scholarly journals Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

1985 ◽  
Vol 101 (2) ◽  
pp. 573-581 ◽  
Author(s):  
E J Neufeld ◽  
P W Majerus ◽  
C M Krueger ◽  
J E Saffitz
Keyword(s):  
Electron Microscope ◽  
Specific Activity ◽  
Cell Types ◽  
Cellular Membrane ◽  
Electron Microscope Autoradiography ◽  
Murine Fibroblasts ◽  
Fibrosarcoma Cells ◽  
Microscope Autoradiography ◽  
Murine Fibrosarcoma ◽  
Short Times

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

Electron microscope autoradiography of isolated molecules

1978 ◽  
Vol 36 (2) ◽  
pp. 192-193
Author(s):  
M. Bouteille ◽  
E. Delain ◽  
N. Angelier
Keyword(s):  
Electron Microscope ◽  
High Efficiency ◽  
Specific Activity ◽  
Molecular Complexes ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Loop Technique ◽  
Silver Grains ◽  
Isolated Molecules

The LIGOP method of electron microscope autoradiography which consists in a combination of coating Ilford emulsion with the loop technique and developing with gold latensification and phenidon has proved to provide small, compact developed silver grains with high efficiency.This has made it possible to use this technique with very small materials such as isolated molecules of molecular complexes.The method was assayed first with 3H-Thymidine labelled T7 phages DNA molecule with 630,000 cpm/μg specific activity (fig. 1). The molecules were spread using the adsorption technique constrasted by rotatory shadowing with platinum and then subjected to autoradiography. The Labelling was sufficient to obtain quantitative data in which the spread molecules were considered as a material comparable to a “hot line”. The efficiency (45%) and the HD value (1600 Å) were calculated.The method was also applied to transcription units of pleurodeles oocytes nucleoli (fig. 2) labelled in vitro with 3H-Uridine.

scholarly journals AN ELECTRON MICROSCOPE AUTORADIOGRAPHIC STUDY OF THE NEURONAL AND EXTRANEURONAL LOCALIZATION OF LABELED AMINE IN THE HEART OF THE BAT AFTER ADMINISTRATION OF TRITIATED NOREPINEPHRINE

1974 ◽  
Vol 62 (3) ◽  
pp. 610-624 ◽  
Author(s):  
Michael D. Gershon ◽  
Martin Hagopian ◽  
Eladio A. Nunez
Keyword(s):  
Electron Microscope ◽  
Specific Activity ◽  
Ventricular Myocardium ◽  
Autoradiographic Study ◽  
Thin Filaments ◽  
Electron Microscope Autoradiography ◽  
Morphological Criteria ◽  
Neural Processes ◽  
Microscope Autoradiography ◽  
Nonrandom Distribution

The localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine (NE) was studied by means of electron microscope autoradiography. Monoamine oxidase was inhibited so that the distribution of amine in both neuronal (Uptake1) and extraneuronal (Uptake2) sites could be analyzed. Labeling was nonrandom in both the atrial and ventricular myocardium. The highest relative specific activity was found in neural processes which showed morphological criteria of terminal adrenergic axons. Analysis of the distribution of label around the labeled axonal varicosities indicated that the radioactive amine was more concentrated peripherally than centrally in these structures. Label was also found over cardiocytes in both atrium and ventricle. The pattern of this labeling indicated that the radioactive amine was associated with myofilaments. In the ventricle, I bands were most heavily labeled, indicating a probable association of radioactive amine with thin filaments. Labeling was prevented by administration of phenoxybenzamine and decreased only in cardiocytes by normetanephrine. The nonrandom distribution of labeled amine within cardiocytes supports the view that Uptake2 represents not only a second mechanism of inactivation of the sympathetic neurotransmitter, but may also be involved in the mediation of some of the action of NE on cardiac muscle.

Synthesis and Localization of Sulphated Mucopolysaccharide in Megakaryocytes and Platelets of the Rat, An Analysis by Electron-Microscope Autoradiography

1972 ◽  
Vol 10 (3) ◽  
pp. 705-717
Author(s):  
G. G. MacPHERSON
Keyword(s):  
Electron Microscope ◽  
Golgi Apparatus ◽  
Blood Platelets ◽  
Total Activity ◽  
Electron Microscope Autoradiography ◽  
Dense Granules ◽  
Level Of Activity ◽  
Microscope Autoradiography ◽  
Almost All

Electron-microscope autoradiography has been used to investigate the synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and blood platelets. Following 10-min incubation of bone marrow with 35S-sulpahte in vitro the majority of the activity in megakaryocytes was associated with the Golgi apparatus, but a substantial proportion was associated with other cytoplasmic organelles, suggesting either rapid transport or sulphation of mucopolysaccharide outside the Golgi apparatus. Three hours after the intravenous injection of 35SO4 only a small proportion of the total activity was associated with the Golgi apparatus, most being associated with demarcation membranes and dense granules, while 12 h after injection almost all the activity was associated with demarcation membranes and granules. A rising proportion of activity localized solely on the demarcation membranes suggested that they may possess some activity of their own. Autoradiographs of blood platelets prepared 72 h after the injection of 35SO4 were analysed. It was shown that most of the activity was associated with the α-granules, but there was strong evidence that the platelet membrane possessed a low level of activity.

Timing of nucleolar DNA replication in Amoeba proteus

1976 ◽  
Vol 22 (3) ◽  
pp. 521-530
Author(s):  
I. Minassian ◽  
L.G. Bell
Keyword(s):  
Electron Microscope ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Central Region ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
G2 Phase

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.

Studies on the Synthesis and Composition of Extracellular Mucilage in the Unicellular Red Alga Rhodella

1974 ◽  
Vol 16 (1) ◽  
pp. 1-21
Author(s):  
L. V. EVANS ◽  
MAUREEN E. CALLOW ◽  
ELIZABETH PERCIVAL ◽  
V. FAREED
Keyword(s):  
Electron Microscope ◽  
Uronic Acid ◽  
Red Alga ◽  
Electron Microscope Autoradiography ◽  
Golgi Bodies ◽  
Microscope Autoradiography ◽  
Reducing Substances

35SO42- has been used to investigate the production of extracellular mucilage by log-phase cells. Uptake of isotope occurs most rapidly in the light, when cells are actively dividing. The mucilage comprises about 50% carbohydrate, 16% protein and 10% sulphate. The major sugar is xylose; uronic acid, a small amount of galactose, glucose (trace) and 2 reducing substances are also present. Methylation studies have established the major linkages. Electron-microscope autoradiography shows that the mucilage is packaged in the Golgi bodies, passing to the plasmalemma in large vesicles. Sulphation of the mucilage occurs in the Golgi cisternae.

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