scholarly journals High-Speed Electron Microscope Autoradiographic Studies of Diffusible Compounds,

1981 ◽  
Vol 29 (1A_suppl) ◽  
pp. 143-160 ◽  
Author(s):  
Vinci Mizuhira ◽  
Michiko Shiihashi ◽  
Yutaka Futaesaku
Keyword(s):  
Electron Microscope ◽  
Exposure Time ◽  
High Speed ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Good Resolution ◽  
Cell Organelles ◽  
Electron Microscope Autoradiography ◽  
Resolution Problem

Three important factors are necessary for successful electron microscope autoradiography (EM-ARG): good resolution, proper preparation of the radioactive isotope (RI) labeled diffusible compounds, and shortened exposure time for ARG. The resolution problem is fundamental to EM-ARG. However, unless the diffusible RI compounds have been fixed correctly in the tissues during preparation, good resolution is useless. It is also necessary to shorten the exposure time for ARG. As yet, a high-speed ARG method for electron microscopy has not been reported, although scintillation ARG methods have been applied to macro- and micro-ARG since 1960. High specific activity, a large amount of radioactivity per unit exposure for radio incorporation (incubation), and careful selection of labeled compounds that concentrate in the DNA or RNA of cell organelles may increase the sensitivity of the emulsion and shorten the exposure time for ARG. For example, labeled thymidine accumulates in nuclear DNA, 3H-SPG (Schizophyllan-produced polyglucan) is incorporated into lysosomal granules, and labeled iodine concentrates in thyroid follicles, often increasing the sensitivity of the emulsion and shortening the exposure time. High-speed ARG yields good data in a very short time, but high-resolution ARG continues to be necessary, even though it requires 4 weeks or more of exposure time. Scintillation autoradiography using tritium seems unstable. We propose a new way to shorten exposure time for EM-ARG, by combining overdevelopment with coating both sides of the grid with emulsion. This method is approximately 100 times more sensitive than the conventional method, and only 4 days of exposure time are required, in contrast to the 1 month usually needed.

Continuous DNA replication in the nucleus of the dinoflagellate Prorocentrum micans (Ehrenberg)

1977 ◽  
Vol 27 (1) ◽  
pp. 81-90
Author(s):  
S.A. Filfilan ◽  
D.C. Sigee
Keyword(s):  
Electron Microscope ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Continuous Process ◽  
Prorocentrum Micans ◽  
Interphase Nuclei ◽  
Electron Microscope Autoradiography ◽  
Dividing Cells ◽  
High Degree ◽  
Very High

The uptake of tritiated thymine into cells of a heterogeneous population of Prorocentrum micans was investigated using light-microscope and electron-microscope autoradiography. Specificity of thymine uptake into DNA was demonstrated by the specific removal of label from wax-embedded material using DNase and by the high degree of localization of nuclear label to chromosomes in the electron-microscope autoradiographs. All nuclei, including both dividing and non-dividing cells, showed a substantial uptake of label, indicating that nuclear DNA synthesis in Prorocentrum micans is a continuous process. The level of DNA synthesis does show considerable variation, however, with very high levels in some interphase nuclei. The continuous replication of nuclear DNA provides further evidence of dinoflagellate affinity to the prokaryotes, and indicates that Prorocentrum micans is a very primitive eukaryote cell.

scholarly journals A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'5' direction

2003 ◽  
Vol 270 (8) ◽  
pp. 1735-1745 ◽  
Author(s):  
Tuan-Nghia Phan ◽  
Nasreen Z. Ehtesham ◽  
Renu Tuteja ◽  
Narendra Tuteja
Keyword(s):  
Pisum Sativum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Dna Helicase

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.

Thymidine-requiring mutants of Dictyostelium discoideum

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

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.

scholarly journals Thymidine-requiring mutants of Dictyostelium discoideum.

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791 ◽  
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.

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 of macromolecules by the epithelial surfaces ofSchistosoma mansoni: an autoradiographic study

Parasitology ◽  
1979 ◽  
Vol 78 (3) ◽  
pp. 295-310 ◽  
Author(s):  
R. A. Wilson ◽  
P. E. Barnes
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
High Specific Activity ◽  
Autoradiographic Study ◽  
Secretory Protein ◽  
Membrane Turnover ◽  
Electron Microscope Autoradiography ◽  
Glycoprotein Synthesis ◽  
Gut Epithelium ◽  
Rapid Mass

SUMMARYThe use of tritiated leucine as a marker for protein synthesis and of tritiated glucosamine as a marker for polysaccharide/glycoprotein synthesis, is described. Adult worms were pulse-labelled by incubation in medium containing the substrate. Labelled worms were then incubated in chase medium, without labelled substrate, for varying lengths of time before fixation. The distribution of label which had been incorporated into macromolecules in the worm tissues, was examined by light and electron microscope autoradiography. It was estimated that the tegument and tegument cell bodies were the source of 67–80%, and the gut epithelium of 20–33%, of exportable leucine-containing protein. Conversely, the gut epithelium was the source of 72%, and the tegument cells 28%, of exportable glucosamine-containing polysaccharide. The specific activity of labelled protein reached a peak in the tegument cytoplasm after 1.5 h of chase incubation. Half of the labelled protein was secreted into the worm's environment by 3 h of chase incubation. The half-life of secretory protein in gut cells appears to be around 2 h. Labelled protein disappears from the gut lumen relatively rapidly but labelled polysaceharide remains in the lumen at high specific activity for at least 24 h. The major carbohydrate labelled may be the glycocalyx on the luminal surface of the gut epithelial cells. The results suggest that the bulk of worm secretions have a rapid turnover with a half-life of a few hours. Against this background of rapid mass secretion a slower process of membrane turnover would be difficult to detect and quantitatively small.

Vacuum System to Minimize the Specimen Contamination of High-Performance EM

1977 ◽  
Vol 35 ◽  
pp. 68-69
Author(s):  
N. Yoshimura ◽  
K. Shirota ◽  
T. Etoh
Keyword(s):  
Electron Microscope ◽  
High Speed ◽  
High Performance ◽  
High Vacuum ◽  
Vacuum System ◽  
Pump System ◽  
Pumping System ◽  
Diffusion Pump ◽  
Almost All ◽  
Cascade Type

One of the most important requirements for a high-performance EM, especially an analytical EM using a fine beam probe, is to prevent specimen contamination by providing a clean high vacuum in the vicinity of the specimen. However, in almost all commercial EMs, the pressure in the vicinity of the specimen under observation is usually more than ten times higher than the pressure measured at the punping line. The EM column inevitably requires the use of greased Viton O-rings for fine movement, and specimens and films need to be exchanged frequently and several attachments may also be exchanged. For these reasons, a high speed pumping system, as well as a clean vacuum system, is now required. A newly developed electron microscope, the JEM-100CX features clean high vacuum in the vicinity of the specimen, realized by the use of a CASCADE type diffusion pump system which has been essentially improved over its predeces- sorD employed on the JEM-100C.

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

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