scholarly journals Rapid isolation of plasma membranes in high yield from cultured fibroblasts

1977 ◽  
Vol 168 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D Thom ◽  
A J Powell ◽  
C W Lloyd ◽  
D A Rees
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
High Yield ◽  
Chemical Compositions ◽  
Differential Centrifugation ◽  
Rapid Preparation ◽  
Cultured Fibroblasts ◽  
Good Agreement

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2—lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.

scholarly journals myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

1986 ◽  
Vol 236 (1) ◽  
pp. 37-44 ◽  
Author(s):  
D M Delfert ◽  
S Hill ◽  
H A Pershadsingh ◽  
W R Sherman ◽  
J M McDonald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Membrane Vesicles ◽  
Heavy Fraction ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Inositol 1,4,5 Trisphosphate ◽  
Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

scholarly journals Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

1980 ◽  
Vol 86 (1) ◽  
pp. 21-28 ◽  
Author(s):  
M S Klempner ◽  
R B Mikkelsen ◽  
D H Corfman ◽  
J André-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Human Neutrophil ◽  
Plasma Membranes ◽  
Hydrolytic Enzymes ◽  
Membrane Vesicles ◽  
Galactose Oxidase ◽  
High Yield ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Equilibrium Ultracentrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

scholarly journals Laminin in cultured mouse C1300 neuroblastoma cells: immunocytochemical localization by pre- and postembedding electron microscope procedures.

1983 ◽  
Vol 31 (6) ◽  
pp. 755-764 ◽  
Author(s):  
P Liesi
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Plasma Membranes ◽  
Neuroblastoma Cells ◽  
Immunocytochemical Localization ◽  
Adhesion Protein ◽  
Antibody Method ◽  
Ultrathin Sections ◽  
Intercellular Connections

Laminin was localized in cultured mouse C1300 neuroblastoma cells by applying the peroxidase-antiperoxidase technique in preembedding electron microscopy. The results were compared to those obtained by indirect immunofluorescence and by the colloidal gold second antibody method on Epon-embedded ultrathin sections. Laminin was found in the cell membranes and within the rough endoplasmic reticulum as well as in intracytoplasmic vacuoles. Plasma membranes of the neuroblastoma cells showed a patchy localization of laminin that was apparently involved in cell-to-substrate attachment and in gap junction-like intercellular connections. Under normal conditions, the Golgi cisternae contained no laminin. Pretreatment of cells with micromolar concentrations of monensin, however, lead to an accumulation of laminin within the Golgi cisternae. These results support a role for laminin as an adhesion protein in cultured neuroblastoma cells and indicate that laminin is transported through the Golgi complex.

Structure of Cultured Fibroblasts from Dermatosparaxic Calves

1975 ◽  
Vol 12 (1) ◽  
pp. 16-31 ◽  
Author(s):  
J. A. Yaeger ◽  
R. L. Church ◽  
M. L. Tanzer
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Endoplasmic Reticulum ◽  
Cell Lines ◽  
Lipid Droplets ◽  
Cultured Fibroblasts ◽  
Fibrillar Material ◽  
Golgi Region ◽  
Clonal Cell Lines ◽  
Extracellular Fibrils

Clonal cell lines from the dermis of a dermatosparaxic calf were grown in tissue culture. After fixation in a mixture of glutaraldehyde and osmium, they were prepared for electron microscopy. Most cells contained a well-developed Golgi region, lysosomes, mitochondria, and dilated cisternae of rough endoplasmic reticulum. They also contained numerous, large bundles of intracellular filaments, many lipid droplets and extensive arrays of vesicles. Cultures accumulated substantial amounts of extracellular fibrillar material. The fibrils were loosely packed and indistinctly cross-banded. Bundles of intracellular filaments were commonly parallel in adjacent cells and also parallel to extracellular fibrils. These cytoplasmic features may result from the inability of the secreted collagen to form normal fibrils.

scholarly journals Isolation of plasma and nuclear membranes of thymocytes. I. Enzymatic composition and ultrastructure

1978 ◽  
Vol 77 (1) ◽  
pp. 211-231 ◽  
Author(s):  
A Monneron ◽  
J d'Alayer
Keyword(s):  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Enrichment Factors ◽  
Viscous Medium ◽  
High Yield ◽  
Equilibrium Density ◽  
Plasma Membrane Vesicles ◽  
Thin Sections ◽  
Nuclear Membranes ◽  
Membrane Components

The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, gamma-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase. Plasma membranes and light nuclear membranes appeared as pure unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.

scholarly journals Electron microscope localization of acetylcholinesterase and butyrylcholinesterase in the ciliary ganglion of the cat.

1984 ◽  
Vol 32 (8) ◽  
pp. 849-861 ◽  
Author(s):  
R Davis ◽  
G B Koelle ◽  
U J Sanville
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Superior Cervical Ganglion ◽  
Extracellular Space ◽  
Plasma Membranes ◽  
Ganglion Cells ◽  
Ciliary Ganglion ◽  
Cervical Ganglion ◽  
High Concentrations

Ciliary ganglia (CG) of cats were stained for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by the bis-(thioacetoxy) aurate (I), or Au(TA)2, method for examination by electron microscopy. Acetylcholinesterase was localized along the axolemmas of the preganglionic fibers and their terminals and on the plasmalemmas of the perikarya and dendrites of the ganglion cells, as in the cat superior cervical ganglion (SCG). In contrast to the SCG, AChE was also found in significant amounts in the rough endoplasmic reticulum of the CG cells and dendrites, and in varying but high concentrations in channels of extracellular space in the complex capsular region surrounding the perikarya and dendrites. Butyrylcholinesterase was confined chiefly to the dendritic and perikaryonal plasma membranes of the ganglion cells, as in the SCG. Lysosomes and mitochondria were stained chiefly for non-cholinesterase enzymes, as indicated by the physostigmine-treated controls. The significance of these distributions is discussed.

Electrogenic ATP-dependent Cl- transport by plasma membrane vesicles from Aplysia intestine

1988 ◽  
Vol 254 (1) ◽  
pp. R127-R133 ◽  
Author(s):  
G. A. Gerencser
Keyword(s):  
Plasma Membrane ◽  
Transport Process ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Cl Transport ◽  
Vesicular Membrane

A Cl--stimulated adenosinetriphosphatase (ATPase) activity and an ATP-dependent Cl- transport process were found in Aplysia enterocyte plasma membranes. In an attempt to further elucidate this transport process plasma membrane vesicles from Aplysia enterocytes were prepared utilizing differential centrifugation and sucrose density gradient techniques. Electrogenicity of the ATP-dependent Cl- transport was confirmed in three ways. First, an inwardly directed valinomycin-induced K+ diffusion potential, making the vesicle interior electrically positive, enhanced ATP-driven Cl- uptake compared with vesicles lacking the ionophore. Second, ATP plus Cl- increased intravesicular negativity measured by lipophilic triphenylmethylphosphonium distribution across the vesicular membrane. Third, both vanadate and thiocyanate inhibited the ATP plus Cl--dependent intravesicular negativity. These results are consistent with the hypothesis that the active electrogenic Cl- transport mechanism in Aplysia intestine could be a Cl--stimulated ATPase found in the enterocyte plasma membrane.

scholarly journals Electron Microscopy of the Development of Tracheoles in Drosophila Melanogaster

1963 ◽  
Vol s3-104 (65) ◽  
pp. 135-140
Author(s):  
S. AHMAD SHAFIQ
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Drosophila Melanogaster ◽  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Developmental Stages ◽  
Membrane Structures ◽  
Flight Muscles

The tracheoblasts associated with the flight-muscles of Drosophila were studied by electron microscopy. During the developmental stages the cytoplasm of such tracheoblasts shows extensive membrane structures arranged in whorls. It seems that these membranes become aligned in pairs, spread out in tracheoblast cytoplasm, and form the walls of the new tracheolar vessels. The membranous whorls appear to have no obvious relationship with the usual endoplasmic reticulum, Golgi apparatus or plasma membranes. They are probably produced from certain large granules distributed irregularly in the cytoplasm of the young tracheoblasts. Membranes limiting the tracheoles from the tracheoblast cytoplasm (the so-called mestracheons) are not usually seen in the younger stages. They are sometimes seen after the cuticular lining of the tracheoles has been formed.

scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1970 ◽  
Vol 44 (3) ◽  
pp. 484-491 ◽  
Author(s):  
D. James Morré ◽  
R. L. Hamilton ◽  
H. H. Mollenhauer ◽  
R. W. Mahley ◽  
W. P. Cunningham ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Complex Network ◽  
Rat Liver ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Rat Hepatocytes ◽  
Secretory Vesicles ◽  
Differential Centrifugation

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.

scholarly journals Thermoelastic properties and γ’-solvus temperatures of single-crystal Ni-base superalloys

2021 ◽  
Vol 56 (12) ◽  
pp. 7637-7658
Author(s):  
O. M. Horst ◽  
D. Schmitz ◽  
J. Schreuer ◽  
P. Git ◽  
H. Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Crystal ◽  
Chemical Compositions ◽  
Thermoelastic Properties ◽  
Resonant Ultrasound Spectroscopy ◽  
Transmission Electron ◽  
Resonant Ultrasound ◽  
Elastic Coefficients ◽  
Good Agreement

Abstract The present work shows that thermal expansion experiments can be used to measure the γʼ-solvus temperatures of four Ni-base single-crystal superalloys (SX), one with Re and three Re-free variants. In the case of CMSX-4, experimental results are in good agreement with numerical thermodynamic results obtained using ThermoCalc. For three experimental Re-free alloys, the experimental and calculated results are close. Transmission electron microscopy shows that the chemical compositions of the γ- and the γʼ-phases can be reasonably well predicted. We also use resonant ultrasound spectroscopy (RUS) to show how elastic coefficients depend on chemical composition and temperature. The results are discussed in the light of previous results reported in the literature. Areas in need of further work are highlighted. Graphical abstract

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