Cryoultramicrotomy of plant protoplasts

1978 ◽  
Vol 36 (2) ◽  
pp. 36-37
Author(s):  
Francis A. Williamson
Keyword(s):  
Cell Wall ◽  
Freezing Point ◽  
Lectin Binding ◽  
Enzymatic Digestion ◽  
Allium Porrum ◽  
General Description ◽  
Plant Protoplasts ◽  
Sodium Cacodylate ◽  
Concentrated Suspension ◽  
Specimen Holder

IntroductionNaked plant cells (protoplasts) have been used for the study of lectin binding to the plasma membrane, and of the initial stages of cell wall formation (2,3 and refs, therein). In this paper, I describe methods for the cryoultramicrotomy of plant protoplasts and give a general description of their ultrastructure. In addition, labelled lectins have been applied to cryo-sections to locate intracellular binding sites, and the superior resolution of the negatively stained cryo-sections has been applied to a preliminary study of cell wall fibril formation.Materials and MethodsProtoplasts were prepared from meristematic leaves of leek (Allium porrum) by enzymatic digestion (3). After washing, the cells were fixed in 1% glutaraldehyde in 0.55 M sorbitol and 1 mM CaCl2 buffered at pH 7.2 with 10 mM sodium cacodylate or HEPES. They were then washed (16 h) in 100 mM sodium cacodylate pH 7.2 and infused with 1 M sucrose in the same buffer for 3 h. A very concentrated suspension of the cells was mounted on a specimen holder and frozen in nitrogen slush (liquid nitrogen at its freezing point).

scholarly journals Effects of Electro- and Chemical Fusion Treatment on Cell Wall Formation and Cell Division of Plant Protoplasts.

1994 ◽  
Vol 63 (4) ◽  
pp. 706-713
Author(s):  
Salak PHANSIRI ◽  
Hiroshi MIYAKE ◽  
Eizo MAEDA ◽  
Takeshi TANIGUCHI
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Cell Wall Formation ◽  
Plant Protoplasts ◽  
Wall Formation

Cell-wall-bound peroxidase activity in roots of mycorrhizal Allium porrum

1988 ◽  
Vol 109 (1) ◽  
pp. 119-124 ◽  
Author(s):  
P. SPANU ◽  
P. BONFANTE-FASOLO
Keyword(s):  
Cell Wall ◽  
Peroxidase Activity ◽  
Allium Porrum

scholarly journals Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study.

1980 ◽  
Vol 85 (2) ◽  
pp. 199-212 ◽  
Author(s):  
J Molano ◽  
B Bowers ◽  
E Cabib
Keyword(s):  
Cell Wall ◽  
Fluorescence Microscopy ◽  
Lectin Binding ◽  
Strong Association ◽  
Fluorescein Isothiocyanate ◽  
Chemical Study ◽  
Total Radioactivity ◽  
Lytic Enzymes ◽  
Derivatives Of ◽  
Lateral Walls

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.

Lomasome biogenesis and function in armillaria mellea

1978 ◽  
Vol 36 (2) ◽  
pp. 402-403
Author(s):  
B. Ch. Behboodi
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Osmium Tetroxide ◽  
Higher Plants ◽  
Cell Wall Formation ◽  
Extensive Investigation ◽  
Sodium Cacodylate ◽  
Armillaria Mellea ◽  
Synthetic Media ◽  
And Function

IntroductionBorder bodies or lomasomes are the aggregation of membranes and vesicles located between the plasma membrane and the cell wall of many fungi, algae, and higher plants. Despite extensive investigation, the biogenesis as well as function of these structures is not yet known. The purpose of this investigation was to describe the biogenesis of lomasomes in Armillaria mellea and to provide some observations on their function related to cell wall formation.Materials and MethodsVarious thalli of fungi as non-aggregated hyphae, pseudosclerotes, rhizomorphs and carpophores were grown either on orange or synthetic media as described previously. The thalli were fixed in 4% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4), and 0.15 M sucrose for 4 h at 4°. They were postfixed with 1% osmium tetroxide in the same buffer for 2 h at 4° and embedded in Epon according to the Luft procedure. Cytochemical studies using thiocarbohydrazide-silver proteinate were performed according the Thiéry.

Simultaneous Freeze-Etching of Eight Specimens

1969 ◽  
Vol 27 ◽  
pp. 398-399
Author(s):  
Russell L. Steere ◽  
Michael Moseley
Keyword(s):  
Freezing Point ◽  
Specimen Holder ◽  
Cold Stage ◽  
Freeze Etching

A specimen cap for the Denton Freeze-Etching Module has been modified (Fig. 1) to permit the easy manipulation and simultaneous freeze-etching of eight specimens. A slot has been cut in one side of the cap to permit the insertion of a brass washer and a thin copper retainer plate above the threads, but below the supporting top of the cap. The inside rims of the washer are elevated, and the end of each specimen holder flange is beveled down and out to the base to form a wedge which can be forced under the washer. Small caps are placed over the specimen in each holder. Specimens are frozen by rapid immersion in Freon 22. The assembled cap is then mounted on a stage, standing in liquid Freon near its freezing point and slightly loosened. The eight frozen specimens, with caps in place, are picked up individually with tweezers and wedged under the washer, lifting it in the process. These operations can be accomplished with cold stage and cap under a dissecting microscope, if desired.

scholarly journals Two Types of Bacteria Adherent to Bovine Respiratory Tract Ciliated Epithelium

1993 ◽  
Vol 30 (1) ◽  
pp. 12-19 ◽  
Author(s):  
A. T. Hastie ◽  
L. P. Evans ◽  
A. M. Allen
Keyword(s):  
Cell Wall ◽  
Microscopic Examination ◽  
Lectin Binding ◽  
Electron Microscopic Examination ◽  
Tracheal Epithelium ◽  
Ciliated Cells ◽  
Electron Microscopic ◽  
Department Of Agriculture ◽  
Immunogold Staining ◽  
Transmission Electron

Two hundred sixty tracheas were obtained from a Philadelphia abattoir under permit from the Department of Agriculture; the tracheas were excised from predominantly Holstein calves of both sexes that weighed approximately 250 kg. Tracheas were transported in normal saline to the laboratory at Thomas Jefferson University, Philadelphia, Pennsylvania. Evidence of bacteria adherent to the tracheal epithelium was found in specimens from 20/24 of these tracheas. The epithelium from each of five tracheas was placed in glutaraldehyde fixative for transmission electron microscopic examination. Epithelium from each of 12 other tracheas was placed in formaldehyde fixative for light microscopic examination. Microscopically, 13 of these 17 bovine tracheal epithelia were observed to contain bacteria located longitudinally parallel to and between cilia and microvilli of ciliated cells. Preparations of ciliary axonemes isolated from the epithelium of seven additional bovine tracheas also contained these bacteria in sections viewed by a transmission electron microscope. These bacteria had two different ultrastructural morphologies: filamentous with a trilaminar-structured cell wall and short with a thick, homogeneously stained cell wall beneath a regularly arrayed surface layer. The short bacillus had surface carbohydrates, including mannose, galactose, and N-acetylgalactosamine, identified by lectin binding. The filamentous bacillus was apparently externally deficient in these carbohydrates. Immunogold staining revealed that the filamentous bacillus was antigenically related to cilia-associated respiratory (CAR) bacillus, which has been identified in rabbit and rodent species. Significantly decreased numbers of cilia were obtained from tracheal epithelium heavily colonized by the filamentous bacilli, suggesting a pathologic change in ciliated cells.

Isolation and lectin-binding properties of barley epidermal and mesophyll protoplasts

1994 ◽  
Vol 72 (11) ◽  
pp. 1688-1691 ◽  
Author(s):  
C. Struck ◽  
R. Rohringer ◽  
R. Heitefuss
Keyword(s):  
Concanavalin A ◽  
Lectin Binding ◽  
Gradient Centrifugation ◽  
Fluorescein Isothiocyanate ◽  
Enzymatic Digestion ◽  
Arabinogalactan Protein ◽  
Fluorescence Labeling ◽  
Hordeum Vulgare L ◽  
Mesophyll Protoplasts ◽  
Soybean Lectin

Protoplasts from primary leaves of barley (Hordeum vulgare L.) were obtained by enzymatic digestion and fractionated by discontinuous density gradient centrifugation to yield highly enriched fractions of mesophyll and epidermal protoplasts. A characterization of both protoplast types resulted in a clear differentiation of the outer protoplast surfaces. The protoplasts were examined for affinity to various lectins by agglutination tests and by labeling with lectin – fluorescein isothiocyanate conjugates. Both types of protoplasts agglutinated with soybean lectin. Fluorescein isothiocyanate-labeled soybean lectin was uniformly distributed on the protoplast surface. Mesophyll protoplasts, but not protoplasts from the epidermis, were agglutinated by Concanavalin A. Both types of protoplasts exhibited fluorescence labeling with Concanavalin A – fluorescein isothiocyanate conjugate. This label often showed a patchy distribution on the protoplast surface. Tetragonolobus lectin and β-D-glucosyl Yariv artificial antigen agglutinated mesophyll but not epidermal protoplasts. One of three tested monoclonal antibodies with specificity for arabinogalactans had affinity to the surface of mesophyll and epidermal protoplasts. Key words: agglutination, arabinogalactan protein, cell surface, epidermal protoplasts, fluorescence labeling, lectin.

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