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At least 109 records · Page 6Linked to original sources
PubMed1967

An artefact in radioautography due to binding of free amino acids to tissues by fixatives.

The binding of labeled free amino acids to liver and to purified protein by commonly used fixatives was investigated. Glutaraldehyde caused 25% of free leucine to be bound to serum albumin in solution, whereas formaldehyde bound only 0.5%. Liver slices were incubated for 2 min in the presence of labeled leucine and of puromycin, which permits absorption of leucine into the cell but inhibits incorporation into protein. Both counting and radioautographic techniques showed that glutaraldehyde bound 30 times, and osmic acid six times, as much free amino acid as did formaldehyde. By comparing liver slices incubated with and without puromycin for 2 min, it was calculated that in radioautographs prepared after fixation with glutaraldehyde, osmic acid, or formaldehyde 63, 25, and 4% respectively of the grains were due to binding of free amino acid. Formaldehyde, freshly prepared from paraformaldehyde, gives good preservation and is the recommended fixative for radioautography. When levels of free substrate in a tissue are high at the time fixative is added, the amount of binding of free substrate induced by the fixative should be included as a control in radioautographic experiments.

Aldehydes↗
PubMed1967

Absolute sensitivity of electron microscope radioautography.

A calibration method is described for measuring absolute radioautographic sensitivities under various experimental conditions. Sensitivities to (3)H and (35)S radiation, i.e. ratio of developed grains to radioactive decays in the specimen, were determined with Ilford L4 and Kodak NTE emulsions. The highest values obtained in monolayers of emulsion were (1/8) for (3)H and 1/21 for (35)S. The influence of various experimental parameters on sensitivity is described, and the possibilities for quantitative electron microscope radioautography are discussed.

Autoradiography↗
PubMed1967

Thymidine-3H electron microscope radioautography of osteogenic cells in the fetal rat.

The proximal tibial epiphyses of 18-21-day-old fetal rats have been studied by thymidine-(3)H radioautography. The results reveal that the label is incorporated into two types of osteogenic cells: (a) a spindle cell type (A cells) with characteristics generally associated with matrix production, including an extensive development of the endoplasmic reticulum and the presence of large intracellular accumulations of a dense, finely granular material, morphologically identifiable as glycogen; and (b) a rounded cell type (B cells) with morphological features similar in degree and kind to those of the developing neutrophilic leukocyte, including an abundance of free ribosomes and mitochondria and a complex Golgi apparatus associated with dense specific granules, morphologically identifiable with primary lysosomes. These results, along with the occurrence of recognizable, labeled, immature, perivascular forms of both of these A and B type cells, lead to the conclusion that the specialization of osteogenic cells into osteoclasts and osteocytes may involve separate pathways of cytodifferentiation.

Animals↗
PubMed1967

[Quantitative study by radioautography and electron microscopy of the utilization of DL-leucine-H3 by duck hypophyseal cells in organ culture].

The synthesis, intracellular transport, storing, and excretion of proteins by duck hypophyseal cells in organ culture were studied with tritiated DL-leucine and high resolution radioautography (pulse-labeling experiments). Quantitative study of the radioautographs allowed a determination of the relative proportions of cytoplasmic radioactivity located in each cellular compartment (ergastoplasm, Golgi apparatus, and protein granules) as well as the variations in these proportions as a function of time. The number of labeled protein granules as opposed to the total number of granules in the cell was also determined (RSg). These data were separately analyzed for the two types of cells present in the explants: prolactin cells and "MSH" cells. The synthetic process follows a course common to both cell types, each of which is distinguished by its particular modalities. The labeled proteins, synthesized within several minutes in the ergastoplasm, are concentrated in the Golgi zone within 30 min. They then migrate out of this area, the emptying of which is accomplished in about 4 hr. These proteins become equally distributed between the protein granules, on the one hand, and the cytoplasm ("sedentary" proteins), on the other. The RSg reaches its maximum when the Golgi zone is emptied, but this figure remains very low (3%). The RSg then decreases slowly (1% in 40 hr). It is concluded that hypophyseal cells are able to store protein in their granules and that their processes of synthesis and excretion are not continuous. The prolactin cells differ from the "MSH" cells in that they have a slower migration of newly synthesized proteins, and these proteins pass via the dilated ergastoplasmic cisterns in which they may possibly be stored.

Animals↗
PubMed1967

High-resolution radioautography of galactose-3H accumulation in rings of hamster intestine.

Radioautography of water-soluble substances has posed a major technical problem for the past decade. Utilizing silicone-impregnated plastic sections of frozen-dried tissue, a quantitative method was developed for studying distribution of (3)H-labeled galactose, mannitol, and phlorizin. The content of a 2-micro band may be measured with an accuracy of +/-20% by light microscopy; radioautographs may also be prepared for the electron microscope. Results with intestinal tissue incubated 1-10 min in vitro and, then, frozen rapidly indicate that the first step in galactose absorption is uphill transport into the brush border of the columnar epithelium. Correction of galactose content for the mannitol space in the brush border suggests that the sugar pump is located at the surface of the microvilli. Further evidence for the surface locus of the glucose-galactose pump was obtained with phlorizin (next paper, reference 40). The galactose content of columnar cell cytoplasm always equalled that of microvilli and no transcellular diffusion gradient could be detected; during the first minutes of incubation, however, a gradient did exist between nucleoplasm and cytoplasm. Downhill exit of galactose from columnar cells may have proceeded either directly across basal membranes to adjacent lamina propria or indirectly via open intercellular spaces. Lastly, even in the absence of muscularis, the connective tissue of the lamina propria constituted enough of a diffusion barrier so that it served as a secondary accumulating compartment for galactose under present in vitro conditions.

Animals↗
PubMed1968

Localization of hyaluronic acid in synovial cells by radioautography.

Cultured human synovial cells secrete hyaluronic acid (HA) into the culture medium. Glucosamine-6-(3)H was shown to be a direct and relatively specific precursor of HA-(3)H by the following observations: the susceptibility of nondialyzable radioactivity in the medium to hyaluronidase, its migration with hexuronic acid on zone electrophoresis in polyvinyl chloride, its exclusion from Sephadex G-200, and the localization of radioactivity to glucosamine after hydrolysis of the labeled polysaccharide. The presence of intracellular HA-(3)H was established by sequential extraction of labeled cells and by radioautography of synovial cell cultures digested with hyaluronidase in situ. When cells were exposed to medium lacking glucose, glucosamine-(3)H-uptake was enhanced; and this made possible electron microscopic radioautographic studies. These studies demonstrate the early and continued presence of HA-(3)H within the Golgi apparatus.

Autoradiography↗
PubMed1968

Localization of tritiated norepinephrine in vascular sympathetic axons of the rat intestine and mesentery by electron microscope radioautography.

The distribution of infused tritiated norepinephrine (NE-(3)H) in small mesenteric arteries and intestinal arterioles in rats was investigated with electron microscopic radioautography. Silver grains, indicating the presence of the tritium label on the sections, were found lying mainly over axon bundles, but some were present over collagen and smooth muscle cells. Axons with the highest concentrations of silver grains had been sectioned at points where they were naked of Schwann cell sheath, were dilated into varicosities, and contained small granular vesicles. This finding was taken as confirmatory circumstantial evidence that the small granular vesicles were the sites of uptake and storage of NE. The short interval between the start of infusion and the fixation of the tissue appeared to rule out any process other than a direct uptake of NE by the peripheral axons. If axonal sites of uptake of NE-(3)H correspond to sites of release of NE, then the evidence suggests that such sites of release are widespread over the terminal part of the axon and are not confined to those parts of the axon which are in close contact with smooth muscle cells. Since the fixation and embedding procedures will remove NE which is not strongly bound to tissues, the localization of NE-(3)H in the radioautographs does not necessarily correspond to the distribution of all the NE present in vivo.

Animals↗
PubMed1969

Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography.

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-(32)P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10(7) sites per endplate, in the diaphragm, about 3 x 10(7). Reaction with DFP-(32)P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10(7) and 1.1 x 10(7)per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.

Acetylcholinesterase↗
PubMed1969

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. II. The distribution of DFP-reactive sties at motor endplates of a vertebrate twitch muscle.

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-(3)H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per micro(3) of cleft tissue or 12,000 sites per micro(2) of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)-a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.

Acetylcholinesterase↗
PubMed1970

The intrarenal distribution of tritiated para-aminohippuric acid determined by a modified technique of section freeze-dry radioautography.

Section freeze-dry radioautography has been used to examine the intrarenal distribution of a water-soluble organic acid (para-aminohippuric acid (PAH-3H)) under constant-infusion, steady-state conditions in mouse and rat kidney in vivo. The technique described here has the following advantages: (a) Sectioning and freeze-drying are accomplished in a closed cryostat at temperatures below -40 degrees C; (b) Handling of the section is facilitated by mounting of the section-to-be on adhesive-coated Saran Wrap prior to cutting; (c) Unembedded freeze-dried sections are attached to photographic film at ambient temperature in the dark room; (d) Fixation follows completion of radioautographic exposure and precedes photographic development; (e) Permanent close contact is maintained between tissue and film. Morphologic preservation compared favorably with that obtained by optimal fixation techniques, which, however, permit diffusion. Cellular accumulation of PAH-3H during secretion was demonstrated in the proximal tubule under steady-state conditions in vivo. The cellular concentration of PAH-3H was uniform throughout the length of the proximal tubule in mouse and rat kidney.

Aminohippuric Acids↗
PubMed1971

Resolution in electron microscope radioautography. II. Carbon 14.

Experimental resolution values, half distances (HD), were determined for electron microscope radioautography with (14)C as the source of radioactivity. These were about a factor of 1.5-2 times higher than for tritium. Grain distributions normalized in units of HD were found to fit the "universal" curves previously obtained for tritium.

Autoradiography↗
PubMed1972

Distribution of leucine- 3 H during axoplasmic transport within regenerating neurons as determined by electron-microscope radioautography.

The distribution of leucine-(3)H in neurons was determined by electron-microscope radioautography after infusion of label into the spinal cord or sensory ganglia of regenerating newts. In the nerve cell bodies 3 days after infusion, the highest concentration of label per unit area occurred over the rough-surfaced endoplasmic reticulum. In the large brachial nerves, the silver grains were not distributed uniformly in the axoplasm, indicating that the labeled materials are restricted in their movement to certain regions of the axon. Almost all of the radioautographic grains observed in myelinated nerves could be accounted for by the presence of a uniformly labeled band occupying the area 1500-9000 A inside the axolemma. This region of the axon was rich in microtubules and organelles while the unlabeled central core of the axon contained mainly neurofilaments. This observation supports the hypothesis that microtubules are related to axonal transport. In small, vesicle-filled nerve terminals in the blastema, labeled material was restricted to a thin zone a short distance beneath the plasma membrane while the central region of the terminal was largely unlabeled. The peripheral pattern of labeling in the nerve endings is consistent with successive addition of newly synthesized proteins at the periphery of the growth cone and release of substances such as trophic factors at the nerve terminal.

Animals↗
PubMed1972

Protein synthesis in the visual cells of the honeybee drone as studied with electron microscope radioautography.

Protein synthesis was studied in the visual cells of an insect (honeybee drone, Apis mellifera) by electron microscope radioautography. After a single injection of tritiated leucine, the radioactivity first appears in the cytoplasm of the visual cell which contains ribosomes. Later, part of this radioactivity migrates to the rhabdome, the visual cell region which is specialized in light absorption. A maximal concentration of radioactivity is reached there 48 hr after the injection of leucine. This pattern of protein synthesis and transport resembles that described in vertebrate visual cells (rods and cones), where newly synthesized proteins have been shown to contribute to the renewal of the photoreceptor membrane.

Animals↗
PubMed1974

Synthesis, migration, and release of precursor collagen by odontoblasts as visualized by radioautography after (3H)proline administration.

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30-40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280-350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.

Animals↗
PubMed1982

Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography.

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.

Animals↗
PubMed1972

Ligand-induced movement of lymphocyte membrane macromolecules. I. Analysis by immunofluorescence and ultrastructural radioautography.

The fate of different complexes on the membrane of thymocytes and spleen lymphocytes was studied with the use of both immunofluorescence and ultrastructural radioautography. The complexes of anti-immunoglobulin (Ig) with the surface Ig of B lymphocytes were present all around the membrane at 4 degrees C; an increase in temperature produced a rapid aggregation of the complex into a cap which was readily interiorized in vesicles. Ultrastructural details of this process were given. The movement of the complexes depended upon the amount of anti-Ig and the temperature. The complexes of anti-lymphocyte antibody with surface antigen(s) did not result in formation of a single large aggregate (or cap) unless an anti-antibody was brought into the reaction. The caps formed by this trilayered complex were not interiorized. Concanavalin A (Con A) bound to cell surface carbohydrate moieties and the complexes of Con A readily formed a cap and were interiorized. Finally, antibodies to H-2 determinants did not form in most instances a single cap aggregate even when anti-antibodies were used. With time the H-2 complexes tended to form several large aggregates with some endocytosis.

Animals↗
PubMed1991

Localization of [125I]endothelin-1 in rat tissues observed by electron-microscopic radioautography.

[125I]Endothelin-1 (ET-1) was administered intravenously into rats, and binding sites in various tissues were examined by electron-microscopic radioautography. Labeling was observed predominantly on the glomerular fenestrated endothelial cells of kidney, alveolar capillary endothelial cells of lung, fat-storing cells in the sinusoids of liver, and subepithelial myofibroblasts of the small intestine. At first, silver grains were observed mostly on the plasmalemma of these cells and with time translocated into the cytoplasm of these cells. Myofibroblasts of the small intestine were cultured to study the cellular effects of ET. By fura-2 fluorescence method, transient increase and oscillation of intracellular Ca2+ level were observed in the cultured myofibroblasts following the application of ET-1 or endothelin-3 (ET-3). These results suggest that ET induces diverse physiological reactions in specific cells of various tissues.

Animals↗
PubMed1984

A combined light and electron microscopic method for the visualization of the same in vitro neuron by radioautography and serial sections.

We report here on a technical improvement which makes it possible to study, at the ultrastructural level, a dopaminergic neuron which has been previously identified by light microscopy. Primary cultures of virtually pure mesencephalic neurons from mouse embryos were obtained. These cultures were kept for 6 days, then incubated with tritiated dopamine, fixed and embedded in Epon. The dopaminergic neurons were firstly visualized by radioautography directly through Epon blocks in toto by light microscopy. In a second step, ultrathin sections of the identified dopaminergic cells were prepared and the neurons observed at the electron microscopy level. The dopaminergic nature of these neurons was regularly checked by radioautographic control on some selected ultrathin sections.

Animals↗