scholarly journals Localization of GTP-stimulated core glycosylation to fused microsomes.

1983 ◽  
Vol 96 (6) ◽  
pp. 1791-1796 ◽  
Author(s):  
J Paiement ◽  
J J Bergeron
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Membrane Fusion ◽  
Electron Micrographs ◽  
Microsomal Membrane ◽  
High Concentrations ◽  
Silver Grains ◽  
Acid Labile

Purified rough microsomes from liver maximally incorporated N-acetyl-[3H]glucosamine into endogenous acceptors from UDP-N-acetyl-[3H]glucosamine substrate, providing the associated ribosomes were removed and 0.5 mM GTP was added. These conditions also led to the coalescence of microsomes into large fused membranes. By measurement of membrane profiles on electron micrographs, a correlation was observed between GTP-stimulated glycosylation and microsomal membrane length (r2 = 0.92). Membrane fusion was not observed in the absence of GTP, with sugar transfer inhibited by greater than 90% for acid-resistant acceptors (protein), and approximately 50% for acid-labile acceptors (lipid-linked intermediates). When radiolabeled acceptors were localized by electron microscope radioautography, high concentrations of silver grains (83 grains/100 microns membrane length) were observed over fused membranes with lower grain densities observed over unfused membranes in the same preparation (20 grains/100 microns). These studies directly link microsomal membrane fusion to GTP-stimulated core glycosylation. The observations extend the suggestion of Godelaine et al. (1979, Eur. J. Biochem. 96:17-26) that physiological levels of GTP promote the translocation of substrate across endoplasmic reticulum membranes which, we propose, occurs via a membrane fusion phenomenon.

Quantitative electron microscope autoradiographs of 125I-cholecystokinin in pancreatic acini

1982 ◽  
Vol 243 (4) ◽  
pp. G291-G296 ◽  
Author(s):  
J. A. Williams ◽  
H. Sankaran ◽  
E. Roach ◽  
I. D. Goldfine
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Specific Binding ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Acini ◽  
Time Points ◽  
Basolateral Plasma Membrane ◽  
Specific Receptors ◽  
Silver Grains

To morphologically evaluate the interaction of cholecystokinin (CCK) with its receptors on pancreatic acinar cells, we incubated isolated mouse acini at 37 degrees C with radioiodinated CCK and then prepared quantitative electron microscope autoradiographs. Specific binding of CCK to acini was one-half maximal at 2 min of incubation and maximal after 10 min. The cell-associated radioactivity was extracted and analyzed on Sephadex G-50. After 2 min, 90% of the total cellular radioactivity remained as intact CCK; after 30 min, the intact radioactivity decreased to 65% of total. At 2 min, the fraction of bound hormone that fixed to acini was 84% of total; this amount decreased to 78% after 30 min. Thus, the majority of radioactivity in the autoradiographs at both time points was intact CCK; however, at 30 min, a small amount was also degraded hormone. After both 2 and 30 min of incubation, silver grains were highly concentrated over the basolateral plasma membrane. A significant number of grains were in the cell interior at both time points, increasing from 13% of total grains at 2 min to 42% at 30 min. At both times, the largest fraction of internalized grains was localized over the endoplasmic reticulum. At 30 min, a significant concentration of CCK grains was observed over multivesicular bodies. The present study demonstrates, therefore, that CCK binds to specific receptors on the basolateral surface of pancreatic acinar cells. After binding, the hormone is internalized, locates predominantly on the endoplasmic reticulum, and is then degraded.

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.

scholarly journals Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Isei Tanida ◽  
Yoko Furuta ◽  
Junji Yamaguchi ◽  
Soichiro Kakuta ◽  
Juan Alejandro Oliva Trejo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Electron Micrographs ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Histone H2b ◽  
Thin Sections ◽  
Red Fluorescent Proteins ◽  
Scanning Electron

AbstractIn-resin CLEM of Epon embedded samples can greatly simplify the correlation of fluorescent images with electron micrographs. The usefulness of this technique is limited at present by the low number of fluorescent proteins that resist CLEM processing. Additionally, no study has reported the possibility of two-color in-resin CLEM of Epon embedded cells. In this study, we screened for monomeric green and red fluorescent proteins that resist CLEM processing. We identified mWasabi, CoGFP variant 0, and mCherry2; two green and one red fluorescent proteins as alternatives for in-resin CLEM. We expressed mitochondria-localized mCherry2 and histone H2B tagged with CoGFP variant 0 in cells. Green and red fluorescence was detected in 100 nm-thin sections of the Epon-embedded cells. In the same thin sections, we correlated the fluorescent signals to mitochondria and the nucleus using a scanning electron microscope. Similar results were obtained when endoplasmic reticulum-localized mCherry2 and histone H2B tagged with CoGFP variant 0 were expressed in the cells. Two-color in-resin CLEM of two cytoplasmic organelles, mitochondria and endoplasmic reticulum, was also achieved using mitochondria-localized mCherry2 and endoplasmic reticulum-localized mWasabi. In summary, we report three new fluorescent protein-alternatives suitable for in-resin CLEM of Epon-embedded samples, and achieved Epon-based two-color in-resin CLEM.

scholarly journals Membrane fusion and glycosylation in the rat hepatic Golgi apparatus.

1982 ◽  
Vol 92 (1) ◽  
pp. 147-154 ◽  
Author(s):  
J Paiement ◽  
R A Rachubinski ◽  
N M Ng Ying Kin ◽  
R A Sikstrom ◽  
J J Bergeron
Keyword(s):  
Electron Microscope ◽  
Membrane Fusion ◽  
Complete Medium ◽  
Transferase Activity ◽  
Golgi Membrane ◽  
Assay Medium ◽  
Galactosyl Transferase ◽  
Cacodylate Buffer ◽  
Triton X ◽  
Silver Grains

When purified Golgi fractions were incubated with UDP-[3H]galactose in the absence of Triton-X-100, radioactivity was incorporated into an endogenous lipid and several peptide acceptors. Electron microscope analysis of Golgi fractions incubated in the endogenous galactosyl transferase assay medium revealed extensive fusion of Golgi saccules. Systematic removal of constituents in the galactosyl transferase assay medium showed enhanced (minus beta-mercaptoethanol) or reduced (minus ATP, minus sodium cacodylate buffer or minus MnCl2) fusion of Golgi membranes compared to the complete medium, Stereologic analysis revealed a correlation between membrane fusion and galactosyl transferase activity (r = 0.99, P less than 0.001). Electron microscope radioautography was carried out after incubation of Golgi fractions with UDP-[3H]galactose. Silver grains were not observed over trans elements of Golgi but were revealed mainly over large fused saccules with the number of silver grains being proportionate to membrane fusion (r = 0.92, P less than 0.001). Bilayer destabilization at points of Golgi membrane fusion may act to translocate galactose across the Golgi membrane and thereby provide a fusion regulated substrate for terminal glycosylation.

Ultrastructure and development of phenolic-storing cells in cotton roots

1976 ◽  
Vol 54 (17) ◽  
pp. 2074-2082 ◽  
Author(s):  
W. C. Mueller ◽  
C. H. Beckman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Light Microscopy ◽  
Electron Micrographs ◽  
Root Tip ◽  
Fixation Procedure ◽  
Rate Of Development ◽  
Temperature Development ◽  
Endodermal Cells ◽  
Surrounding Parenchyma

The development of phenolics in the radicle of cotton was determined with the nitroso reaction and light microscopy. In all cotton cultivars tested, phenolics could be detected in the endodermis at a distance of 1.0 to 2.0 mm from the root tip and in the root cap 0.5 mm from the root tip. By 3.0 mm the phenolic-storing cells in the endodermis were mature. The rate of development of the phenolic-storing endodermal cells was similar in all cultivars but varied with temperature, with maximum rates at 35 °C. At this temperature, development and maturation of the phenolic-storing cells occurred in 30 min. Phenolics could first be detected with the electron microscope in the vacuoles of endodermal cells 1.0 mm from the root tip; these cells were essentially mature 3.0 mm from the root tip. Except for the phenolics in the vacuoles, developing phenolic-storing cells were identical with surrounding parenchyma and contained abundant endoplasmic reticulum and ribosomes, mitochondria, dictyosomes, and large, variable plastids. Although several fixation methods were used and the appearance of the cells varied according to the fixation procedure, no positive origin for the phenolic material could be detected in the electron micrographs.

Utilizing Dark Field Electron Microscopy to Produce Lantern Slides

1974 ◽  
Vol 32 ◽  
pp. 116-117
Author(s):  
J. N. Meador ◽  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Electron Microscope ◽  
Electron Micrographs ◽  
Dark Field ◽  
Limiting Factor ◽  
Clinical Laboratories ◽  
Field Electron ◽  
Time Element ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Dark Field Micrographs

The electron microscope is being utilized more and more in clinical laboratories for pathologic diagnosis. One of the major problems in the utilization of the electron microscope for diagnostic purposes is the time element involved. Recent experimentation with rapid embedding has shown that this long phase of the process can be greatly shortened. In rush cases the making of projection slides can be eliminated by taking dark field electron micrographs which show up as a positive ready for use. The major limiting factor for use of dark field micrographs is resolution. However, for conference purposes electron micrographs are usually taken at 2.500X to 8.000X. At these low magnifications the resolution obtained is quite acceptable.

Copper Localization in Cirrhotic Rat Liver by Scanning Electron Microscopy

1969 ◽  
Vol 27 ◽  
pp. 38-39 ◽  
Author(s):  
J. C. Russ ◽  
E. McNatt
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Copper Acetate ◽  
Lead Citrate ◽  
Dense Bodies ◽  
Transmission Electron ◽  
Electron Mode ◽  
Scanning Electron

In order to study the retention of copper in cirrhotic liver, rats were made cirrhotic by carbon tetrachloride inhalation twice weekly for three months and fed 0.2% copper acetate ad libidum in drinking water for one month. The liver tissue was fixed in osmium, sectioned approximately 2000 Å thick, and stained with lead citrate. The section was examined in a scanning electron microscope (JEOLCO JSM-2) in the transmission electron mode.Figure 1 shows a typical area that includes a red blood cell in a sinusoid, a disse, and a portion of the cytoplasm of a hepatocyte which contains several mitochondria, peribiliary dense bodies, glycogen granules, and endoplasmic reticulum.

Ultrastructural comparison of prolactin adenomas of pituitary in men and women

1988 ◽  
Vol 46 ◽  
pp. 346-347
Author(s):  
R.C. Caughey ◽  
U.P. Kalyan-Raman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Phosphate Buffer ◽  
Pituitary Adenomas ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Men And Women ◽  
Transmission Electron

Prolactin producing pituitary adenomas are ultrastructurally characterized by secretory granules varying in size (150-300nm), abundance of endoplasmic reticulum, and misplaced exocytosis. They are also subclassified as sparsely or densely granulated according to the amount of granules present. The hormone levels in men and women vary, being higher in men; so also the symptoms vary between both sexes. In order to understand this variation, we studied 21 prolactin producing pituitary adenomas by transmission electron microscope. This was out of a total of 80 pituitary adenomas. There were 6 men and 15 women in this group of 21 prolactinomas.All of the pituitary adenomas were fixed in 2.5% glutaraldehyde, rinsed in Millonig's phosphate buffer, and post fixed with 1% osmium tetroxide. They were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole's non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin.

Smooth endoplasmic reticulum in perfusion and immersion-fixed Leydig cells

1990 ◽  
Vol 48 (3) ◽  
pp. 82-83
Author(s):  
R.T.F. Bernard ◽  
R.H.M. Cross
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Steroid Hormones ◽  
Transmission Electron Microscope ◽  
Leydig Cells ◽  
Smooth Endoplasmic Reticulum ◽  
Random Mixture ◽  
Transmission Electron

Smooth endoplasmic reticulum (SER) is involved in the biosynthesis of steroid hormones, and changes in the organisation and abundance of this organelle are regularly used as indicators of changes in the level of steroidogenesis. SER is typically arranged as a meshwork of anastomosing tubules which, with the transmission electron microscope, appear as a random mixture of cross, oblique and longitudinal sections. Less commonly the SER appears as swollen vesicles and it is generally suggested that this is an artefact caused during immersion fixation or during immersion of poorly-perfused tissue.During a previous study of the Leydig cells of a seasonally reproducing bat, in which tissue was fixed by immersion, we noted that tubular SER and vesicular SER often occured in adjacent cells and sometimes in the same cell, and that the abundance of the two types of SER changed seasonally. We came to doubt the widelyheld dogma that vesicular SER was an artefact of immersion fixation and set out to test the hypothesis that the method of fixation does not modify the ultrastructure of the SER.

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