the silica. We also identified a distinct material consisting of glucose polymer with variable localization depending on the species. In some species this component was directly involved in the morphogenesis of silica structure while in others it appeared to be only a structural component of the cell wall. A novel glucose-rich structure located between daughter cells during division was also identified. This work for the first time correlates the structure, composition, and localization of insoluble organic matrices associated with diatom cell walls. Additionally we identified a novel glucose polymer and characterized its role during silica structure formation.     

THE FINE STRUCTURE AND DEVELOPMENT OF THE COMPANION CELL OF THE PHLOEM OF ACER PSEUDOPLATANUS

At maturity the companion cell of the phloem of the sycamore Acer pseudoplatanus has a large nucleus, simple plastids closely sheathed with rough endoplasmic reticulum, and numerous mitochondria. The cytoplasm contains numerous ribosomes, resulting in a very electron-opaque cytoplasm after permanganate fixation. Bodies similar to the spherosomes of Frey-Wyssling et al. (4) are collected in clusters and these also contain bodies of an unidentified nature similar to those found by Buttrose (1) in the aleurone cells of the wheat grain. The pores through the wall between the companion cell and sieve tube are complex and develop from a single plasmodesma. Eight to fifteen plasmodesmata on the companion cell side communicate individually with a cavity in the centre of the wall which is linked to the sieve tube by a single pore about twice the diameter of an individual plasmodesma. This pore is lined with material of an electron opacity equivalent to that of material bounding the sieve plate pores. The development of the cell organelles, the possible role played in the phloem tissue by the companion cell, and the function of the complex pores contained in its wall are discussed.     

Anatomical structure of <Emphasis Type="Italic">Camellia oleifera</Emphasis> shell

The main product of Camellia oleifera is edible oil made from the seeds, but huge quantities of agro-waste are produced in the form of shells. The primary components of C. oleifera fruit shell are cellulose, hemicellulose, and lignin, which probably make it a good eco-friendly non-wood material. Understanding the structure of the shell is however a prerequisite to making full use of it. The anatomical structure of C. oleifera fruit shells was investigated from macroscopic to ultrastructural scale by stereoscopic, optical, and scanning electron microscopy. The main cell morphology in the different parts of the shell was observed and measured using the tissue segregation method. The density of the cross section of the shell was also obtained using an X-ray CT scanner to check the change in texture. The C. oleifera fruit pericarp was made up of exocarp, mesocarp, and endocarp. The main types of exocarp cells were stone cells, spiral vessels, and parenchyma cells. The mesocarp accounted for most of the shell and consisted of parenchyma, tracheids, and some stone cells. The endocarp was basically made up of cells with a thickened cell wall that were modified tracheid or parenchyma cells with secondary wall thickening. The most important ultrastructure in these cells was the pits in the cell wall of stone and vessel cells that give the shell a conducting, mechanical, and protective role. The density of the shell gradually decreased from exocarp to endocarp. Tracheid cells are one of the main cell types in the shell, but their low slenderness (length to width) ratio makes them unsuitable for the manufacture of paper. Further research should be conducted on composite shell-plastic panels (or other reinforced materials) to make better use of this agro-waste.     

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Small angle neutron scattering on an absolute intensity scale and the internal surface of diatom frustules from three species of differing morphologies

The internal nanostructure of the diatoms Cyclotella meneghiniana, Seminavis robusta and Achnanthes subsessilis was investigated using small angle neutron scattering (SANS) to examine thin biosilica samples, consisting of isotropic (powder) from their isolated cell walls. The interpretation of SANS data was assisted by several other measurements. The N₂ adsorption, interpreted within the Branuer–Emmet–Teller isotherm, yielded the specific surface area of the material. Fourier transform infrared (FTIR) and Raman spectroscopy indicates that the isolated material is amorphous silica with small amounts of organic cell wall materials acting as a filling material between the silica particles. A two-phase (air and amorphous silica) model was used to interpret small angle neutron scattering data. After correction for instrumental resolution, the measurements on two SANS instruments covered an extended range of scattering vectors 0.0011 nm^?1 < q < 5.6 nm^?1, giving an almost continuous SANS curve over a range of scattering vectors, q, on an absolute scale of intensity for each sample. Each of the samples gave a characteristic scattering curve where log (intensity) versus log (q) has a ?4 dependence, with other features superimposed. In the high-q regime, departure from this behaviour was observed at a length-scales equivalent to the proposed unitary silica particle. The limiting Porod scattering law was used to determine the specific area per unit of volume of each sample illuminated by the neutron beam. The Porod behaviour, and divergence from this behaviour, is discussed in terms of various structural features and the proposed mechanisms for the bio-assembly of unitary silica particles in frustules.     

Morphogenetic processes inAttheya decora (Bacillariophyceae,Biddulphiineae)

The valva of the diatomAttheya decora is formed within a silica deposition vesicle which enlarges centrifugally by the fusion of small vesicles. The silica deposition vesicle can already be seen when the spindle has not yet disappeared completely. Valva formation seems to begin with the shaping of an organic matrix within a silica deposition vesicle. Later, this material silicifies. The complicated shape of the labiate process is preformed by the silica deposition vesicle, the inner membrane of which is associated with electron dense material on both faces. The horns are formed when the expanding silica deposition vesicle has reached the cell corners. They are elaborated without participation of microtubules. Swelling of local depositions of polysaccharides seems to provide the forces that spread the silicified horns during daughter cell separation and to cause the local spontaneous plasmolyses under the valva and along the cell flanks in the region of the intercalary bands. The inner organic wall layers and the organic continuations of the intercalary bands are formed on the surface of the plasmalemma; each of the continuations is produced simultaneously with the intercalary band belonging to it and becomes attached to the latter when the silica deposition vesicle opens.     

Ultrastructure of the nematode pathogen, Bacillus penetrans

The ultrastructure and development of Bacillus penetrans in root-knot nematodes, Meloidogyne spp., was studied with a transmission electron microscope. Host infection was by a germ tube from the cup-shaped sporangium containing the endospore. The prokaryotic vegetative cells contained septa formed by an ingrowth of the inner layer of the trilaminate cell wall and were associated with mesosomes. Structure of the endospore was similar to other bacteria with a spore protoplast enclosed within two cortical layers and three spore coats. An exosporium which may function in attachment and host specificity surrounded the endospore. Ultrastructural changes accompanying sporulation were similar to those reported for other endospore-forming bacteria but with some parasite specialization. The filamentous vegetative growth was characteristic of some Actinomycetales. Endospore development at the apices of dichotomously branched filaments of the thallus resembled the genus Actinobifida.     

Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.     

THE POLYMORPHIC DIATOM PHAEODACTYLUM TRICORNUTUM: ULTRASTRUCTURE OF ITS MORPHOTYPES1,2

The ultrastructure of the oval, fusiform and triradiate morphotypes of Phaeodactylum tricornutum Bohlin is described. The organization and structure of the cytoplasmic organelles is similar in all three morphotypes, except that the vacuoles occupy the extra volume created by the arms of the fusiform and triradiate cells. The frustule in fusiform and triradiate cells is organic; in the oval type it may be organic or one of the valves may have a silica frustule surrounded by an organic wall. In all cells, the organic cell wall has up to 10 silica bands (13 nm wide) embedded in its surface in the girdle region, lacks girdle bands, and has an outer corrugated cell wall layer, except in the girdle region. Cell division, organic wall formation and silica deposition are described in detail. Four types of oval cells are also described. The relation to other diatoms is discussed.     

ULTRASTRUCTURE OF A CHAIN-FORMING DIATOM PHAEODACTYLUM TRICORNUTUM1

The ultrastructure of a chain-forming clone of the polymorphic diatom Phaeodactylum tricornutum Bohlin has been studied by scanning and transmission electron microscopy. Both fusiform and tri-radiate cells are capable of forming chains. The cells, lacking any silica shell, are attached to each other at the central region of the theca, leaving the arms free. Neither homogenization nor sonication completely disrupts the chains. The attachment is due to fusion of the cell wall in the central region of the cell during cell wall deposition. This fusion results from failure of the cytoplasmic cleavage furrow to separate the plasma membranes of the two daughter cells sufficiently so that a single wall is deposited instead of two separate walls. Possible explanations for this are discussed.     

Reproductive biology of the antarctic opisthobranch Philine gibba Strebel

Monthly samples (August 1974 to February 1976) and cultured material have been used to describe ovotestis maturation, spawning, and larval development of the Antarctic opisthobranch Philine gibba Strebel. Ovotestis maturation took one year and mature individuals (mean shell length 12 ± 1.4 mm) spawned a single egg mass in April–May containing 975–4760 eggs, 379 μm in diameter.Development was non-pelagic but an active veliger was observed within the egg capsule. Juveniles (mean shell length 375 ± 55 μm), which hatched from August to October, resembled adults except for the presence of eye spots and an exposed shell. The mantle enclosed the shell by February when shell length was 890 ± 30 μm.     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

北京鸭产卵期输卵管管状腺细胞超微结构研究

用电子显徽镜对北京鸭输卵管管状腺细胞进行观察。鸭输卵管由五部分组成:漏斗、蛋白分泌部、峡部、壳腺和阴道。蛋白分泌部的管状腺细胞有四种类型。A型细胞有电子密度深色颗粒;B型细胞充满了无定型低电子密度物质;C型细胞具有非常明显的粗面内质网和高尔基复合体;D型细胞是由致密的颗粒和低电子密度的颗粒所组成,腔内充满分泌颗粒。我们在这篇文章中分析了蛋白分泌周期的四个不同阶段。     

Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography

Cyanobacteria are photosynthetic organisms responsible for ~ 25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.     

Ultrastructural study of mycobacteria in experimentally produced lung lesions of mice.

Mice were infected by iv injection with a virulent strain of Mycobacterium bovis, Ravenel strain, to prepare specimens for electron microscopical observation of their intracellular morphology. Observation was made with ultra-thin sections of the granulomatous lungs at an advanced stage of infection. Many apparently intact bacterial cells were found intracellulary, and the majority of them had lipoidal inclusions enclosed by a membranous structure. Several layers of mycobacterial cell wall were discernible, including a fairly wide space of the electron-transparent zone just beneath the electrondense outmost layer. Mesosomes, nuclear material, small dense granules and cross wall were found in almost the same appearance as those reported of mycobacteria grown in vitro. The bacilli were located mainly within intact or damaged phagosomes which were often filled with amorphous material of various electron densities.     

Ultrastructural localization of polysaccharides in the motile diatomNavicula cuspidata

Summary Generation of movement in benthic diatoms is thought to be intimately associated with secretion at the raphe, a slit in the silica cell wall. The presence and distribution of extracellular substances and their source was investigated cytochemically by transmission electron microscopy. Extracellular material, possibly-acid mucopolysaccharide, was observed consistently within the entire length of the raphe of both valves and also as a sheath enveloping the silica frustule. Such quantities of extracellular material are absent in conventionally fixed motile diatoms. Numerous cytoplasmic vesicles, with fibrillar contents, distributed peripherally but concentrated along the raphe and at the cell poles, react strongly with a polysaccharide specific stain; their distribution in the cell and polysaccharide content suggest these may be the source of raphe and sheath material. Results support the most recent theories on the mechanism of locomotion in outline only; the details cannot be clarified. Localization procedures using alcian blue and silver staining of peroxidised sections are discussed briefly.