scholarly journals Visualization of nucleosomes in thin sections by stereo electron microscopy.

1980 ◽  
Vol 87 (3) ◽  
pp. 833-836 ◽  
Author(s):  
A L Olins ◽  
D E Olins ◽  
H Zentgraf ◽  
W W Franke
Keyword(s):  
Electron Microscopy ◽  
Ionic Strength ◽  
Electron Micrographs ◽  
High Magnification ◽  
Thin Sections ◽  
Nucleosome Structure ◽  
The Reformation ◽  
The Stability ◽  
Chicken Erythrocytes ◽  
Low Ionic Strength

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.

Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins

1984 ◽  
Vol 246 (6) ◽  
pp. H865-H875 ◽  
Author(s):  
C. K. Manjunath ◽  
G. E. Goings ◽  
E. Page
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Electron Micrographs ◽  
Rat Heart ◽  
Major Protein ◽  
Thin Sections ◽  
Cytoplasmic Surface ◽  
Sds Page ◽  
Junction Protein ◽  
Major Protein Band

Gap junctions were purified from rat hearts in the presence of absence of proteolysis inhibitors and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy of thin sections. In absence of proteolysis inhibitors or in presence of ethylenediaminetetraacetic acid or leupeptin, gap junctions contained a single major protein band at relative molecular weight (Mr) 29,500 and minor bands at Mr 44,000–47,000, 17,750, and 16,500 and showed smooth cytoplasmic surfaces in electron micrographs. SDS-PAGE of junctions prepared with phenylmethylsulfonylfluoride (PMSF) showed markedly decreased intensity of the Mr 29,500 band and increased intensity of bands at Mr 44,000, 45,500, and 47,000; electron microscopy of these gap junctions showed presence of a fuzzy layer on their cytoplasmic surfaces. Urea (8 M) could not remove this fuzzy layer. In electron micrographs of rat ventricular myocytes, cytoplasmic surfaces of gap junctions were fuzzy. We conclude that rat heart gap junction protein consists of an intramembrane component (Mr 29,500) that extends into the “gap” and a cytoplasmic surface component (Mr 14,500–17,500) that corresponds to the fuzzy layer and is hydrolyzable by a serine protease.

scholarly journals The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.

1989 ◽  
Vol 109 (4) ◽  
pp. 1537-1547 ◽  
Author(s):  
J H Sinard ◽  
W F Stafford ◽  
T D Pollard
Keyword(s):  
Electron Microscopy ◽  
Light Scattering ◽  
Ionic Strength ◽  
Analytical Ultracentrifugation ◽  
Myosin Ii ◽  
Alkaline Ph ◽  
Predominant Species ◽  
Assembly Mechanism ◽  
Rapid Equilibrium ◽  
Low Ionic Strength

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.

Aminoacyl-tRNA synthetases of Halobacterium cutirubrum: preliminary fractionation studies

1970 ◽  
Vol 48 (8) ◽  
pp. 944-946 ◽  
Author(s):  
E. Griffiths
Keyword(s):  
Ionic Strength ◽  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Trna Synthetase ◽  
Partial Purification ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Fractionation Procedure ◽  
The Stability ◽  
Low Ionic Strength

The stability, in solutions of low ionic strength, of aminoacyl-tRNA synthetases from the extremely halophilic bacterium Halobacterium cutirubrum was studied as a preliminary to their fractionation. The enzymes differed considerably in their sensitivity to such solutions. Conditions were found where reactivation from the salt-free and inactive state could be achieved. Removal of both K+ and Mg2+ together generally resulted in better stability than the removal of K+ alone. A low temperature (4°) was also important for stability in buffers of low ionic strength. In some cases the L-amino acid substrates afforded protection against inactivation in the salt-free state. Gel filtration in low ionic strength medium was found to work well as a fractionation procedure; a partial purification of phenylalanyl-tRNA synthetase was effected in this way. The use of other conventional protein fractionation procedures is now possible.

scholarly journals Proteolytic removal of three C-terminal residues of actin alters the monomer-monomer interactions

1993 ◽  
Vol 289 (3) ◽  
pp. 897-902 ◽  
Author(s):  
M Mossakowska ◽  
J Moraczewska ◽  
S Khaitlina ◽  
H Strzelecka-Golaszewska
Keyword(s):  
Electron Microscopy ◽  
Steady State ◽  
Ionic Strength ◽  
Rate Constants ◽  
Initial Rate ◽  
Critical Concentration ◽  
Rate Of Polymerization ◽  
Low Ionic Strength ◽  
Hydrolysis Of

Homogeneous preparations of actin devoid of the three C-terminal residues were obtained by digestion of G-actin with trypsin after blocking proteolysis at other sites by substitution of Mg2+ for the tightly bound Ca2+. Removal of the C-terminal residues resulted in the following: an enhancement of the Mg(2+)-induced hydrolysis of ATP in low-ionic-strength solutions of actin; an increase in the critical concentration for polymerization; a decrease in the initial rate of polymerization; and an enhancement of the steady-state exchange of subunits in the polymer. Electron microscopy indicated an increased fragility of the filaments assembled from truncated actin. The results suggest that removal of the C-terminal residues increases the rate constants for monomer dissociation from the polymer ends and from the oligomeric species.

scholarly journals An Effective Method of Preparing Sections of Bacillus polymyxa Sporangia and Spores for Electron Microscopy

1960 ◽  
Vol 7 (2) ◽  
pp. 373-376 ◽  
Author(s):  
Pauline E. Holbert
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Micrographs ◽  
Epoxy Resins ◽  
Bacillus Polymyxa ◽  
Thin Sections ◽  
Similar Images ◽  
Embedding Medium ◽  
First Time ◽  
Diamond Knife

Bacillus polymyxa sporangia and spores were prepared for examination in the electron microscope by methods whose critical features were apparently: judicious use of vacuum, to encourage complete penetration of the embedding medium; the use of epoxy resins as embedding media; and cutting of the thin sections with a diamond knife. Electron micrographs of material prepared in this manner exhibit undeformed sporangial sections. Some of the structures revealed have been shown before, though perhaps less distinctly; other structures are revealed here for the first time. While this single study does not pretend to elucidate all the complexities of sporulation in bacteria, these and similar images should make this possible, and some mention of the preparatory techniques that lead to them seems advisable at this time.

Tannic Acid as Fixative and Stain for Amyloid Tissues

1977 ◽  
Vol 35 ◽  
pp. 542-543
Author(s):  
O. G. Rodgers ◽  
T. Shirahama ◽  
A. S. Cohen
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Tannic Acid ◽  
Electron Micrographs ◽  
Amyloid Fibrils ◽  
Fibrillar Structure ◽  
High Magnification ◽  
Animal Kingdom ◽  
Morphologic Characteristics ◽  
Liver And Kidney

Amyloid is a protein deposited in a variety of organs in the disorder amyloidosis which is widely distributed in the animal kingdom. The identification of this substance and diagnosis of the disease is largely dependent on its morphologic characteristics and tinctorial properties by light microscopy and the fibrillar structure by electron microscopy (1). In electron micrographs prepared conventionally, very often the amyloid fibrils show low electron density, making it difficult to identify small airyloid deposits when scanning by low magnification, and difficult to analyze ultrastructural details at high magnification. After testing various fixatives and stains to achieve good fixation and staining of the anyloid fibrils, we have found that the use of tannic acid as a fixative (2) may satisfy these objectives.Various tissues (skin, tongue, prostate, spleen, liver and kidney) were obtained at biopsy, surgery or autopsy from patients with primary, secondary, myelcma-associated or hereditary amyloidosis. Spleen, liver and kidney from CBA/J mice in which amyloidosis was induced by daily casein injections were also used.

Neutron scattering studies of nucleosome structure at low ionic strength

Biochemistry ◽  
1983 ◽  
Vol 22 (21) ◽  
pp. 4916-4923 ◽  
Author(s):  
Edward C. Uberbacher ◽  
Venkatraman Ramakrishnan ◽  
Donald E. Olins ◽  
Gerard J. Bunick
Keyword(s):  
Ionic Strength ◽  
Neutron Scattering ◽  
Nucleosome Structure ◽  
Low Ionic Strength

Europium tartronate and mandelate ion association in water

1967 ◽  
Vol 45 (14) ◽  
pp. 1643-1647 ◽  
Author(s):  
P. G. Manning
Keyword(s):  
Ionic Strength ◽  
Solvent Extraction ◽  
Stability Constant ◽  
Stability Constants ◽  
Ion Association ◽  
Tridentate Ligand ◽  
Radiotracer Method ◽  
The Stability ◽  
Two Stages ◽  
Low Ionic Strength

Stepwise stability constants have been determined for the 1:1 and 1:2 Eu3+:mandelate− and Eu3+:tartronate2− complexes in water. Measurements were made at low ionic strength and the temperature was 25 °C. The solvent-extraction–radiotracer method was used.For the mandelate system at an ionic strength of 0.104, K1 = 5.0 × 102, K2 = 1.58 × 102, and K1:K2 = 3.1. The K1:K2 ratios suggest monodentate ligandcy.The stepwise stability constants for the two stages of tartronate ion association are: K1 = 7.1 ( ± 15%) × 104 and K1K2 = 4.2 ( ± 5%) × 108. The magnitudes of the stability constants suggest that tartronate is a tridentate ligand. The stability constant ratios are discussed with reference to the ratios for piperidinedicarboxylate and iminodiacetate complexes.

scholarly journals Stereo electron microscopy of the 25-nm chromatin fibers in isolated nuclei.

1979 ◽  
Vol 81 (1) ◽  
pp. 260-265 ◽  
Author(s):  
A L Olins ◽  
D E Olins
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Electron Micrographs ◽  
Chicken Erythrocyte ◽  
Thin Sections ◽  
Isolated Nuclei ◽  
Chromatin Fibers

Thin sections (0.1-0.25 micron) of isolated chicken erythrocyte nuclei were examined at various tilt angles. Stereo pairs of electron micrographs document the parallel alignment of 25-nm chromatin fibers adjacent to the nuclear envelope, and demonstrate a fiber substructure consistent with close-packed arrays of nucleosomes.

scholarly journals The stability and aggregation properties of human liver acid β-d-galactosidase

1981 ◽  
Vol 193 (3) ◽  
pp. 773-779 ◽  
Author(s):  
C M Heyworth ◽  
E F Neumann ◽  
C H Wynn
Keyword(s):  
Ionic Strength ◽  
Molecular Form ◽  
Active Form ◽  
Gel Filtration ◽  
British Library ◽  
Fluorescence Excitation ◽  
Dimeric Form ◽  
The Stability ◽  
Low Ionic Strength ◽  
Beta Galactosidase

1. A method is described for following continuously the action of beta-galactosidase on 4-methylumbelliferyl beta-D-galactoside at pH 4.5, in which 4-methylumbelliferone production is measured at fluorescence excitation and emission wavelengths of 324 and 444nm respectively. 2. Initial-rate studies show that the presence of salt activates beta-galactosidase up to 100 mM, but is inhibitory above that concentration. The enzyme is very unstable at 37 degrees C and low ionic strength, but stability increases with ionic strength. 3. The stability of the enzyme at 37 degrees C decreases markedly with rising pH in the range 5.9–8.0. 4. Gel-filtration patterns demonstrate that there is a marked tendency to polymerization with increasing ionic strength. The gel-filtration pattern shows decreasing amounts of dimer with increasing pH. 5. The correlation between activity, stability and molecular form of beta-galactosidase is discussed. It is suggested that the dimeric form of the enzyme is the most stable and active form. The implications of this finding for the assay of beta-galactosidase under physiological conditions for prenatal diagnosis are discussed. 6. Evidence for the possible occurrence of a 36 000-mol.wt. from of beta-galactosidase is presented. 7. A computer program for the calculation of initial rates has been deposited as Supplementary Publication SUP 50114 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.

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