Superficial cell-wall layers on Spirillum "Ordal" and their in vitro reassembly

1976 ◽  
Vol 22 (4) ◽  
pp. 567-582 ◽  
Author(s):  
T. J. Beveridge ◽  
R. G. E. Murray
Keyword(s):  
Cell Envelope ◽  
Amorphous Layer ◽  
Superficial Layer ◽  
Optical Diffraction ◽  
Hexagonal System ◽  
Thin Sections ◽  
Regular Structures ◽  
Packed Layer ◽  
Freeze Etching

The cell envelope of Spirillum sp. strain "Ordal" (possibly a variety of S. anulus) demonstrated multiple superficial wall layers which were diverse in their macromolecular arrays. Negative staining and freeze-etching techniques revealed an outer hexagonally packed layer and an inner tetragonally packed layer. However, both thin sections and freeze-etched cleavages of the wall showed that each of these regular structures rested upon a backing layer, and that there was a delicate amorphous layer overlying the outer hexagonal array. Rotary integration, optical diffraction, and reconstruction of image were used to clarify measurements of each array and to verify the validity of a diagrammatic model of the outer hexagonal system. The integrity of these layers required suitable cations (Ca2+ appeared essential) and pH ([Formula: see text] dissociated most superficial layers). These observations aided in the development of a low-pH cationic-substitution technique, in which Na+ replaced essential Ca2+, for extraction of the layers from the cell surface. Dialysis to remove Na+ and restoration of Ca2+ initiated in vitro reassembly of the superficial layer components until regularly structured assembly products were formed.

Fine structure of the macroconidia of Fusarium solani

1975 ◽  
Vol 53 (19) ◽  
pp. 2134-2146 ◽  
Author(s):  
J. P. Tewari ◽  
W. P. Skoropad
Keyword(s):  
Cell Wall ◽  
Fusarium Solani ◽  
Superficial Layer ◽  
Dense Layer ◽  
Inoculum Potential ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Innermost Layer ◽  
Freeze Etching ◽  
Myelin Figures

Ultrastructure of the macroconidia of Fusarium solani as visualized by transmission (ultrathin sectioning and freeze-etching) and scanning electron microscopy is described. The cell wall has four layers. The innermost layer is electron-lucid followed by an electron-dense layer. The next outer layer is spongy in appearance followed by a superficial layer consisting of fine filamentous processes. Freeze-etch replicas of conidia directly removed from the sporodochia and still suspended in the mucilaginous material (in which they are produced) frequently show the conidia connected by the superficial filamentous processes in the cell wall. This agglutination of the conidia is likely to increase the inoculum potential of this pathogen at the sites of infection. Structure of various membrane systems in the cells is described. The endoplasmic reticulum is fairly extensive and fenestrated. Thin sections of routinely fixed conidia show myelin figures. However, such structures were not seen in replicas of conidia that were freeze-etched without use of chemical fixation or cryoprotection.

The biosynthesis of cellulose by Acetobacter xylinum and Acetobacter acetigenus

1977 ◽  
Vol 23 (6) ◽  
pp. 701-709 ◽  
Author(s):  
J. Ross Colvin ◽  
Gary G. Leppard
Keyword(s):  
Cell Envelope ◽  
Acetobacter Xylinum ◽  
Cellulose Microfibrils ◽  
Cellulose Synthesis ◽  
Electron Microscopic ◽  
Air Drying ◽  
Thin Sections ◽  
Morphological Aspects ◽  
Transmission Electron ◽  
Freeze Etching

The morphological aspects of biosynthesis of cellulose by Acetobacter xylinum and Acetobacter acetigenus were studied by transmission electron microscopy of both freeze-etch replicas and sections of cellulose-free cells in suspension culture before and subsequent to the induction of cellulose synthesis. Also examined were freshly synthesized, thoroughly washed, cellulose pellicles. Thin sections of rapidly dividing, glucose-metabolizing cells of both species showed irregular features on the cell surface including a small polar invagination which sometimes contained or was associated with fibrils as fine as 3 nm in diameter of a substance which stains with electron-microscopic counterstains. Cellulose microfibrils in thin sections of freshly synthesized pellicles were coated with a surface material which also stained with the same counterstains (uranyl ions and lead ions). The effect of air-drying on freshly synthesized cellulose was striking. When examined by freeze-etching, thoroughly washed, never air-dried pellicles of both species showed a nascent form of cellulose fibril which consisted of a central, dense core surrounded by a sheath of amorphous gel. This sheath may be up to 100 nm wide. When the pellicle was air-dried and rehydrated before freeze-etching, the amorphous sheath was rare and shrunken but ordinary microfibrils of classical dimensions were visible. The sheath and core are sometimes closely associated with the envelope of the cells of both species. These observations can be interpreted in the context of recent advances in cellulose synthesis by assuming that chains of an initial, highly hydrated, intermediate polyglucan are released from the cell and that such chains associate to form a nascent fibril external to the cell but associated with the cell envelope. Air-drying of nascent fibrils converts them to classical microfibrils and this conversion is considered here in molecular terms.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

1994 ◽  
Vol 52 ◽  
pp. 276-277
Author(s):  
Mary Beth Downs ◽  
Wilson Ribot ◽  
Joseph W. Farchaus
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Cell Envelope ◽  
Single Species ◽  
Supporting Cell ◽  
Surface Layers ◽  
Rabbit Igg ◽  
In Vitro Assembly ◽  
Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

Studies on Thrombolysis with Streptokinase

1971 ◽  
Vol 25 (02) ◽  
pp. 354-378 ◽  
Author(s):  
R Gottlob ◽  
L Stockinger ◽  
U Pötting ◽  
G Schattenmann
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Whole Blood ◽  
Jugular Vein ◽  
Thin Sections ◽  
The Third ◽  
Morphological Findings ◽  
Blood Clots ◽  
Arteries And Veins

SummaryIn vitro whole blood clots of various ages, experimental thrombi produced in the jugular vein of rabbits and human thrombi from arteries and veins were examined in semi-thin sections and by means of electron microscopy.In all types of clots examined a typical course of retraction was found. Retraction starts with a dense excentrical focus which grows into a densification ring. After 24 hours the entire clot becomes almost homogeneously dense; later a secondary swelling sets in.Shortly after coagulation the erythrocytes on the rim of the clot are bi-concave discs. They then assume the shape of crenate spheres, turn into smooth spheres and finally become indented ghosts which have lost the largest part of their contents. In the inner zone, which makes up the bulk of the clot, we observed bi-concave discs prior to retraction. After retraction we see no crenations but irregularly shaped erythrocytes. Once the secondary swelling sets in, the cross-section becomes polygonal and later spherical. After extensive hemolysis we observe the “retiform thrombus” made up of ghosts.Experimental and clinical thrombi present the same morphology but are differentiated from in vitro clots by: earlier hemolysis, immigration of leukocytes, formation of a rim layer consisting of fibrin and thrombocytes, and the symptoms of organization. Such symptoms of organization which definitely will prevent lysis with streptokinase were found relatively late in experimental and clinical thrombi. Capillary buds and capillary loops were never found in clinical thrombi prior to the third month.The morphological findings agree with earlier physical and enzymatic investigations. The observation that phenomena of reorganization occur relatively late and frequently only in the rim areas of large thrombi explains why lytic therapy is possible in some of the chronic obliterations.

Mineral Density and Fluoride Content of in vitro Remineralized Lesions

1988 ◽  
Vol 67 (3) ◽  
pp. 577-581 ◽  
Author(s):  
Y. Jima ◽  
T. Koulourides
Keyword(s):  
Complete Recovery ◽  
Fluoride Content ◽  
Mineral Density ◽  
Acid Dissolution ◽  
Thin Sections ◽  
Partially Saturated ◽  
In Vitro Investigation ◽  
Bovine Enamel ◽  
Caries Lesions

This in vitro investigation studied the remineralization of experimental caries lesions in bovine enamel by use of three methods: (1) surface microhardness, (2) microradiography, and (3) abrasion biopsy for mineral density and fluoride content. The lesions were produced by a two-day exposure to 0.01 mol/L lactic acidlsodium hydroxide buffer partially saturated with 3.0 mmol/L Ca, 1.8 mmol/L P, in 1% CMC, at pH 4.0 and 37°C. The lesions were exposed to a remineralizing solution containing 3.0 mmol/L Ca, 1.8 mmol/L P, and 3 ppm F in 1% CMC at pH 7.0 and 37°C for two, six, and ten days, with solution changes every two days. The data derived from the three methods are presented in sequence from the baseline and at days two, six, and ten of the remineralizing treatment. Microhardness measurements showed hardness recoveries of 35.9, 78.9, and 87.5%, respectively. Microradiography suggested complete recovery with the ten-day remineralization. Abrasion biopsy of successive 10-μm layers to a depth of 100 μm indicated 15.2, 39.8, and 68.8% mineral density recoveries, with fluoride content of the surface layer increasing from a baseline of 300 ppm to 4600, 9000, and 9800 ppm F for the 2, 6, 10 days of remineralization, respectively. Subsequent acid-etching of thin sections from the ten-day-remineralized specimens showed that the fluoride-enriched remineralized area was more resistant to acid dissolution than was the underlying nonnal enamel.

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