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At least 1,279 records · Page 71Linked to original sources
PubMed1979

An electron microscope comparison of plasma membrane vesicles from meristematic and mature soybean root tissue.

Plasma membrane vesicles were isolated from homogenates of meristematic and mature soybean root tissue by differential sucrose gradient centrifugation. Vesicles were positively identified by the phosphotungstic acid-chromic acid procedure (PACP). The two preparations were comparable in size class distribution, mitochondrial contamination, and per cent plasma membrane vesicles present. Purity levels were estimated to be greater than 75%. The specificity of PACP was observed for a variety of cell types from both regions. Some variability in PACP staining was offset by careful modulation of the stain protocol and was found to be independent of developmental stage in subcellular fractions. Patchy or discontinuous staining, observed in both intact tissue and in subcellular fractions from both regions, was found to be a function of stain time.

Journal Article↗
PubMed1980

Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES.

PLASMA MEMBRANE PREPARATIONS FROM SOYBEAN ROOT AND HYPOCOTYL CONTAINED THE FOLLOWING FREE STEROLS: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.Electron microscope studies indicated that all root plasma membrane preparations were equivalent in terms of relative purity. Hypocotyl membrane preparations contained significantly greater levels of contaminating membrane components.Root plasma membrane fractions were between 70 and 80% pure as determined by staining with the phosphotungstic acid-chromic acid procedure (PACP). Staining was most definitive for vesicles present in complete cross-section. Electron micrographs showed that vesicles treated with concanavalin A (Con A)-ferritin were extensively labeled at the outer surface indicating the presence of mannosyl and/or glucosyl residues at the vesicle surface. Densities of ferritin were highest on vesicles present in oblique section. PACP and Con A-ferritin were thus complementary with respect to topological specificity.The percentage of Con A-ferritin-labeled and/or PACP-stained vesicles in plasma membrane root preparations was greater than 80%. Con A did not bind in purified tonoplast preparations, and binding was reduced in regions of low PACP reactivity in a root membrane fraction containing a lowered proportion of plasma membrane. Con A specificity for the plasma membrane in subcellular membrane preparations is discussed.

Journal Article↗
PubMed1982

A comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes.

Plasma membrane-associated ATPase obtained from cauliflower (Brassica oleraceae L.) florets isolated and assayed by several different procedures was stimulated 150 to 400% by K(+). In contrast, winter wheat (Triticum aestivum L. cv. Kharkov 22 MC) shoot and root ATPase obtained by the same methods exhibited only 10 to 25% stimulation by K(+). The level of K(+)-stimulation of the wheat enzyme was not significantly increased by purifying the crude microsomal membrane fraction using sucrose density gradients. ATPase associated with density gradient-purified cauliflower membranes was inhibited by Ca(2+), high ATP concentration in the presence of low Mg(2+), and by several metabolic inhibitors. In contrast, the wheat enzyme was largely unaffected by all of these treatments. The plasma membranes of intact wheat and cauliflower cells gave a positive reaction with the plasma membrane-specific, phosphotungstic acid-chromic acid stain (PACP). A high proportion of the cauliflower membrane vesicles in the putative plasma membrane-enriched fraction stained with PACP, whereas only a small proportion of the wheat membrane vesicles reacted positively with PACP. These results indicate that a plasma membrane-enriched fraction has been isolated successfully from cauliflower floret tissue, but that none of the procedures used effectively separate plasma membranes from homogenates of wheat shoots and roots.

Journal Article↗
PubMed1983

Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants.

A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilizing an aqueous two-polymer phase system is outlined. Mainly, the plant used was Orchard grass (Dactylis glomerata L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-15 millimolar Tris-maleate (pH 7.3), and 30 millimolar NaCl. In this system, the plasma membranes and the other membranes were preferentially partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a single partition (i.e. about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase. The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mg(2+)-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a high specificity for Mg(2+) and ATP. KCl stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be applicable to a wide variety of plants and plant tissue including green leaves.

Journal Article↗
PubMed2001

Novel filaments 5 nm in diameter constitute the cytosolic ring of the plastid division apparatus.

The plastid division apparatus (called the plastid-dividing ring) has been detected in several plant and algal species at the constricted region of plastids by transmission electron microscopy. The apparatus is composed of two or three rings: an outer ring in the cytosol, an inner ring in the stroma, and a middle ring in the intermembrane space. The components of these rings are not clear. FtsZ, which forms the bacterial cytokinetic ring, has been proposed as a component of both the inner and outer rings. Here, we present the ultrastructure of the outer ring at high resolution. To visualize the outer ring by negative staining, we isolated dividing chloroplasts from a synchronized culture of a red alga, Cyanidioschyzon merolae, and lysed them with nonionic detergent Nonidet P-40. Nonidet P-40 extracted primarily stroma, thylakoids, and the inner and middle rings, leaving the envelope and outer ring largely intact. Negative staining revealed that the outer ring consists of a bundle of 5-nm filaments in which globular proteins are spaced 4.8 nm apart. Immunoblotting using an FtsZ-specific antibody failed to show immunoreactivity in the fraction containing the filament. Moreover, the filament structure and properties are unlike those of known cytoskeletal filaments. The bundle of filaments forms a very rigid structure and does not disassemble in 2 M urea. We also identified a dividing phase-specific 56-kD protein of chloroplasts as a candidate component of the ring. Our results suggest that the main architecture of the outer ring did not descend from cyanobacteria during the course of endosymbiosis but was added by the host cell early in plant evolution.

Actin Cytoskeleton↗
PubMed1979

An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses.

A brief historical account of the development and applications of the negative staining techniques to the study of the structure of viruses and their components as observed in the electron microscope is presented. Although the basic method of surrounding or embedding specimens in opaque dyes was used in light microscopy dating from about 1884, the equivalent preparative techniques applied to electron microscopy were comparatively recent. The combination of experiments on a sophisticated bacterial virus and the installation of a high resolution electron microscope in the Cavendish Laboratory, Cambridge, during 1954, subsequently led to the analysis of several important morphological features of animal, plant and bacterial viruses. The implications of the results from these early experiments on viruses and recent developments in negative staining methods for high resolution image analysis of electron micrographs are also discussed.

Animals↗
PubMed1993

Viruses accumulate spontaneously near droplet surfaces: a method to concentrate viruses for electron microscopy.

Virus particles suspended in a drop of water tend to concentrate at or near its surface with the air. The concentrated, and probably more purified, particles may then be collected on a film-coated grid for negative staining and electron microscopy. This is a useful method, simpler than others (e.g. high-speed centrifugation, Lyphogel, or precipitation by (NH4)2SO4) which are used to process clinical specimens for diagnosis where virus particles may be too dilute in the original sample. It is shown, by freeze-fracturing for electron microscopy, that Orf virus particles do accumulate at and just below the surfaces of drops. Various physical effects which may cause the particles to accumulate are considered. Results from a computer model suggest that Brownian motion alone could be adequate to transport a useful quantity of the particles in the body of a 2-mm-diameter hemispherical drop to its surface if the particles do not clump and if they remain trapped at the surface when they reach it. In practice, transport by Brownian motion is likely to be augmented by swirling, convection and other effects within drops.

Bacitracin↗
PubMed1997

Nemaline bodies of skeletal muscle fibers of the neck in a case of pharyngeal cancer.

Nemaline bodies were found in the regenerating muscle fibers of the neck of a 74-year-old Japanese male with pharyngeal cancer. Histologically, the nemaline bodies were needle-shaped structures and hematoxophilic with phosphotungstic acid-hematoxylin stain. Electron microscopic examination suggested that they arise as extensions of the Z bands. Although nemaline bodies occur in a variety of pathological conditions other than nemaline myopathy, their occurrence in regenerating skeletal muscle fibers invaded by cancer has not been reported previously. The pathogenesis of the nemaline body formation in the present case is discussed.

Aged↗