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PubMed1975

Analysis by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes.

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.

Animals↗

PubMed1985

Muscarinic acetylcholine receptors in rat gastric mucosa. A radioautographic study using a potent muscarinic antagonist, 3H-pirenzepine.

The muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.

Animals↗

PubMed1980

A radioautographic study on the incorporation of 14C-bupranolol (beta-blocking agent) into the rabbit eye.

Localization of instilled 14C-bupranolol was studied in the rabbit eye by means of light microscopic radioautography. After the administration of 50 microliter of 14C-bupranolol into the eyes of two rabbits, both eyes were enucleated at intervals of 15 and 30 min respectively, and radioautographic procedures were carried out on cryostat sections by means of both dry-mounting and wet-mounting methods. Although with the former it was possible to demonstrate many radioautographic silver grains in the specimens, with the latter it was not possible to detect their localization. On the dry-mounting procedure, radioautographic grains were observed mainly in the epithelium of the ciliary body 15 and 30 min after the administration of 14C-bupranolol. The numbers of grains were counted on the 15 and 30 min specimens. They were decreased in the conjunctiva, and cornea 30 min after the administration. On the contrary, they were remarkably increased in the epithelium of the ciliary body. From these results it is concluded that the 14C-bupranolol first penetrates into the epithelium of the conjunctiva and cornea, then moves rapidly to the ciliary body, and that only very small amount of 14C-bupranolol is changed into the insoluble form. Also, these results suggest that bupranolol plays a role in the control of aqueous production in the ciliary processes.

Animals↗

PubMed1985

Quantitative radioautographic study of intracellular localization of calmodulin antagonist, W-7, in Chinese-hamster ovary cells.

The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 microM), 2 (33.4 microM), 5 (83.5 microM) and 10 microCi/ml (167 microM) tritiated W-7. Some cells were preincubated in 10, 50 and 100 microM unlabelled W-7 for 30 min and then labelled with 2 or 5 microCi/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 micron-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.

Animals↗

PubMed1983

Stimulation and inhibition by magnesium ions of intrinsic protein phosphorylating systems in synaptosomal membrane fragments from rat brain.

Synaptosomal membrane fragments from rat brain were incubated with [gamma-32P]ATP in the presence of cyclic AMP or Ca2+ plus calmodulin and a range of Mg2+ concentrations. Incorporation of 32P into membrane polypeptides was examined by electrophoresis and radioautography. Cyclic AMP-stimulated reactions were stimulated by low concentrations and inhibited to varying degrees by high concentrations of Mg2+ in the range 1-50 mM. In general the Ca2+ plus calmodulin-stimulated reactions were maximally active in the range 30-50 mM Mg2+, but the Ca2+ plus calmodulin dependent phosphorylation of Protein I was progressively inhibited by concentrations of Mg2+ above 5 mM. These results emphasize the importance of establishing optimum Mg2+ concentrations in the study of specific membrane protein phosphorylating systems.

Animals↗

PubMed1979

Functional activity of uremic erythroblast incubated in autologous and homologous plasma.

The uptake of 3H-thymidine, 3H-uridine and 3H-leucine in the erythroid precursors of patients with chronic renal failure (CRF) was examined by radioautography. The pattern of incorporation of the radioactive precursors was similar to that observed in erythroblasts of control subjects, i.e., the uptake decreased with cell maturation. CRF erythroblasts incubated with normal, homologous plasma, showed significant increase in the uptake of the radioactive precursors, compared to the activity of these cells incubated in autologous plasms, the only exception being the incorporation of 3H-leucine in the proerythroblasts, in which the increase was not statistically significant. These results suggest that the impaired function of CRF erythroblasts related to DNA, RNA and protein synthesis is due not to a defective mechanism in the cells themselves, but most probably to the effect of factors present in uremic plasma, the nature of which remains to be detected.

Adult↗

PubMed1982

Regeneration and retrograde degeneration of axons in the rat optic nerve.

The retinal stump of the rat optic nerve was examined histologically 1-64 weeks after intracranial section of the nerve with or without grafting of autologous peripheral nerve segments. Single unmyelinated axons and bundles of unmyelinated axons appeared in cut optic nerves and were most abundant 2-4 weeks after section. With light and electron microscope radioautography after injection of tritiated amino acids into the globe, it was confirmed that many unmyelinated fibres arose from the optic nerve rather than from nearby peripheral nerves and it was estimated that some axons regenerated as far as 0.5 mm. At or near the end of retinofugal axons, structures resembling growth cones were seen at 2 weeks and vesicle-containing swellings similar to synapses were found at 1-2 months. Outgrowth from optic nerve axons was not obviously enhanced by peripheral nerve grafts although a few retinofugal axons became ensheathed by Schwann cells. Retrograde axonal degeneration was rapid in both cut and grafted optic nerves, the number of nerve fibres near the globe falling to less than 10% of normal after 4 weeks. A few myelinated and unmyelinated fibres were still present 64 weeks after nerve transection. In conclusion, some cut axons in the rat optic nerve display a transient regenerative response before undergoing retrograde degeneration.

Animals↗

PubMed1984

Regeneration of long spinal axons in the rat.

To investigate regeneration of long spinal axons, the right lateral column of the rat spinal cord was cut at high cervical, low cervical, midthoracic or lumbar level, and one end of an autologous sciatic nerve segment was grafted to the spinal cord at the site of incision. Three to six months after operation, the origin of axons in the grafts was traced retrogradely with horseradish peroxidase injected into the grafts and, in some cases, anterogradely with radioautography of tritiated amino acids injected into the brainstem. Axons from each of the major lateral spinal tracts arising in the brainstem as well as axons ascending from the lower spinal cord succeeded in growing into low cervical grafts. However, long descending axons rarely regenerated after midthoracic or lumbar injury; axons ascending from lumbar segments of the spinal cord usually failed to enter high cervical grafts. Differences in axonal regrowth at the four segmental levels were not simply attributable to dwindling of axonal number in fibre tracts. Axonal regeneration from Clarke's column or the red nucleus was observed only with lesions causing atrophy of many neurons. There was no obvious example of a fibre tract in the lateral spinal columns from which axons failed to regenerate nor from which axons regenerated exceptionally well. Under the conditions of these experiments, the distance from cell body to injury appeared to be an important determinant of axonal regeneration.

Animals↗

PubMed1985

Localization of 5-hydroxytryptamine to neurons and endoneurial mast cells in rat sensory ganglia.

5-Hydroxytryptamine (5-HT) localization has been studied in the spinal and trigeminal ganglia of adult rats using immunofluorescence, peroxidase-antiperoxidase immunocytochemistry and [3H] 5-HT uptake radioautography, the latter two at the ultrastructural level. Endoneurial mast cells, identified by alcian blue staining, formed 8 and 14% of all mast cells and neurons in spinal and trigeminal ganglia respectively and had a median diameter of 7.6 microns. Light and electron microscopic immunocytochemistry showed that these mast cells contained 5-HT-like immunoreactivity. Some 75% of them accumulated exogenous [3H]5-HT in vitro. A population of small-diameter neurons, which did not stain with alcian blue, was also labelled with anti-5-HT serum and accumulated [3H]5-HT. The possible roles of 5-HT in sensory ganglia are discussed.

Alcian Blue↗

PubMed1985

VIP neurons as prime synaptic targets for serotonin afferents in rat suprachiasmatic nucleus: a combined radioautographic and immunocytochemical study.

Cellular relationships between serotonin (5-HT) axon terminals and neurons containing vasoactive intestinal peptide (VIP) were characterized by combined radioautography and immunocytochemistry in rat suprachiasmatic nucleus (SCN). Light microscopic immunoradioautographs showed significant overlap between (3H)5-HT uptake sites and VIP-immunoreactive elements in the ventral half of the SCN. Of the 255 (3H)5-HT-labelled axonal profiles detected in a systematic electron microscopic survey of single thin sections from this area, 75 (30%) were directly apposed to VIP-immunoreactive nerve cell bodies and/or dendrites. Radioautographically labelled 5-HT varicosities often showed well-differentiated, symmetrical or asymmetrical synaptic junctions, 60% of which were established on VIP-immunoreactive nerve cell bodies or dendrites. In a separate sampling of 198(3H)5-HT-labelled terminals seen in apposition with VIP-immunoreactive elements, 50 showed a junctional complex at the site of contact. Postsynaptic immunoreactive elements were mostly dendrites but also included nerve cell bodies. Despite the methodological limitations inherent to the present double labelling approach, these data strongly support the view that VIP neurons are prime synaptic targets for 5-HT afferents in the SCN. VIP/5-HT interactions are thus likely to play an important functional role in this nucleus and may in particular subserve the 5-HT mediated regulation of certain circadian rhythms, including that of pituitary hormone secretion.

Afferent Pathways↗

PubMed1976

Potential of Schwann cells from unmyelinated nerves to produce myelin: a quantitative ultrastructural and radiographic study.

In adult mice, most fibres in the cervical sympathetic trunk (CST) are unmyelinated whereas a large proportion of sural nerve fibres are myelinated. This study of nerve grafts in syngeneic mice was designed to determine if Schwann cells originating from the unmyelinated CST would produce myelin when in contact with regenerating axons of the sural nerve. Quantitative microscopy of triated thymidine-labelled CST segments grafted to unlabelled sural nerve stumps revealed that, one month after grafting, previously unmyelinated grafts contained many myelinated fibres. By phase and electron microscope radioautography, nearly 40% of the myelin-producing cells in the reinnervated graft were shown to have originated in the unmyelinated CST. These findings indicate that Schwann cells originating from unmyelinated fibres are able to differentiate into myelin producing cells.

Animals↗

PubMed1975

Experimental necrosis and arrest of proliferation of Schwann cells by cytosine arabinoside.

In developing rat cervical sympathetic trunks, Schwann cells proliferate intensely during the first week after birth but axonal populations do not increase. Thus, experimental inhibition of DNA synthesis should affect Schwann cells and spare axons whose cell bodies are not dividing. The present investigation was aimed at determining the effects of an inhibitor of DNA synthesis-cytosine arabinoside (ara-C)--on axons and Schwann cells in developing nerves. Ara-C (60 mg kg(-1) body weight) was injected subcutaneously to newborn rats every six hours for 36 hours. At intervals from 2--4 days of age, animals were given tritiated thymidine (4 muCi per gram body weight) to label Schwann cells in synthesis phase. One hour later rats were killed by systemic perfusion of phosphate buffered glutaraldehyde. Cervical sympathetic trunks (CST's) and sciatic nerves were removed and processed for radioautography and electron microscopy (EM). Labelled and unlabelled Schwann cell nuclei were counted to determine the labelling index (LI%) for each nerve. By the end of ara-C treatment there was almost complete absence of labelling but LI's rose sharply 24 hours after discontinuing ara-C. By EM axons appeared normal; Schwann cells, however, showed prominent nuclear and cytoplasmic changes consisting of nuclear degeneration, dilatation of rough endoplasmic reticulum and increased cytoplasmic density. Ara-C, by suppressing proliferation and causing necrosis of Schwann cells but sparing axons, results in a developmental alteration of axon-Schwann cell relationships. It is suggested that the pathogenetic mechanism involves an inbalance in DNA/RNA synthesis metabolism.

Animals↗

PubMedDate not supplied

Structure and nature of macrophages participating in the wallerian degeneration of nerve fibers.

The posttraumatic processes of Wallerian degeneration of nerves have been illuminated in detail. The dynamics of the breakdown of axons and the myelin sheaths of nerve fibers has been established, as have been the periods of the changes in the composition of myelin, and the reactive changes in the Schwann cells and the connective tissue structures in the makeup of the nerve as well as the formation of "foam" cells have been described. The controversial questions which have been raised in these studies regarding the role of the cellular elements (the Schwann cells, the endoneurial fibroblasts, the cells of the epi- and perineurium) during Wallerian degeneration remain unresolved until the present time. In particular, the question as to which cells participate in the cleanup of the products of the breakdown of the myelin sheaths, and as to the character of the inflammatory infiltration in Wallerian degeneration and the degree of the participation of the various cellular elements in the destructive and reparative processes, has not been elucidated. Some investigators believe that the Schwann cells accomplish the cleanup of the products of the breakdown of the myelin sheaths. There are also data suggesting that the macrophages are of considerable significance in the cleanup of the products of the breakdown of nerve fibers of both the PNS and the CNS following their injury. It has been demonstrated by means of monoclonal antibodies to macrophages, radioautography, and immunocytochemical methods that these macrophages have a hematogenous origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Ultrastructural relationships of serotonin and GABA terminals in the rat suprachiasmatic nucleus. Evidence for a close interconnection between the two afferent systems.

Serotonin (5-HT) and gamma-aminobutyric acid (GABA) nerve endings were identified in the rat suprachiasmatic nucleus (SCN) by combined [3H]5-HT uptake radioautography and glutamate decarboxylase (GAD) immunocytochemistry at the electron microscope level. In areas of good overlap between radioautographic and immunocytochemical labellings, there were no axonal varicosities exhibiting both labellings, indicating that 5-HT and GABA are not co-localized in the SCN. The systematic survey in these areas of all profiles that had accumulated [3H]5-HT and of all GAD-immunoreactive varicosities allowed the analysis of 247 of the former and 896, i.e. an almost four-fold greater number, of the latter. This seems concordant with the view that GABA endings would be the most numerous of all classes of nerve terminals so far identified in the SCN. More than 22% of the [3H]5-HT labelled profiles showed the membrane specialization typically associated with synaptic junctions. Thereby, it was possible to evaluate that about 45% of the 5-HT terminals actually form a synapse in the SCN. Some 37% of the GAD-positive varicose profiles which could be formally interpreted also showed well differentiated synaptic contacts, suggesting that the GABAergic innervation of the SCN could be entirely junctional. Whereas 5-HT terminals usually innervated only one dendritic or somatic element, a convergence of several GABAergic terminals onto the same postsynaptic target also receiving a 5-HT input was frequently observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1977

Localization of 5-hydroxytryptamine and protein(s) in the secretion granules of the rudimentary photoreceptor cells in the pineal of Lacerta.

In order to localize 5-hydroxytryptamine (5-HT) stores, the pineal organ of Lacerta was examined by electron microscopy following argentaffin and chromaffin reactions. The results obtained with these ultracytochemical reactions were correlated with those obtained previously using the Falck and Hillarp method and electron microscopic radioautography. The secretion granules (diameter: 60-340 nm) of the secretory rudimentary photoreceptor (SRP) cells are the locus of the argentaffin and chromaffin reactions. The reactivity of the granules disappeared after p-CPA or reserpine administration and increased after nialamide injection. From the previous and present results it can be concluded that 5-HT in the pineal organ of Lacerta is stored in the secretion granules of SRP cells and coexists with a proteinaceous and/or possibly a glycoproteinaceous component. Whether this secretion is neurohormonal in nature is still not clear.

Animals↗

PubMed1987

Biochemical and radioautographic evidence for dopaminergic afferents of the locus coeruleus originating in the ventral tegmental area.

The dopamine (DA) content of the locus coeruleus (LC) and the uptake of tritiated DA in the presence of desmethylimipramine into fresh vibratome sections of the LC-area were determined in control rats and in rats whose ventral tegmental area (VTA) had been destroyed by local application of 6-hydroxydopamine (6-OHDA). Destruction of the VTA reduced the DA content and the number of dopaminergic fibers visualized by radioautography in the LC area. This indicates that the DA containing afferents of the LC originate, at least partly, in the VTA.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1985

Epithelial cell proliferation in human fundic and antral mucosae. Influence of superselective vagotomy and relationship with gastritis.

Epithelial cell proliferation in the fundic and antral mucosae was studied in 19 duodenal ulcer patients, 11 patients having undergone fundic superselective vagotomy for duodenal ulcer, and 10 controls. This was achieved through in vitro incorporation of tritiated thymidine in mucosal biopsies and radioautography. Except for increased fundic mucosal height, duodenal ulcer patients did not differ from controls for all parameters studied. In vagotomized patients, as compared to the other two groups, the labeling index was significantly enhanced in the innervated antral mucosa where atrophic gastritis developed, but there was no change in the labeling index and no worsening of mucosal inflammation in the denervated fundic mucosa. The only abnormality in the latter was a striking expansion, towards the surface, of the proliferative area within the fundic pit. The labeling indices and the degree of gastritis in gastric mucosae are significantly correlated in control and duodenal ulcer patients. After superselective vagotomy, this correlation still existed in antral mucosa (r = 0.88, P less than 0.001) but not in fundic mucosa. If findings in antral mucosa, after superselective vagotomy, seemed related to gastritis lesions, those in fundic mucosa were not and may indicate an alteration due to the vagotomy per se.

Adolescent↗

PubMed1998

Immunohistochemical localization of taurine in various tissues of the mouse.

The localization of taurine was investigated in several tissues of the mouse. Immunohistochemical methods using a polyclonal antibody for taurine derived from rabbits was used in these studies. This method was used since it is a simple procedure and the results are clear and reliable. Tissues were fixed with paraformaldehyde, embedded in paraffin and treated in a microwave oven before using an avidin-biotin-complex method (ABC method). Control staining was accomplished by employing absorption staining using various amino acids: taurine, arginine, cysteine, hypotaurine and others. For purposes of comparison, radioautography (RAG) with 3H-taurine was performed to confirm the reliability of the immunohistochemical staining compared with the localization of the 3H-taurine incorporation in endothelial cells of the blood vessels of several tissues. In this investigation, immunoreactivity was broadly observed in many tissues: Purkinje cells of the cerebellum, glia cells of brain tissue, cardiac muscle cells, matrices of the bone, mucus granules of goblet cells of the intestines, and brown adipose cells of the fetus. Although the meaning of this widespread localization of taurine can not be explained completely, we surmise that taurine may have a different function in each of the tissues. In addition, taurine reactivity was observed in cell nuclei which was evidence of the presence of taurine in the nuclei.

Age Factors↗