Characterization of a protein-lipopolysaccharide complex released from cell walls of Pseudomonas aeruginosa by ethylenediaminetetraacetic acid

1969 ◽  
Vol 15 (7) ◽  
pp. 743-748 ◽  
Author(s):  
S. W. Rogers ◽  
H. E. Gilleland Jr. ◽  
R. G. Eagon
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Cell Walls ◽  
Ethylenediaminetetraacetic Acid ◽  
Gradient Centrifugation ◽  
Hollow Spheres ◽  
Gel Filtration ◽  
A Chain ◽  
Ultracentrifugal Analysis

Results from analytical ultracentrifugal analysis, Sephadex gel filtration, isopycnic density-gradient centrifugation, and polyacrylamide disc-gel electrophoresis revealed that ethylenediaminetetraacetic acid liberated a protein–lipopolysaccharide complex from cell walls of Pseudomonas aeruginosa with an estimated molecular weight of not less than 160 000 and probably about one million. Electron microscopy of this complex revealed spherules and rodlets. The diameter of the former was approximately 70 ± 10 Å while the dimensions of the latter were 70 ± 10 Å × 200 ± 50 Å. The rodlets appeared to be composed of three or more spherules arranged in a chain-like fashion. Electron microscopy of protein-free lipopolysaccharide revealed predominantly hollow spheres from 300 Å to 1500 Å in diameter, morphologically resembling membrane sacculi. It is proposed that the protein–lipopolysaccharide complex, but not the protein-free lipopolysaccharide, is representative of the in situ form of native endotoxin.

LYSIS OF CELL WALLS AND INTACT CELLS OF PSEUDOMONAS AERUGINOSA BY ETHYLENEDIAMINE TETRAACETIC ACID AND BY LYSOZYME

1965 ◽  
Vol 11 (2) ◽  
pp. 193-201 ◽  
Author(s):  
R. G. Eagon ◽  
K. Jane Carson
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Cell Walls ◽  
Chelating Agents ◽  
Tetraacetic Acid ◽  
Intact Cells ◽  
Divalent Metals ◽  
Ethylenediamine Tetraacetic Acid

It was shown that Pseudomonas aeruginosa strain 64 was extensively Iysed by EDTA alone and that destruction of cells was as complete as when lysozyme was used in combination with EDTA as evidenced by microscopic examinations and viability tests of the bacterial suspensions. Lysozyme alone caused no appreciable lysis. Lysozyme was not completely without effect. Cells that were preincubated with lysozyme, washed, and resuspended in the presence of EDTA lysed at the same rate and to the same extent as in a system containing both EDTA and lysozyme. Electron microscopy revealed that, after incubation with lysozyme, cells showed altered physical appearances manifested by "ballooning" and bulging of the cell walls. Incubation of cells with rhodamine-labelled lysozyme indicated that lysozyme is bound to the cellular surface and that it is readily removed by washing.There is evidence that EDTA acts through its chelation properties. Calcium was chelated in greatest quantity. EDTA was replaceable by structurally related polyacetic acid chelating agents but not by other chelating agents or by detergents. It is concluded that the binding of divalent metals, which may function to form cross-linkages, is essential for the integrity of the cell walls of P. aeruginosa.

Biochemical and Structural Changes in Mitochondria and Other Cellular Components of Pea Cotyledons During Germination

1972 ◽  
Vol 50 (7) ◽  
pp. 725-737 ◽  
Author(s):  
T. Solomos ◽  
S. S. Malhotra ◽  
S. Prasad ◽  
S. K. Malhotra ◽  
Mary Spencer
Keyword(s):  
Electron Microscopy ◽  
Structural Changes ◽  
Gradient Centrifugation ◽  
Microscopic Analysis ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cellular Components ◽  
Further Development

Integrated studies comprising biochemical and electron microscopic analysis suggested that the increase in respiratory activity of pea cotyledon mitochondria during germination results from further development of the original mitochondria present in dormant seeds. Electron microscopy of isolated mitochondria as well as mitochondria in situ has revealed that membranes are scarce in the mitochondria present in dormant seeds. Mitochondrial cristae become well developed during the initial stages of germination. Crude mitochondrial preparations from pea cotyledons were fractionated by sucrose density gradient centrifugation and analyzed through electron microscopy. These studies showed that, at all stages of germination, "peroxisome"-like structures were present in the fractions of higher sucrose densities than that containing mitochondria. Biochemical studies revealed that the activities of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) and peroxidase (guaicol:H2O2 oxidoreductase, EC 1.11.1.7) were associated mainly with these fractions and their activities increased during germination.

Synthesis, Characterization and Catalytic Properties of Monometal/SiO2 and Bimetal/SiO2 Hollow Spheres with Mesoporous Structure

NANO ◽  
2017 ◽  
Vol 12 (12) ◽  
pp. 1750148 ◽  
Author(s):  
Xinzhi Sun ◽  
Fanglin Du
Keyword(s):  
Electron Microscopy ◽  
Hollow Spheres ◽  
Mesoporous Structure ◽  
Hydrogen Atmosphere ◽  
Hydrothermal Conditions ◽  
Reaction Conditions ◽  
Transmission Electron ◽  
One Step ◽  
Temperature Programmed

Monometallic M1(M[Formula: see text] Ni/Cu/Fe/Co) silicates and bimetallic Ni–M2(M[Formula: see text] Cu/Fe/Co) silicates hollow spheres with mesoporous structure and the controllable morphology have been synthesized successfully via one-step sacrificial template method under hydrothermal conditions. The catalysts were obtained by reducing the corresponding silicates in situ under the hydrogen atmosphere at a certain temperature. All the silicates and the catalysts M1/SiO2 and Ni–M2/SiO2 hollow spheres have been characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer–Emmett–Teller (BET) and temperature-programmed reduction (TPR) thoroughly and systematically. The morphology and reaction conditions of bimetallic Ni–M2 silicates hollow spheres depend on the second metal M2, which has been verified by SEM, TEM and XRD. From the results, it can be concluded that bimetallic silicates possess better physical properties in favor of the catalytic activity. Bimetallic Ni–M2/SiO2 hollow spheres had higher catalytic property than the monometallic M1/SiO2 and the conversion of nitrobenzene could reach 100% within 3[Formula: see text]h using Ni–Cu/SiO2 and Ni–Fe/SiO2 hollow spheres as catalysts.

scholarly journals Incorporation of In Situ Synthesized Nano-Copper Modified Phenol-Formaldehyde Resin to Improve the Mechanical Properties of Chinese Fir: A Preliminary Study

Polymers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 876
Author(s):  
Fan Li ◽  
Cuiyin Ye ◽  
Yanhui Huang ◽  
Xianmiao Liu ◽  
Benhua Fei
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Cell Walls ◽  
Phenol Formaldehyde ◽  
Environmental Scanning Electron Microscopy ◽  
Phenol Formaldehyde Resin ◽  
Chinese Fir ◽  
Formaldehyde Resin ◽  
The Impact

Phenol-formaldehyde (PF) resin, modified using nano-copper with varying contents (0 wt%, 1 wt%, 3 wt%), was manufactured to improve the mechanical properties of Chinese fir. The morphology, chemical, micromechanical and micromechanical properties of the samples were determined by transmission electron microscopy (TEM), atomic force microscopy (AFM), environmental scanning electron microscopy (ESEM), Fourier transform infrared spectroscopy (FTIR), nanoindentation (NI) and traditional mechanical testing. The TEM and AFM results indicated that the in situ synthesized nano-copper particles were well-dispersed, and spherical, with a diameter of about 70 nm in PF resin. From the FTIR chemical changes detected by FTIR inferred that the nano-copper modified PF resin penetrated into the Chinese fir cell walls and interacted with the acetyl groups of hemicellulose by forming a crosslinked structure. Accordingly, the micro-mechanical properties of the Chinese fir cell walls were enhanced after treatment with nano-copper modified PF resin. The filling of the PF-1-Cu resin (1 wt% nano-copper) in the wood resulted in 13.7% and 22.2% increases in the elastic modulus (MOE) and hardness, respectively, of the cell walls. Besides, the impact toughness and compressive strength of the Chinese fir impregnated with PF-1-Cu resin were 21.8% and 8.2% higher than that of the PF-0-Cu resin. Therefore, in situ synthesized nano-copper-modified PF resin is a powerful treatment method for Chinese fir due to improved diffusive properties and reinforcement of the mechanical properties.

scholarly journals Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions

1986 ◽  
Vol 240 (1) ◽  
pp. 41-48 ◽  
Author(s):  
D J Thornton ◽  
I A Nieduszynski ◽  
K Oates ◽  
J K Sheehan
Keyword(s):  
Electron Microscopy ◽  
Femoral Head ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mixed Salt ◽  
Electron Microscopic ◽  
Femoral Head Cartilage ◽  
Microscopic Studies

Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a ‘beaded’ structure and a contiguous filamentous ‘tail’ at one end and others which appeared as round ‘blobs’, some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the ‘blob-like’ appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.

scholarly journals Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons.

1979 ◽  
Vol 82 (2) ◽  
pp. 336-346 ◽  
Author(s):  
R J Lasek ◽  
N Krishnan ◽  
I R Kaiserman-Abramof
Keyword(s):  
Electron Microscopy ◽  
Sucrose Gradient ◽  
Chelating Agents ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Intermediate Filament Proteins ◽  
Protein Subunits ◽  
Giant Axons ◽  
Discontinuous Sucrose Gradient ◽  
Sepharose 4B

Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.

Action of ethylenediaminetetraacetic acid, tris(hydroxymethyl)aminomethane, and lysozyme on cell walls of Pseudomonas aeruginosa

1968 ◽  
Vol 14 (8) ◽  
pp. 913-922 ◽  
Author(s):  
S. T. Cox Jr. ◽  
R. G. Eagon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Cell Walls ◽  
Ethylenediaminetetraacetic Acid ◽  
Individual Cell ◽  
Intact Cell ◽  
Divalent Cations ◽  
Total Cell ◽  
The Individual ◽  
Phosphate Groups

Incubation of isolated cell walls of Pseudomonas aeruginosa with tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), and lysozyme, either singly or in combinations, results in partial solubilization of the cell walls.When cell walls were incubated with a combination of Tris and EDTA, approximately 30% of the total cell wall and 32% of the lipopolysaccharide fraction were solubilized. The following percentages of the individual cell wall constituents were solubilized: protein, 19%; carbohydrate, 35%; lipid, 5%; ash, 54%; and, phosphorus 23%. When lysozyme was included with Tris and EDTA, approximately 36% of the total cell wall and 86% of the lipopolysaccharide fraction were solubilized. Solubilization of the individual cell wall constituents was similarly increased.Phospholipids, which make up 7–8% of the intact cell wall of P. aeruginosa, were not released by incubation of the cell walls with these agents. Fatty acids in addition to those found in the lipopolysaccharide, however, were detected in the solubilized cell wall materials.Incubation systems composed of Tris, of EDTA, and of Tris and lysozyme solubilized cell walls to a lesser extent than incubation systems of Tris and EDTA, and of Tris, EDTA, and lysozyme. Incubation of cell walls in water, furthermore, was effective in solubilizing approximately 8% of the cell walls. Muramic acid containing materials were solubilized only in the presence of lysozyme.Materials liberated from cell walls which were incubated in systems containing EDTA exhibited single symmetrical Schlieren peaks characterized by uncorrected sedimentation constants of approximately 7 S, suggesting the release of homogenous subunits from the cell walls.Intra- and inter-molecular cross-linking by divalent cations via phosphate groups contained in lipoprotein and lipopolysaccharide components of the cell wall of P. aeruginosa is proposed.

scholarly journals Resolution and analysis of ‘native’ and ‘activated’ properdin

1987 ◽  
Vol 243 (2) ◽  
pp. 507-517 ◽  
Author(s):  
T C Farries ◽  
J T Finch ◽  
P J Lachmann ◽  
R A Harrison
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
Alternative Pathway ◽  
Gel Filtration ◽  
Alternative Activation ◽  
Functional Heterogeneity ◽  
Native Population ◽  
Physiological Event ◽  
Effective Screening

A rapid and reproducible procedure for the resolution of ‘native’ and ‘activated’ forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only ‘native’ properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of ‘native’ into ‘activated’ properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the ‘native’ population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. ‘Activated’ properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin ‘activation’ is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the ‘native’ population.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

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