scholarly journals Peptides of human bronchial mucus glycoproteins. Size determination by electron microscopy and by biosynthetic experiments

1987 ◽  
Vol 248 (1) ◽  
pp. 189-195 ◽  
Author(s):  
T Marianne ◽  
J M Perini ◽  
J J Lafitte ◽  
N Houdret ◽  
F R Pruvot ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Length Distribution ◽  
Molecular Size ◽  
Pulse Labelling ◽  
Bronchial Mucosa ◽  
Chase Period ◽  
Bronchial Mucus ◽  
Multiple Treatments

Secreted human bronchial mucins, directly collected from macroscopically healthy bronchial mucosa, were prepared in the presence of six proteinase inhibitors, and analysed by electron microscopy. These mucins were similar in length distribution to molecules prepared from sputum [Slayter, Lamblin, Le Treut, Galabert, Houdret, Degand & Roussel (1984) Eur. J. Biochem. 142, 209-218], although they were a little longer, their lengths ranging up to about 1,650 nm. This length corresponds to an extended mucin peptide of about 450 kDa. In order to compare these peptide lengths with the molecular size of biosynthetic precursors, an antiserum raised against trifluoromethanesulphonic acid-treated highly glycosylated regions of human bronchial mucins was used to isolate mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling with [3H]threonine or [3H]glucosamine. A main precursor labelled with [3H]threonine and with an apparent molecular mass of about 400 kDa was detected by fluorography following SDS/polyacrylamide-gel electrophoresis. This band was observed as early as 20 min; it was more intense after a 40 min chase and had disappeared after a chase period of 280 min in unlabelled medium, presumably owing to glycosylation. Much fainter bands at about 200 kDa and between 200 and 400 kDa, also labelled with [3H]threonine, were observed mainly after a 40 min chase and had disappeared after a 280 min chase. None of these bands was labelled with [3H]glucosamine, nor did they disappear after multiple treatments with immobilized lectins. After a 280 min chase, [3H]threonine-labelled material appeared in the stacking gel, which also contained [3H]glucosamine label. The results indicate that the 200-400 kDa species are mucin precursors, whose size is comparable with that obtained by electron microscopy for respiratory mucins collected directly from the macroscopically healthy bronchial mucosa.

scholarly journals Electron microscopy of cervical, gastric and bronchial mucus glycoproteins

1986 ◽  
Vol 239 (1) ◽  
pp. 147-153 ◽  
Author(s):  
J K Sheehan ◽  
K Oates ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Gastric Mucus ◽  
Bronchial Mucus ◽  
Mucus Gel ◽  
Macromolecular Architecture ◽  
Mucus Glycoproteins ◽  
Low Shear

Mucus glycoproteins (mucins) were obtained from human cervical and pig gastric mucus as well as from chronic-bronchitic sputum after low-shear extraction. The mucus gel was solubilized in guanidinium chloride supplemented with proteinase inhibitors, and the macromolecules were purified by using isopycnic density-gradient centrifugation. The macromolecules were spread in monolayers of benzyldimethylalkyl-ammonium chloride and studied with electron microscopy after staining with uranyl acetate and/or shadowing with platinum/carbon. The mucins appeared as flexible linear threads with lengths varying from approx. 200 nm to about 400 nm. No regularly branched or star-shaped structures were observed. The macromolecular architecture of cervical, respiratory and gastric mucins is thus similar.

scholarly journals Kerogen nanoscale structure and CO2 adsorption in shale micropores

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aleksandra Gonciaruk ◽  
Matthew R. Hall ◽  
Michael W. Fay ◽  
Christopher D. J. Parmenter ◽  
Christopher H. Vane ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Chemical Composition ◽  
Focused Ion Beam ◽  
Gas Adsorption ◽  
Ion Beam ◽  
Molecular Size ◽  
Gas Sorption ◽  
Nanoscale Structures ◽  
Adsorption Sites ◽  
Nanoscale Structure

AbstractGas storage and recovery processes in shales critically depend on nano-scale porosity and chemical composition, but information about the nanoscale pore geometry and connectivity of kerogen, insoluble organic shale matter, is largely unavailable. Using adsorption microcalorimetry, we show that once strong adsorption sites within nanoscale network are taken, gas adsorption even at very low pressure is governed by pore width rather than chemical composition. A combination of focused ion beam with scanning electron microscopy and transmission electron microscopy reveal the nanoscale structure of kerogen includes not only the ubiquitous amorphous phase but also highly graphitized sheets, fiber- and onion-like structures creating nanoscale voids accessible for gas sorption. Nanoscale structures bridge the current gap between molecular size and macropore scale in existing models for kerogen, thus allowing accurate prediction of gas sorption, storage and diffusion properties in shales.

Effect of detergents and chemicals on purified vaccinia virus: analysis by SDS polyacrylamide gel electrophoresis and electron microscopy

1977 ◽  
Vol 23 (3) ◽  
pp. 240-252 ◽  
Author(s):  
J. Boisvert ◽  
T. Yamamoto
Keyword(s):  
Electron Microscopy ◽  
Vaccinia Virus ◽  
Gel Electrophoresis ◽  
Alkali Treatment ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ammonium Bromide ◽  
Molecular Weights ◽  
Viral Particles

Vaccinia virus particles were dissociated into their constituent polypeptides and analysed by sodium dodecyl sulfate (SDS) gel electrophoresis. Thirty-three distinct polypeptide bands were identified and their molecular weights ranged between 11 000 and 150 000 daltons.Specific staining of gels containing polypeptides of dissociated virions revealed the presence of eight glycopeptides. No lipopeptides were detected.Analysis of chemical extracts (urea, guanidine hydrochloride, and alkali treatment) of the virus by SDS gel electrophoresis indicated that a total of 10 to 14 different polypeptides ranging in molecular weights from 11 000 to 70 000 daltons were solubilized.Analysis of detergent extracts and of the remains of extracted viral particles has shown that the detergent Nonidet P-40 (NP-40) solubilized a total of 11 polypeptides of which 6 were glycopeptides. The other detergents sodium deoxycholate (SDC) and cetyl trimethyl ammonium bromide (CTAB) were not as selective, both solubilizing more than 25 of the polypeptides composing the virus. Gel electrophoresis results also indicated that most of the small molecular weight (11 000–70 000 daltons) polypeptides were readily solubilized by NP-40, SDC, and CTAB, while those with molecular weights of 70 000 daltons and higher were not well solubilized.The effects of detergents were also analysed by electron microscopy. Evidence was obtained for subpopulations of viral particles having different susceptibility to detergent extraction.

scholarly journals Purification and Three-Dimensional Electron Microscopy Structure of the Neisseria meningitidis Type IV Pilus Biogenesis Protein PilG

2007 ◽  
Vol 189 (17) ◽  
pp. 6389-6396 ◽  
Author(s):  
Richard F. Collins ◽  
Muhammad Saleem ◽  
Jeremy P. Derrick
Keyword(s):  
Electron Microscopy ◽  
Neisseria Meningitidis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Pilus Biogenesis

ABSTRACT Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 Å in length and 80 Å in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower “waist” region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Effects of Drugs on Mucus Glycoproteins and Water in Bronchial Secretion

1979 ◽  
Vol 7 (5) ◽  
pp. 434-442 ◽  
Author(s):  
T C Medici ◽  
P Radielovic
Keyword(s):  
Water Content ◽  
Water Transport ◽  
Animal Experiments ◽  
Osmotic Gradient ◽  
Bronchial Mucosa ◽  
Bronchial Secretion ◽  
Epithelial Surface ◽  
Bronchial Mucus ◽  
Mucus Glycoproteins

The result of chemical analysis of the bronchial secretion is simple; up to 95% of the secretion is made up of water, and up to 5% is composed of ash, protein, carbohydrate, lipid, nitrogen and desoxyribonucleic acid. More complicated is the question of how bronchial secretion is formed and of which active biological components it is composed. Bronchial secretion is the result of the different processes, secretion, transudation, exudation and exfoliation from a highly differentiated bronchial mucosa. To those substances secreted belong, amongst others, constituents important for the flow properties and the transportability of the secretion: the bronchial mucus glycoproteins and water. The bronchial glycoproteins are the most important group, constituting 50–80% of the macromolecules. They are formed and secreted by the bronchial mucosa. The synthesis and secretion of bronchial glycoproteins are influenced by drugs in different ways. Beta-adrenergic stimulants do not alter these processes in in vitro studies on human glands, although an increase in mucus of glycoprotein production has been demonstrated in animal experiments and indirectly in man. Cyclic adenosine monophosphate and the methylxanthines stimulate mucus glycoprotein production, anticholinergic agents reduce but do not completely supress this process. Anti-allergic agents do not alter the production of bronchial glycoproteins with the exception of the corticosteroids which partially inhibit the synthesis and secretion. Neither expectorants nor mucolytic agents influence the production of mucus glycoproteins in human bronchial glands as opposed to animal experiments, in which these compounds produce an increase in the output of the bronchial fluid. Water constitutes 95% of the bronchial secretion and the water content considerably influences mucociliary function. An osmotic gradient, the result of active sodium and chloride ion transport across the bronchial epithelium, ensures on the one hand that water diffuses through epithelium on to the epithelial surface where it forms the serous sol layer in which the cilia beat. On the other hand water is probably transported in the same way across the mucosal glands where it mixes with the extremely hydrophilic mucus glycoproteins. The ion and water transport is influenced by drugs. Acetylcholine, histamine and terbutaline stimulate the ion and thereby water transport. Atropine, diphenylhydramine, an H1-antagonist, propranolol, a beta-blocker andfurosemide inhibit these transport mechanisms. Whether ketotifen, a new antihistaminic drug used in the treatment of bronchial asthma, will affect these processes, decreasing the water content of bronchial mucus, remains to be seen.

Binding of Tetrahymena dynein to axonemes of Marsilea vestita lacking the outer dynein arm

1985 ◽  
Vol 73 (1) ◽  
pp. 299-310
Author(s):  
J.S. Hyams
Keyword(s):  
Electron Microscopy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Valuable Insight ◽  
Marsilea Vestita ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
And Function ◽  
Relationship Of ◽  
The Relationship ◽  
Insight Into

Axonemes from the heterosporous water fern Marsilea vestita were fixed in the presence of tannic acid and examined by thin-section electron microscopy. Transverse sections revealed the normal 9+2 configuration except for the absence of the outer of the two dynein arms. Both arms were normally preserved in parallel preparations of Chlamydomonas axonemes. Isolated dynein from the ciliated protozoon Tetrahymena bound to Marsilea axonemes at the site normally occupied by the outer arm. Dynein binding was partially reversed by ATP as judged by both electron microscopy and polyacrylamide gel electrophoresis. This system should provide a valuable insight into the biochemistry and function of the inner dynein arm and the relationship of the two arms to motility in more conventionally equipped axonemes.

Intracellular fibres in cultured cells: analysis by scanning and transmission electron microscopy and by SDS-polyacrylamide gel electrophoresis

1978 ◽  
Vol 31 (1) ◽  
pp. 369-392
Author(s):  
J.A. Trotter ◽  
B.A. Foerder ◽  
J.M. Keller
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Dimensional Structure ◽  
3T3 Cells ◽  
Transmission Electron ◽  
Ionic Detergent ◽  
Over The Top

The 3-dimensional structure of the fibrous cytoskeleton of 3T3 cells was examined by scanning electron microscopy of cells extracted with the non-ionic detergent Triton X-100. Detergent-extracted cells consist of the nucleus and an extensive system of fibres, the largest of which correspond to stress fibres visible by phase-contrast microscopy. The system of fibres, which is coterminous with the borders of the native cell, remains firmly adherent to the substratum. The major fibres branch into smaller fibrils which appear to end by ravelling out into fine filaments that constitute a matted network in a plane very close to that of the substratum. In the nuclear region all the major fibres pass over the top of the nucleus, where they may also branch into a system of fine fibrils. Thin-section transmission electron microscopy in conjunction with heavy meromyosin treatment of extracted cells shows the fibres to be composed of native F-actin. Intermediate filaments are also present, and are prominent in the matted network, together with actin filaments. The major proteins of the residue are identified by SDS-polyacrylamide gel electrophoresis as actin, a 56000 Dalton peptide, and histones. Also present are myosin heavy chain, peptides of 225,000 and 250,000, and minor bands at 60,000 and 94,000 Daltons. The non-ionic detergent extracts 70% of the cellular protein, including 50% of the actin and 75% of the myosin. The Triton-insoluble fraction of 3T3 cells appears to constitute, in addition to the nucleus, a stable cytoskeletal system, composed largely of contractile proteins and 10-nm filaments, which functions in maintenance of cell shape, in substratum adhesion, and in positioning the nucleus within the cell.

Catabolism of streptokinase and polyethylene glycol-streptokinase: evidence for transport of intact forms through the biliary system in the mouse

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 73-79 ◽  
Author(s):  
FH Brucato ◽  
SV Pizzo
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Gel Electrophoresis ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gi Tract ◽  
Substantial Fraction ◽  
Sds Page ◽  
Derivatives Of

Abstract The catabolism of streptokinase (SK) and polyethylene glycol derivatives of SK (PEG-SK) were studied in mice. The clearance and catabolism of SK:plasmin (SK:Pm) and PEG-SK:Pm activator complexes were also investigated. Native 125I-SK cleared rapidly (t1/2 = 15 minutes) from the circulation, with the majority of the ligand accumulating in the liver and gastrointestinal (GI) tract and a substantial fraction also localizing in the kidneys. SK, which was removed from the plasma by the liver, was secreted into bile and then the GI tract. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that 125I-SK recovered from liver and bile was homogeneous and of the same molecular weight (mol wt approximately 50,200) as native SK. PEG-125I-SK cleared slowly (t1/2 greater than 200 minutes), with more than 80% of the preparation localizing in liver and GI tract. The PEG-125I-SK secreted into the bile was also intact. The bile containing 125I-SK was incubated with stoichiometric amounts of plasminogen and electrophoresed under nondenaturing conditions. This study demonstrated that the secreted SK was able to form SK:Pg complexes. SDS-PAGE also showed activation of 125I-Pg that was incubated with recovered bile containing the SK. 125I-SK:Pm catabolism was also studied. In these experiments, the mol wt approximately 42,000 fragment obtained when SK is cleaved by plasmin was found in the bile. This fragment of 125I-SK was not recovered as part of a complex with plasmin, consistent with our previous observations that catabolism of SK:Pm involves transfer of the plasmin to plasma proteinase inhibitors while SK is catabolized independently. By contrast, when PEG-125I-SK:Pm was injected into mice, only intact PEG-125I-SK was found in the bile, consistent with our previous observations that the PEG derivatization blocks its degradation by plasmin.

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