Peter L. Jeffrey

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).

Antigenicity of isolated synaptosomal membranes

Abstract An antiserum has been prepared against an isolated synaptosomal membrane preparation obtained from chick forebrain. Immunohistochemical localization studies showed that the antiserum reacts with nerve terminal membranes and preterminal axonal membranes in the central nervous system. These antigens were not detected in extracts of non-neural tissues or in the perikarya of neurons. We suggest that some components of the synaptic membrane may be derived from the preterminal axonal membrane.

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.

Distribution of Thy-1 in the avian nervous system: Immunohistochemical and absorption analyses with a monoclonal antibody

A monoclonal antibody against chicken Thy-1 has been used to study the histochemical localisation of Thy-1 in chicken nervous and lymphoid tissues and to quantitate the relative amounts of Thy-1 in different brain subregions, subcellular fractions and non-neural tissues. An indirect ELISA using chicken brain membranes as a target established that the highest levels of Thy-1 were present in chicken forebrain, followed by midbrain, brainstem, spinal cord, cerebellum, retina and sciatic nerve. Analyses of subcellular fractions of chicken forebrain revealed a generalised localisation of Thy-1 on membranes comprising both the junctional and extrajunctional components of the synaptosome. Consistent with this finding are the immunohistochemical studies on cryostat sections where Thy-1 was localised to certain axonal and synaptic regions of chicken nervous tissue. Strong monoclonal antibody binding was found in the molecular layer and white matter of chicken cerebellar sections with fibrous staining on the axons running through the granule cell layer. No continuous staining could be seen on the perikaryal membranes of Purkinje cells or granule cells and no staining was present within the cell bodies. The Bergmann glia of the cerebellum were Thy-1-negative. The monoclonal antibody showed preferential binding to the inner plexiform and optic fibre layers of the chicken retina, suggesting a retinal ganglion cell localisation for chicken Thy-1, as has been suggested for the rat and mouse homologues. Surprisingly the lymphocytes of both the bursa and thymus gland were Thy-1-negative, however some extracellular staining was observed of interlobular connective tissue of the bursa.

THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum.

The metabolic turnover of the major proteins of the postsynaptic density

We have used the method of Austin, Lowry, Brown and Carter, to measure the steady-state metabolic half-life of tubulin (alpha and beta individually) and actin (beta and gamma together) in the total cytosolic (S3), microsomal (P3), synaptic plasma membrane (SPM) and synaptic junction (SJ) subcellular fractions from 6-day-old and adult chicken forebrain. In the SPM and SJ fractions we also measured the steady-state metabolic half-life of the major postsynaptic density protein (mPSDp). In SPM and SJ fractions from 6-day-old chickens tubulin and actin turned over approximately twice as slowly (t1/2 approximately equal to 24 days) as tubulin and actin in the S3 fraction (t1/2 approximately equal to 13 days). This difference was unlikely merely to be due to association with membranes since the t1/2 values for the proteins were the same in P3 and S3. The estimated t1/2 values for mPSDp were similar to that for tubulin and actin in SPM and SJ fractions. Similar results were obtained in adult chickens except that all t1/2 values in all fractions were approximately 30% larger. The calculated t1/2 values did not change between labelling periods of 4 and 6.5 h suggesting that the lag phase of incorporation of newly synthesized PSD proteins is sufficiently rapid to not produce this result artefactually. When the brain from a non-labelled chicken was homogenized in the presence of the S3 fraction from a labelled chicken and sub-fractionated the relative specific activities of the SPM and SJ fractions produced were 1-2% of those from the labelled brain. These results support the notion that tubulin and actin are intrinsic components of the PSD.

Bilateral enzymic changes in rat nodose ganglia following unilateral cervical vagotomy.

Abstract Aspects of the metabolism of the nodose ganglia of the rat and their relationship to chromatolysis induced by unilateral cervical vagotomy were examined. The development of chromatolysis in the vagotomized ganglion followed a typical pattern. The changes in nucleic acid metabolism were elucidated by measurement of the activity of the pyrimidine biosynthesis specific enzyme: glutamine dependent carbamoyl phosphate synthetase. The activity of this enzyme showed a biphasic response supporting the conclusion that the first rapid increase in the rate of synthesis of ribosomal RNA may be a result of increased activity of the de novo pyrimidine biosynthetic pathway; a biphasic response also occurred in the enzyme activity in the contralateral ganglion. Lysosomal activity during the response was investigated by measuring the activity of acid phosphatase. There was also a bilateral change in the acid phosphatase activities of the two ganglia. This was more marked in the vagotomized ganglion, but followed a biphasic time response. The bilateral nature of the response observed in enzyme activities may reflect the similarities in metabolic requirements of axon regrowth in the vagotomized ganglion and axon collateral growth in the contralateral ganglion.

Effect of denervation on sarcolemmal proteins and glycoproteins of fast and slow mammalian skeletal muscle

We compared the protein and glycoprotein composition of a sarcolemmal membrane fraction isolated from normal and denervated rat extensor digitorum longus (EDL) and soleus muscles. Membranes from EDL and soleus muscles showed significantly different protein compositions. A relatively small number of glycoproteins, which were all minor proteins, accounted for the majority of concanavalin A (ConA) and Ricinus communis agglutinin (RCA120) binding. These glycoproteins appear to be common to EDL and soleus but bound different relative amounts of lectin in the two muscles. A large proportion of the ConA binding sites in EDL, but not soleus, were cryptic (not accessible by ConA unless the membrane structure was disrupted). Denervation had a differential effect on sarcolemma from the two muscles with EDL exhibiting large changes and soleus changing little if at all. Several major proteins changed their relative concentrations after denervation and the relative amount of RCA120 bound to the major glycoproteins also changed. The major ConA-binding glycoproteins did not change in either membrane but denervation resulted in the exposure of most of the cryptic ConA-binding sites in EDL membranes. Endogenous sialyl- and galactosyl-transferase activities in the membrane fractions significantly increased in EDL, but did not change in soleus, suggesting that the turnover of the glycoproteins is increased in EDL after denervation.

The developmental appearance of Thy-1 antigen in the avian nervous system

A monoclonal antibody against chicken Thy-1 glycoprotein was utilised in an indirect binding assay and an immunohistochemical technique to investigate the developmental appearance of Thy-1 in the chicken nervous system. The largest increase of Thy-1 levels in chicken forebrain during embryonic development coincided with the major period of neuronal development and synapse formation but preceded the major period of myelination. Immunohistochemical localisation studies on sections of chicken cerebellum, retina and spinal cord during this period showed that Thy-1 first appeared in regions of tissue which contained differentiated synapses of axons; however, not all such areas were Thy-1 positive. Areas of tissue rich in actively dividing neuroblasts or postmitotic undifferentiated neurons, such as the external granular layer of the cerebellum, showed very little staining. The disappearance of Thy-1-specific staining with age observed in the white matter of sections of cerebellum and spinal cord was due to the masking effect of myelination and not to a loss of Thy-1 from neuronal membranes.

SELECTIVE LABELLING OF TWO PHASES OF AXONAL TRANSPORT OF CHOLESTEROL IN THE CHICK OPTIC SYSTEM

Abstract— In the chick optic system cholesterol is axonally transported in two phases which appear to take their cholesterol from different cellular pools. The intraocular injection of radioactire cholesterol results in the specific labelling of the slow phase which carries cholesterol in the unesterificd form and appears to move at the same rate as the slow phase of protein transport (Rostaset al., 1975). The intraocular injection of radioactive mevalonic acid, a metabolic precursor of cholesterol, results in the preferential labelling of a more rapid phase of axonal transport which also carries cholesterol in the unesterified form and is first detected at the optic tectum 10 h after the injection. It is likely that this rapid phase travels at the same rate as the rapid phase of protein transport and that the delayed arrival at the tectum is due to a lag time in the retina caused by the synthesis of cholesterol and its packaging for transport. Because the individual pools for the two transport phases can be selectively labelled, the retina and optic nerve provide a unique model system in which the metabolic turnover, intracellular compartmentalization and intracellular transport of cholesterol can be studied.