Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

Deoxycytidine Transport and Metabolism in the Central Nervous System

Abstract: The mechanisms by which deoxycytidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [³H]deoxycytidine intraarterially, intravenously, and intraventricularly. After intracarotid injection of deoxycytidine (1.0 μM) into rats, deoxycytidine did not pass through the blood-brain barrier at a faster rate than sucrose. [³H]Deoxycytidine, either alone or together with unlabeled deoxycytidine, was infused at a constant rate into conscious adult rabbits. At 130 min, [³H]deoxycytidine readily entered CSF, choroid plexus, and brain. In brain, approx. 60% of the nonvolatile radioactivity was attributable to [³H]deoxycytidine phosphates. The addition of 0.22 mmol/kg unlabeled deoxycytidine to the infusion syringe decreased the phosphorylation of [³H]deoxycytidine in brain by approx. 50%; the addition of 2.2 mmol/kg of unlabeled deoxycytidine to the infusion syringe decreased the relative entry of [³H]deoxycytidine into CSF and brain by approx. 50 and 75%, respectively. Two hours after the intraventricular injection of [³H]deoxycytidine, [³H]deoxycytidine was rapidly cleared from CSF, in part, to brain, where approx. 65% of the [³H]deoxycytidine was converted to [³H]deoxycytidine phosphates. The intraventricular injection of unlabeled deoxycytidine with the [³H]deoxycytidine decreased the phosphorylation of [³H]deoxycytidine in the brain significantly and also decreased the clearance of [³H]deoxycytidine from the CSF. These results were interpreted as showing that the entry of deoxycytidine from blood into CSF occurs by a saturable transport system within the choroid plexus. Once within the CSF, the deoxycytidine can enter brain, undergo phosphorylation to deoxycytidine phosphates, and subsequently be incorporated into DNA.     

Peripheral nervous system myelin assembly in vitro: perturbation by the ionophore monensin

Abstract: Sciatic nerves from 13-day-old rats were incubated in vitro with [³⁵S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′-cyclic nucleotide phospho-diesterase (myelin), 5′-nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, and bands corresponding to the P₀ and myelin basic proteins were identified by co-migration with unlabelled myelin marker proteins on both one-dimensional SDS-PAGE and two-dimensional nonequilibrium isoelectric focussing/SDS-PAGE systems. Following a 90-min incubation with [³⁵S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P₀ protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P₀ and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P₀: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P₀ were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P₀ glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

Experimental study of synchronization phenomena under periodic light irradiation of a nonlinear chemical system

A photosensitive chemical oscillating reaction, i.e., the Briggs-Rauscher (B.R.) reaction, exhibiting a wealth of nonlinear behavior, when performed in a continuous-flow stirred-tank reactor, and subjected to periodic light irradiation, is studied as an experimental example of entrainment phenomena observable in biological systems. The adaptation patterns under periodic light irradiation are elucidated by means of the response of the system to continuous and single-pulse light irradiation. It is shown that self-oscillating states, excitable steady states and bistable systems can exhibit the same types of synchronization patterns when submitted to periodic external forces with appropriate amplitude and time scale conditions.     

Structural organization of the normal and anoxic heart of Scyllium stellare

Summary The general and ultrastructural organization of the heart of the elasmobranch, Scyllium stellare, was studied in normal and in anoxic animals. The rich coronary supply was revealed three-dimensionally by the use of corrosion casts, showing a thebesian system of coronary arterioles and capillaries in the thin, outer compact layer as well as in the predominant, inner spongy layer of trabeculae.Only the sinus venosus received a neuronal input of large bundles of granule-containing axons terminating at fenestrated regions of the endocardium and suggesting a neurohormonal function.A simple, tubular sarcoplasmic reticulum with flattened junctional cisternae was present in myocardial cells of 1–5 m diameter, which contained one or two bundles of myofibrils. The latter were closely apposed to the inner aspect of the plasmalemma. Mitochondria were located centrally in the cells, which were joined by unfolded desmosomes involving Z-band material.Long periods of anoxia were tolerated without loss of heart function, but at the expense of cytoplasmic glycogen. Lipid granules were abundant in all layers and chambers, notably in animals prepared in the summer. The lipid granules displayed a marked increase in electron density when the heart was incubated in a buffered oxalate solution prior to fixation. A glycogen-sparing effect of the lipids during anoxia was observed.     

The differentiation of glial cells and glia limitans in organ cultures of chick spinal cord

Summary Differentiation of glial cells and the glia limitans in organ cultures of chick spinal cord explanted at early neural tube stages, alone or with adjacent tissues, was studied by electron microscopy. Oligodendrocytes and astrocytes comparable to those seen in the chicken in vivo were observed, mainly in areas of good neuronal differentiation. A glia limitans with basal lamina, comparable to that in vivo, was found when spinal cord was bordered by normally adjacent tissues. When it was surrounded by vitelline membrane only, a characteristic limiting layer of glial processes, but no basal lamina, was seen. Contact with a filter membrane (Millipore) elicited excessive differentiation of glial filaments and modified cell fine structure; no glia limitans was formed. Supported by Grant 5 RO 1 NB 0637 from the United States Public Health Service.     

Light and electronmicroscope study of one of the systems of centrifugal fibers found in the lamina of muscoid flies

Summary By combining the Golgi and the electronmicroscope techniques it has been possible to identify accurately the system of centrifugal fibers which arborizes in the lamina of muscoid flies forming the so-called nervous bags. Each of them originates from a single fiber entering the lamina at the site in which the second order and the long visual fibers leave it. This single fiber represents the peripheral portion of a T-shaped trunk stemming from a small neuronal body located in the external region of the medulla. The central branch terminates within the first synaptic field of this visual center.After entering the lamina the centrifugal fiber ramifies profusely and its branches can be seen climbing and synapsing on the surface of the photoreceptor axon endings. The synaptic loci show characteristic synaptic ribbons located within the nervous bag fibers. This fact suggests that direction of conduction is from the medulla to the lamina. This study has also revealed that the intramedullar terminals of the centrifugal fibers establish intimate contacts with one of the two second order fiber endings.This work was supported by Grant No. 618–67 (Mod No. 67–0618) of the Office of Aerospace Research, United States Air Force and by NIH grant NSO 866901.     

An argyrophil fibrillar system and amitotic nuclear division in pars intermedia cells of the rainbow trout (Salmo irideus)

Summary Argyrophil fibrils were revealed in pars intermedia chromophobe cells of Salmo irideus. For the light microscopical demonstration of the fibrils a recently developed copper sulphate-silver protein technique for Bouin-fixed hypophyses was used.The fibrils, apparently belonging to one fibrillar system, are found only in limited regions of the cytoplasm. They occur in ring-, loop-, or cracknel shapes in the close vicinity of the rounded nucleus in the chromophobe cell, and continue in linear shape into the cell base or cell apex where they may end near the adjacent basement membrane.Electron microscopically, the fibrils are composed of filaments, 70–90 Å in diameter, which are arranged in parallel. Bundles of filaments are frequently found near the nucleus. In addition to perinuclear filaments, bundles of filaments with tiny offshoots occur in different areas of the cytoplasm, extending sometimes as far as the cell membrane which is located near the basement membrane of the pars intermedia epithelium.In cells with a bi- or trilobular, apparently amitotically dividing, nucleus a fibrillar loop or ring usually surrounds the interlobular part(s) of the nucleus. This relation of the fibrils to the dividing nucleus and their consisting of regularly arranged filaments indicates the significance of the fibrillar system in the chromophobe cell. It is thus suggested that the fibrillar system is involved in the constriction of the nucleus during amitosis.The number of chromophobe cells in which fibrils are light microscopically visible varied greatly among the rainbow trout used.Miss M. C. Wentzel is thanked for her technical assistance, Mr. M. Veenhuis for his advice and excellent help with the preparation of the electron micrographs, and Mr. L. Hoekstra for preparing the final drawings. Mr. N. J. Williams' corrections to the English are gratefully acknowledged.