L. Austin

SYNAPTOSOMAL PROTEIN SYNTHESIS IN A CELL‐FREE SYSTEM*

—Synaptosomes were isolated from cerebral cortex of young rats and incubated with 14C‐labelled l‐leucine in vitro. Amino acid incorporation into proteins of the synaptosomal cytoplasm, mitochondria and membrane components was observed. There was no incorporation into proteins of the vesicles. The protein‐synthesizing system was not stimulated by the addition of either ATP or an ATP‐generating system. ATP at all concentrations was inhibitory.

PROXIMO‐DISTAL TRANSPORT OF [14C]NOR‐ADRENALINE AND PROTEIN IN SYMPATHETIC NERVES

Abstract— —Both [14C]noradrenaline and [14C]leucine were injected into the coeliac ganglia of cats in an attempt to label the noradrenaline and protein of the granular vesicles, so that their movement in the splenic nerves could be followed.

Transport of proteins and ribonacleic acid along nerve axons

The distribution of isotope in the sciatic nerve was determined at times up to 22 days after injection of (a) [14C]leucine, or (b) [3H]orotic acid, in the spinal cord of the chicken. The results indicate a distal flow of proteins, RNA precursors and perhaps RNA along the nerve axon.

AN AUTORADIOGRAPHIC STUDY OF THE FLOW OF PROTEIN AND RNA ALONG PERIPHERAL NERVE

Abstract— Bath chicken spinal cord and the posterior chamber of the rat eye have been injected with either [3H]leucine or [3H]orotic acid. The movement of newly synthesized protein and RNA down the sciatic and optic nerves was followed by means of autoradiography. Protein moved down both nerves by axoplasmic flow, the protein being confined to intra‐axonal spaces. RNA appeared to move at about the same rate as protein, but much of the newly formed RNA appeared in structures outside the axons, particularly in Schwann and glial cells. There was, however a considerable proportion of the RNA inside the axons. This RNA may be involved in the direction of protein synthesis at sites along the axonal processes.

Some physical properties of bovine adrenal medullary dopamine -hydroxylase.

(1) Dopamine, β‐hydroxylase (EC 1.14.2.1) was purified from bovine adrenal medullae according to the method of Foldes, Jeffrey, Preston and Austin (1972).

THE BLOCKAGE OF AXOPLASMIC FLOW OF PROTEINS BY COLCHICINE AND CYTOCHALASINS A AND B

Abstract— Colchichine blocks axoplasmic flow of proteins in chicken sciatic nerve. The slow component is more effectively blocked than the fast. The colchicine effect on slow flow is independent of the time delay between colchicine injection and that of the [14C]‐leucine used to measure flow, over a period extending from 2 h after to 9 days before the leucine. It is still effective, but to a lesser degree, after a period of 27 days. There is little effect on the fast component when the colchicine is administered after leucine. When given before leucine the effect is not pronounced up to a time interval of 1 day. Maximum blockage was obtained with longer intervals of up to 27 days. These results are discussed in relation to the involvement of the microtubules in both slow flow and rapid flow.

IN VIVO SYNTHESIS OF STABLE RNA WITHIN THE RAT NODOSE GANGLIA FOLLOWING VAGOTOMY

Abstract— We have investigated the in vivo synthesis of stable RNA in the rat nodose ganglia following unilateral vagotomy. Incorporation of precursor into rRNA, and possibly tRNA, in the injured ganglion increases rapidly after vagotomy, reaching twice the normal value at 2 days post‐crush. In contrast to this, incorporation within the uninjured colateral ganglion decreases to about 65% of normal values after 4 days. But, by 7 days, both ganglia appear to be returning to their normal levels. One phase of DNA synthesis is detected in the injured ganglion, at days 1 and 2. while two phases are found in the contralateral ganglion, at days 2 and 34.

The In Vitro Synthesis of RNA within the Rat Nodose Ganglion Following Vagotomy

Abstract: This report describes the application of an in vitro labelling procedure for the evaluation of changes in the uptake and incorporation of tritiated nucleotides into RNA of the rat nodose ganglion following crush injury of the cervical vagus nerve. Significant changes in the incorporation into 28S, 18S and 4S RNA were observed at 3 and 9 days after injury which confirms and extends our previous in vivo observations where [32P]orthophosphate was used as the precursor. An early stimulation in the uptake of nucleotides, which was maximal at 2 days after injury, was also observed. Evidence is presented which indicates that this data reflects a real increase in RNA synthesis within the injured tissue concomitant with an increase in the uptake of nucleotide precursors which may reflect an increase in the nucleotide pool size. The transient nature of the rRNA synthetic responses and their occurrence prior to the peak of the chromatolytic changes suggest that there may be a shift in the distribution of ribosome types resulting in qualitative changes in protein production rather than an overall increase in protein synthesis resulting from an increased ribosome population.

SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE AND INHIBITORS IN THE HIPPOCAMPUS AND CAUDATE NUCLEUS IN SHEEP BRAIN

—The enzyme dopamine-β-hydroxylase (EC 1.14.17.1) which converts dopamine to noradrenaline was found to be present in substantial amounts in sheep brain hypothalamus and caudate nucleus and was located to the synaptic vesicle fractions in these two brain regions by subcellular fractionation. This dopamine-β-hydroxylase was associated with paniculate matter in these two brain regions since it was resistant to solubilization with butan-1-ol and 0.1% Triton X-100. As highly significant levels of dopamine-β-hydroxylase were present in the caudate nucleus, factors other than a simple lack of this enzyme must operate to maintain the low levels of noradrenaline and high levels of dopamine in the caudate nucleus. Purified adrenal dopamine-β-hydroxylase was substantially inhibited by two factors prepared from sheep brain hypothalamus and caudate nucleus. These were found to be cupric ions and a sulphydryl inhibitor. High levels of the sulphydryl inhibitor of dopamine-β-hydroxylase were found in synaptosomal fractions from sheep brain hypothalamus and caudate nucleus and the levels were comparable in both regions. Upon subfractionation of a synaptosome-containing fraction from the hypothalamus, the inhibitor was located predominantly in the soluble fraction, although there were significant levels in the synaptic vesicle fraction. Therefore, the sulphydryl inhibitor must be considered as a possible regulator of dopamine-β-hydroxylase activity. Free cupric ion concentrations as low as 2·5 μM were found to inhibit purified adrenal dopamine-β-hydroxylase in vitro and the concentration of copper in the soluble tissue component of hypothalamus and caudate nucleus was well above this minimal copper concentration. The percentage content of soluble copper in the caudate nucleus was significantly higher than in the hypothalamus. The importance of the soluble to particulate-bound ratio of copper in brain was shown in studies of the developing rat brain. A rapid increase in the level of copper in brain was found in the first 4 weeks but the level was constant by 2 months of age. The percentage of soluble copper, however, was maximal soon after birth and had declined to a constant figure by 2 months of age. A scheme for the regulation of dopamine-β-hydroxylase activity involving these factors is proposed.

IN VIVO SYNTHESIS OF RAPIDLY-LABELLED RNA WITHIN THE RAT NODOSE GANGLIA FOLLOWING VAGOTOMY

Abstract— The synthesis of rapidly‐labelled RNA was studied in the nodose ganglia following unilateral vagotomy. Early changes were detected in the electrophoretic patterns of RNA from both ganglia following a crush of the right vagus. The right ganglionic response is characterized by two phases, the first involving rapid processing of rRNA with associated changes in ‘message‐like’ RNA (mlRNA). A second change, detected by 14 days, appears to involve an increase in the heterodispersity of mlRNA and decreased processing of rRNA. The left ganglion follows a very similar response to that of the right with the exception of the changes in rRNA. However, the left response lags behind that of the right by at least 1 day. We conclude that extensive changes in gene expression are occurring in both ganglia. The similarity of the responses of both ganglia suggests that axon regeneration (in the right) and collateral sprouting (in the left) may produce analogous changes in gene expression, but not in rRNA synthesis.