WALLERIAN DEGENERATION IN RABBIT OPTIC NERVE: CELLULAR LOCALIZATION IN THE CENTRAL NERVOUS SYSTEM OF THE S-100 AND 14-3-2 PROTEINS1

Abstract— The S-100 and 14-3-2 proteins, which are found only in nervous tissues, were measured in degenerating rabbit optic nerve at 0, 5 10, 20, 40, 60, 80, 100, 150 and 200 days after unilateral enucleation in order to obtain indications of the cellular localization of these proteins in the central nervous system. S-100 increased and 14-3-2 decreased (both approximately 70 per cent) in cut nerves by 200 days of degeneration. Changes in amounts of the proteins were related to cellular alterations which characterize the degenerative process, as demonstrated by electron microscopy. In uncut nerves (intact eye) from these experimental animals, S-100 increased and 14-3-2 decreased slightly at 5 days, after which time the levels of each returned to those approximating the content in corresponding nerves from unoperated control animals. No appreciable change in total soluble proteins was measured in degenerating or intact nerves. Since S-100 increased and 14-3-2 decreased in the degenerating optic nerve as it became relatively enriched in glial constituents but impoverished in axonal content, it is suggested that S-100 is primarily a glial protein and 14-3-2 predominantly a neuronal protein in the central nervous system.     

Glial and Neuronal Marker Proteins in the Silicone Chamber Model for Nerve Regeneration

Abstract: In the present study, neuronal and Schwann cell marker proteins were used to biochemically characterize the spatiotemporal progress of degeneration/regeneration in the silicone chamber model for nerve regeneration. Rat sciatic nerves were transected and the proximal and distal stumps were inserted into a bridging silicone chamber with a 10-mm interstump gap. Using dot immunobinding assays, S-100 protein and neuronal intermediate filament polypeptides were measured in different parts of the nerve 0–30 days after transaction. In the most proximal nerve segment, all the measured proteins were transiently increased. In the proximal and distal stumps adjacent to the transection, the studied proteins were decreased indicating degeneration of the nerve. Within the silicone chamber, the regenerating nerve expressed the Schwann cell S-100 protein already at 7 days, whereas the neurofilament polypeptides appeared later. These observations are corroborated by previous morphological studies. The biochemical method described provides a new and fast approach to the study of nerve regeneration.     

Levels of myo-inositol in normal and degenerating peripheral nerve

—Free inositol was measured in peripheral nerves of the monkey, rabbit, rat, frog and lobster; levels in mammalian nerve were similar, and two to three times greater than in the other species. Concentrations of myo-inositol in rabbit tibial nerve increased from proximal to distal segments; in optic nerve the concentrations decreased with greater distance from the retina. In the early stages of Wallerian degeneration rabbit tibial nerve contained 25 per cent less free myo-inositol, rat nerve 50 per cent less. Rabbit nerves were analysed at 2 and 5 weeks after section; by 5 weeks levels of myo-inositol had increased to 50 per cent above normal. Similar changes were found in degenerating rabbit optic nerve. The combination of galactose feeding and nerve section resulted in reduction of the myo-inositol in rat sciatic nerve to one-fifth of the control value; galactitol in the nerve decreased by 50 per cent after section. The evidence suggests that myo-inositol in nerve is located mainly in Schwann cells or glia.     

NUCLEAR LOCALIZATION OF S-100 PROTEIN

Abstract— S-100 protein has been found in the nuclei isolated from the brain cortex of rabbit. The nuclear S-100 constitutes a small portion (0.55 per cent) of the S-100 present in the cytosol. Most of the large and pale nuclei appear to contain much more S-100 than the small and dark ones. The nuclear membrane is permeable in vitro to S-100 in presence of divalent cations. Three forms of S-100 occur in subnuclear fractions: free S-100, present in the soluble protein fraction; labile-bound S-100, present in the deoxyribonucleoprotein fraction and stable-bound S-100, present in the residual or‘nucleolar’fraction. The localization of the S-100 in those regions of the nucleus that are most active in RNA synthesis provides basic information for further studies on the possible role of this protein on genomic expression in nervous tissue.     

Changes in the concentration of enolase isozymes and S-100 protein in degenerating and regenerating rat sciatic nerve

Levels of enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) and S-100 protein in rat sciatic nerves were determined during their degeneration and regeneration processes. The sciatic nerves were unilaterally crushed or severed. The rats were killed 1, 2, 6, and 8-9 weeks later, and both the proximal and distal portions of the damaged nerves were dissected. Control samples were obtained from the untreated contralateral hindlimbs. Enolase isozymes and S-100 protein in the nerve segments were determined with the enzyme immunoassay method. The control nerves contained about 40, 90, and 30 pmol/mg protein of alpha alpha, alpha gamma, and gamma gamma enolases, respectively, and 0.85 microgram/mg protein of S-100 protein. These levels were not affected by repetitive electrical stimulation of the nerve fibers in vivo. The levels of the nervous system-specific forms of enolase (alpha gamma and gamma gamma) and S-100 protein decreased markedly within a week in the distal portion of the crushed nerve (alpha gamma, 27 pmol/mg; gamma gamma, 5.5 pmol/mg; S-100 protein, 0.36 microgram/mg) with apparently no change in the concentration of alpha alpha enolase. These levels in the proximal portion of the crushed nerve remained unaltered. The sensory and motor functions impaired by the sciatic nerve crush showed a recovery more or less after 4-9 weeks. This recovery was accompanied by a gradual regaining of the specific proteins in the distal portion of injured nerves (alpha gamma, 64 pmol/mg; gamma gamma, 13 pmol/mg; S-100 protein, 0.63 microgram/mg at the 8-9th week).     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

WATER-SOLUBLE AND PENTANOL-EXTRACTABLE PROTEINS IN HUMAN BRAIN NORMAL TISSUE AND HUMAN BRAIN TUMOURS, WITH SPECIAL REFERENCE TO S-100 PROTEIN

Disc electrophoretic separation of water-soluble and pentanol-extractable protein from normal human brain and human brain tumours (glioblastoma, neurinoma and medulloblastoma) on 10 per cent polyacrylamide gels showed minor differences between tissues. After disc electrophoresis ependymomal tumour cells contained high concentrations of a rapidly migrating anodic protein fraction which was immunologically distinct from S-100 protein. After electrophoresis of normal brain grey matter in a continuous buffer system, a rapidly migrating anodic protein fraction which was immunologically distinct from S-100 protein was found, and this protein fraction had a similar relative mobility to that of ependymomal tumour cells. This protein fraction was present to a low extent in human normal white matter, but absent from neurinoma and glioblastoma. In a continuous buffer system at least two separable protein fractions, immunologically equivalent to S-100 protein, were observed in normal human brain. The more anodic of these two fractions was shown to be present in relatively high amounts in neurinomas, and may be of Schwann cell origin. Additional S-100 protein could be extracted from residual material remaining after removal of water-soluble proteins; 2.8-10 per cent of the water-soluble S-100 in normal material, and 0.1-0.6 per cent of that present in tumour material, was extractable from the water-insoluble residue by pentanol.     

Dynamic Changes of Cerebral-Specific Proteins in Full-Term Newborns with Hypoxic–Ischemic Encephalopathy

The aim of this study was to observe the dynamic changes of serum brain-derived neurotrophic factor (BDNF), S-100B, and Tau proteins levels in full-term newborns with hypoxic–ischemic encephalopathy (HIE) and to discuss their significance in brain damage. Serum samples of 28 full-term newborns diagnosed with HIE and 20 controls were obtained in the first 24 h of life. Another serum samples were also taken, respectively, at 3 and 7 days of life in HIE group. The concentrations of BDNF, S-100B, and Tau proteins were measured by the enzyme-linked immunosorbent assay method. Mean concentrations of BDNF, S-100B, and Tau proteins among different time period and in different grades of HIE group were calculated and compared. Compared with the control group, serum BDNF and proteins S-100B levels in HIE group were significantly elevated in 24 h after birth (P < 0.05) and their concentrations were also significantly higher among patients with mod-severe HIE compared to those with mild HIE at 24 h and 7 days after asphyxia (P < 0.05). Regardless of whether mod-severe HIE or mild HIE, there were no significant difference of serum BDNF and proteins S-100B among the three different time periods. There was no difference in Tau protein levels between HIE group and control group, also no difference between mod-severe HIE group and mild HIE group. BDNF and proteins S-100B are up-regulated early in asphyxia neonates with HIE; and the released amount of BDNF and proteins S-100B from nerve center system correlate with the extent of encephalopathy.     

CHANGES IN CENTRAL NORADRENALINE NEURONSADMINISTRATION AFTER SYSTEMIC 6-HYDROXYDOPAMINE ADMINISTRATION

—Intravenous injection of a large dose of 6-hydroxydopamine (100 mg/kg) to adult rats caused a significant and long-lasting reduction (about 30 per cent) of the in oirro uptake of [³H]NA in the cerebral cortex and spinal cord, while no changes were seen in the hypothalamus. The endogenous NA in whole brain was similarly reduced (about 20 per cent). Fluorescence histochemistry revealed catecholamine accumulations which are degenerative signs, induced by 6-hydroxydopamine, in axons of the dorsal NA bundle innervating the cerebral cortex. It is concluded that the blood–brain barrier in adult rats is not completely protective with respect to the neurotoxic action of systemically injected 6-hydroxydopamine, which can produce degeneration of a significant number of NA nerve terminals in the cerebral cortex and spinal cord. Previous studies have shown that 6-hydroxydopamine caused a permanent and selective degeneration of a large number of central NA nerve terminals when injected systemically up to 1 week after birth, due to an incompletely developed blood-brain barrier. This barrier for 6-hydroxydopamine develops between the 7th and 9th day after birth (Sachs , 1973). In the present study 6-hydroxydopamine was found to cause a small transient reduction in [³H]NA uptake in cerebral cortex of rats between 9 and 28 days of age, while in older rats the damage produced by 6-hydroxydopamine was long-lasting. Thus, the NA nerves ascending to the cerebral cortex seem to possess a regenerative capacity to a 6-hydroxydopamine-induced degeneration up to about 28 days postnatally, but which later disappears or is markedly retarded.     

Changes in carbohydrates,amino acids and proteins in developing seed of chickpea

Developing seeds of chickpea cultivars G-130, L-550 and 850-3/27 grown under field conditions were sampled at different stages of maturity and analysed for soluble sugars, starch, soluble nitrogen, protein nitrogen and amino acids. Fr. wt of seeds of all three cultivars decreased after 28 days of flowering while the dry wt continued to increase. Rapid starch accumulation was observed between 14 and 28 days after flowering. Starch as per cent of seed dry wt started to decrease after 28 days, while starch per seed increased till maturity. The percentage of salt-soluble proteins decreased with maturation of seed. The electrophoretic pattern revealed that deposition of seed storage protein in cotyledons occurred 14 days after flowering. Most of the biochemical activity apparently occurred between 14 and 28 days after flowering.     

TRANSPORT OF 14C INCORPORATED PROTEIN IN THE CORTICOSPINAL TRACT OF THE RAT BRAIN

Abstract— The origin of fibres of a corticospinal pathway in rat brain was located by cortical ablation and Marchi staining and also by electrical stimulation of the motor cortex.
Enzyme changes investigated histochemically over 0–14 days post-injection of 5 μ10.15 m NaCl into the neocortex indicated that very little apparent disturbance of nerve cell metabolism beyond a narrow band adjacent to the path of the microneedle within the cortex had occurred. [¹⁴ C]Leucine as a precursor of protein synthesis was used to study incorporation of the amino acid into protein. At the site of injection the maximum level of labelled protein was recorded at 30 min post-injection, the level decreasing to less than 2 per cent of this at 6 hr.
The subsequent axonal flow of labelled protein along the corticospinal pathway was investigated during the period 15 min to 21 days post-injection. Within 24 hr increasing amounts of labelled proteins were measured caudally, but not more than 6 per cent had migrated beyond 5 mm from the site of injection. At 3 days this percentage had increased to 14.6 per cent, the labelled proteins being distributed in progressively decreasing amounts to a further 13 mm caudal. Very little change from this position was seen during the following 18 days.     

PURIFICATION AND SOME PROPERTIES OF S-100 PROTEIN FRACTIONS FROM SHEEP AND PIG BRAINS

Abstract— The S-100 protein fraction of pig and sheep brain was purified in 40 per cent yield by a modification of the procedure of M oore (1965), which avoided selective loss of S-100 components. The S-100 fraction of both pig and sheep is a mixture of proteins as indicated by acrylamide gel electrophoresis and N -terminal amino acid analysis. Differences in amino acid composition, electrophoretic heterogenity and N -terminal analysis were found.
One fraction (fraction A) was isolated by DEAE-Sephadex chromatography from pig brain S-100 protein fraction. It was considered to be a single protein since it migrated as a single band on acrylamide gel electrophoresis and showed a single symmetrical peak during ultracentrifugal analysis. Only one N -terminal amino acid was detected in fraction A. The amino acid composition of this fraction showed minor but significant differences from that of the complete S-100 protein fraction from pig brain. The S-100 protein fraction of both species, as well as fraction A, had similar s _{20, w} values and similar molecular weights (about 20,000) as indicated by gel filtration. These results, together with the immunological data obtained by other authors, suggest that the proteins of the S-100 fraction are closely related; the heterogeneity of the S-100 protein fraction may be of the same type as the lactate dehydrogenase isoenzymes.     

Induction of rat E and chicken A-I apolipoproteins and mRNAs during optic nerve degeneration

Apolipoprotein synthesis was measured in control optic nerves and optic nerves undergoing Wallerian degeneration. After short term organ culture with radiolabeled amino acid, optic nerve extracts were reacted with antiserum to rat or chicken apolipoproteins. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the degenerating rat optic nerve, apo-E synthesis increased from 0.30 to 0.90% of newly synthesized protein and from 0.45 to 1.4% of secreted protein. A DNA-excess solution hybridization assay was constructed to measure the absolute amount of apo-E mRNA in control and degenerating optic nerves. Paralleling the increase in apo-E protein synthesis, the absolute amount of apo-E mRNA was elevated 3- to 4-fold after enucleation. Similar to rat apo-E, apo-A-I synthesis was increased in degenerating chicken optic nerve. Chicken apo-A-I represented 0.65 and 3.5% of newly synthesized protein from control and enucleated optic nerves, respectively. Apo-A-I increased from 0.85 to 5.5% of secreted protein following enucleation. Using in vitro translation to quantitate relative amounts of chicken apo-A-I mRNA, enucleated optic nerve apo-A-I mRNA content was increased 5-fold. These results suggest that local apolipoprotein synthesis may be involved in the mobilization of myelin cholesterol which occurs during Wallerian degeneration. The similar response of the rat and chicken to increase optic nerve apolipoprotein synthesis during degeneration supports the idea that avian peripheral apo-A-I and mammalian peripheral apo-E may be performing functions common to both classes of animals.     

S-100 protein in Schwann cells of the developing human peripheral nerve

Summary From approximately 7 weeks gestational age in developing human peripheral nerve, as in adult nerve, S-100 protein was found to be expressed solely and uniformly by Schwann cells associated with axons. In embryos younger than 7 weeks S-100 was much less constant and many cells did not show clear staining. The trigger for the initial appearance of the protein at around this age remains unclear although a relationship of S-100 expression in Schwann cells to close axonal contact is suggested. The value of S-100 protein in distinguishing Schwann cells from perineurial cells in normal nerves and nerve sheath tumours remains unclear.     

S-100-Mediated Inhibition of Brain Protein Phosphorylation

The effects of the glial-specific, calcium-binding, S-100 protein on brain membrane and supernatant protein phosphorylation were assessed. S-100 concentrations as low as 5 micrograms/ml caused a marked inhibition of the phosphorylation of a soluble brain protein having a molecular weight of 73,000 daltons (73K). This protein was designated the S-100 protein-modulated phosphoprotein (SMP). Half-maximal inhibition of the phosphorylation of SMP by S-100 was obtained at concentrations of 12 micrograms/ml (0.57 microM). The inhibition of SMP phosphorylation by S-100 was calcium-dependent, with a calculated calcium Ka of 2.0 +/- 0.3 microM. SMP phosphorylation was also inhibited by calmodulin, but only partially and with a much lower potency. The inhibition of SMP phosphorylation by S-100 was not inhibited by fluphenazine, whereas the effect of calmodulin was. SMP was found in many brain areas, with the highest levels seen in the corpus callosum. Various peripheral tissues, such as kidney; liver; and pineal, pituitary, and adrenal glands, did not contain detectable SMP levels. At higher S-100 concentrations, greater than 10 micrograms/ml, the phosphorylation of several other soluble proteins was markedly inhibited. These proteins have molecular weights of 56K, 50K, and 47K. The phosphorylation of these proteins was enhanced by calmodulin. These data suggest that the S-100 protein may function to modulate the phosphorylation of brain proteins in a manner analogous to (although in a reciprocal fashion) that of calmodulin.     

Changes in CLIP3 expression after sciatic nerve injury in adult rats

CLIP3 (cytoplasmic linker protein 3) is a 547 amino acid residue cytoplasmic protein that localises to Golgi stacks and tubulovesicular elements juxtaposed to Golgi cisternae. Composed of three Ank (ankyrin) repeats and two CAP-Gly (cytoskeleton-associated protein-glycine) domains, CLIP3 may function as a cytoplasmic linker protein that is involved in TGN–endosome dynamics. To define the expression and role of CLIP3 during peripheral nervous system degeneration and regeneration, we created an acute sciatic nerve injury (SNI) model in adult rats. Western blot analyses revealed prominent up-regulation of CLIP3 and PCNA (proliferating cell nuclear antigen) protein levels at 3?days after SNI. Immunohistochemistry displayed that the expression of CLIP3 was noticeably increased in the injured nerve. Immunofluorescence further revealed that the CLIP3 and PCNA proteins colocalised respectively with S100 in the cytoplasm of Schwann cells. The expression profile of the SC/neuron co-cultures demonstrated that CLIP3 and PCNA protein levels were markedly expressed during the early stage of myelination. These results suggest that CLIP3 is likely associated with the myelination of proliferating Schwann cells, and nerve tissue regeneration after peripheral nerve injury. CLIP3 and PCNA expression during early myelination may be related to the direct uptake and transport of lipids and cholesterol, which were derived from the degenerating myelin, by Schwann cells to prepare for the formation of myelin sheath-like structures around regenerated axons after SNI.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING PROTEINS IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

—The redistribution of rapidly migrating [³H]leucine-labelled proteins was studied using double ligatures applied to the vagus nerve and single ligatures, applied to the hypoglossal nerves. Rapidly migrating proteins accumulating for 16 h proximal to a distal ligature of the cervical vagus redistributed to give a retrograde accumulation distal to a second ligature. Within 6 h a substantial redistribution occurred indicating a rapid retrograde transport. After 21 h there was a further accumulation with 70 per cent of the labelled material accumulating at the distal end of the isolated nerve segment and 16 per cent accumulating at the proximal end. It was shown that about a half of the retrograde accumulation was dependent on the distal accumulation zone. Rapidly migrating proteins accumulated distal to a ligature applied to the hypoglossal nerve 16 h after labelling of the nerve cell bodies indicating that a rapid retrograde transport of labelled macromolecules occurs from the peripheral parts of the nerve in the tongue. Labelled proteins accumulated proximal to ligatures and transections of both the hypoglossal and vagus nerve when applied 16 h after labelling of the nerve cell bodies, indicating the presence of axonal proteins, migrating at a rate of transport intermediate to that of rapidly and slowly migrating proteins.     

Rapid transport of protein in the optic system of the goldfish

Abstract— Several amino acids, particularly [³H]proline and [³H]asparagine specifically and efficiently labelled rapidly transported proteins in the goldfish optic nerve and tectum after intraocular injection. Studies with these amino acids showed that the rapidly transported proteins moved as a discrete band at a rate which was temperature-dependent, and was equal to 70-100 mm per day at 20°C. Transported protein in the optic tectum was 80 per cent particulate and was found in synaptosomal, mitochondrial, and myelin fractions, but not in purified nuclei or ribosomes.     

Immunocytochemical localization of S-100 protein in astrocytes and Müller cells in the rabbit retina

Summary The localization of S-100 protein was studied in histological sections of retinae from adult rabbits. By use of double-immunolabeling techniques it was shown that most but not all radially oriented vimentin-positive Müller cells were co-labeled by an antiserum to S-100 protein. Glial fibrillary acidic protein-positive astrocytes, which in the rabbit retina are restricted to the medullary rays formed by myelinated optic nerve fibers, consistently showed S-100 protein immunoreactivity. The present report shows that, with respect to S-100 protein staining, Müller cells represent a heterogeneous population of glial elements.     

EFFECTS OF AMPHETAMINE ADMINISTRATION IN VIVO ON IN VITRO PROTEIN SYNTHESIZING SYSTEM FROM RAT BRAIN 1

Abstract— A highly active in vitro protein synthesizing system (S-28) has been prepared from rat brain. Poly (U)-dependent [³H] phenylalanine incorporation by brain S-28 system is significantly inhibited by D-amphetamine. The extent of inhibition by amphetamine is significantly higher than by other biogenic amines such as dopamine and serotonin. At the 100°g level of amphetamine, the inhibition is about 70°. Experiments with ribosomes and soluble enzymes from control and amphetamine-treated systems indicate that the observed inhibition may be due to the effect of the drug on the ribosomes. Kinetic analysis of the reaction mixture in the presence as well as absence of D-amphetamine indicate that this sympathomimetic drug inhibits polysome formation in vitro.