A p-nitrophenyl α-galactoside hydrolase from Pseudomonas atlantica. Localization of the enzyme

1975 ◽  
Vol 21 (10) ◽  
pp. 1476-1483 ◽  
Author(s):  
D. F. Day ◽  
M. Gomersall ◽  
W. Yaphe
Keyword(s):  
Electron Microscopy ◽  
Lactic Acid ◽  
Cytoplasmic Membrane ◽  
Disulfonic Acid ◽  
Staining Procedure ◽  
Specific Staining ◽  
Lactic Acid Dehydrogenase ◽  
Whole Cells ◽  
Track Membranes ◽  
Double Track

A p-nitrophenyl α-galactoside hydrolase is partially released when whole cells of Pseudomonas atlantica are converted to spheroplasts. The p-nitrophenyl α-glactoside hydrolase is completely inactivated by treatment of whole cells with diazonaphthalene – disulfonic acid (NDS), a reagent which does not penetrate the cytoplasmic membrane. Under the conditions used no inactivation of lactic acid dehydrogenase was observed. A specific staining procedure for this enzyme for use in electron microscopy was developed. The results with this technique in conjunction with the results of spheroplasting and NDS localization suggest that p-nitrophenyl α-galactoside hydrolase is located in or on the double-track membranes, primarily on the outer double track.

Localization of L-asparaginase in Pseudomonas aeruginosa

1971 ◽  
Vol 17 (8) ◽  
pp. 1025-1028 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lactic Acid ◽  
Cytoplasmic Membrane ◽  
Nadh Oxidase ◽  
Disulfonic Acid ◽  
Lactic Acid Dehydrogenase ◽  
Whole Cells ◽  
Acid Dehydrogenase ◽  
Membrane Enzyme ◽  
Asparaginase Activity

Whole cells of Pseudomonas aeruginosa possess L-asparaginase activity. The enzyme is released by suspension in 0.2 M MgCl2 and resuspension in 0.1 M Tris buffer, pH 8.4. Under these conditions neither the plasma membrane enzyme, NADH oxidase, nor the scluble enzyme, lactic acid dehydrogenase, are released. The L-asparaginase and the periplasmic alkaline phosphatase are completely inactivated by treatment of whole cells with diazonaphthalene–disulfonic acid, a reagent which does not penetrate the cytoplasmic membrane. Under the conditions used no inactivation of NADH oxidase or lactic acid dehydrogenase was observed. L-Asparaginase is also released by converting whole cells to spheroplasts. The results suggest that L-asparaginase of P. aeruginosa is located exterior to the cytoplasmic membrane, possibly in the periplasmic space.

The effects of phospholipid depletion on the cleavage pattern of the cell walls of frozen Gram-negative bacteria

1973 ◽  
Vol 19 (8) ◽  
pp. 1056-1057 ◽  
Author(s):  
A. Forge ◽  
J. W. Costerton
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Cytoplasmic Membrane ◽  
Gram Negative Bacteria ◽  
Cleavage Pattern ◽  
Gram Negative ◽  
Whole Cells ◽  
The Cross ◽  
Double Track ◽  
Chloroform Methanol

Extraction of whole cells of the marine pseudomonad (B-16) with chloroform–methanol causes the disappearance of the cleavage planes, and the cross-sectioned profile of both the cytoplasmic membrane and the double-track layer of the cell wall.

scholarly journals A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

1998 ◽  
Vol 46 (10) ◽  
pp. 1203-1209 ◽  
Author(s):  
Françoise Jaunin ◽  
Astrid E. Visser ◽  
Dusan Cmarko ◽  
Jacob A. Aten ◽  
Stanislav Fakan
Keyword(s):  
Electron Microscopy ◽  
Dna Replication ◽  
Salt Concentration ◽  
Chinese Hamster ◽  
S Phase ◽  
Tween 20 ◽  
Proliferating Cells ◽  
Specific Staining ◽  
Double Labeling ◽  
Chinese Hamster Cells

We describe a colloidal gold immunolabeling technique for electron microscopy which allows one to differentially visualize portions of DNA replicated during different periods of S-phase. This was performed by incorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chinese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate blocking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific staining of DNA with osmium ammine show that labeling corresponds well to replicated DNA. Different patterns of labeling distribution, reflecting different periods of DNA replication during S-phase, were characterized. Cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated with the nucleolus. The good correlation between our observations and previous double labeling results in immunofluorescence also proved the technique to be reliable.

scholarly journals Electron microscopy snapshots of single particles from single cells

2018 ◽  
Vol 294 (5) ◽  
pp. 1602-1608 ◽  
Author(s):  
Xiunan Yi ◽  
Eric J. Verbeke ◽  
Yiran Chang ◽  
Daniel J. Dickinson ◽  
David W. Taylor
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Protein Complexes ◽  
Signal To Noise Ratio ◽  
Single Cells ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Whole Cells ◽  
Potential Applications

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

scholarly journals Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid

PLoS Biology ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. e3001020
Author(s):  
Eloïse Bertiaux ◽  
Aurélia C. Balestra ◽  
Lorène Bournonville ◽  
Vincent Louvel ◽  
Bohumil Maco ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Crucial Role ◽  
Reduced Form ◽  
Malaria Parasites ◽  
Whole Cells ◽  
Apicomplexan Parasites ◽  
Dynamic Assembly

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

Lactic acid dehydrogenase as a marker of renal injury during renal artery stenting

2009 ◽  
Vol 10 (3) ◽  
pp. 199
Author(s):  
Satjit Adlakha ◽  
Amanda Sinclair ◽  
Steven Haller ◽  
Pamela Brewster ◽  
Mark Burket ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Renal Artery ◽  
Renal Injury ◽  
Lactic Acid Dehydrogenase ◽  
Acid Dehydrogenase ◽  
Renal Artery Stenting
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