Posttranslational Protein Modification by Polyamines in Intact and Regenerating Nerves

A 150,000-g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N'-dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase-mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N'-dimethylcasein) showed labeling of 18-, 46- and 200-kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein-bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.     

The role of nerve lysosomal enzymes in the pathogenesis of denervation atrophy. Electromyographic and histochemical study in rats

Section of sciatic nerves of rats produced fibrillations within 3 days. Foci of hyalination leading to necrosis corresponded to segments of muscles containing end plates. The electrolyte content, mainly Ca, was increased, NADH2-TR activity was decreased and membrane ATP-ase was increased. The known increase in hydrolytic enzyme activities in denervated muscles was due to spilling of lysosomal enzymes from degenerating axons at the myoneural junction. This explains the discrepancy between morphological studies indicating paucity of lysosomes in normal muscles and the high hydrolytic enzyme activities in denervation. We propose that denervation changes are at least partly due to the effect of lysosomal spillage from degenerating axons.     

The role of nerve lysosomal enzymes in the pathogenesis of denervation atrophy

Summary Section of sciatic nerves of rats produced fibrillations within 3 days. Foci of hyalination leading to necrosis corresponded to segments of muscles containing end plates. The electrolyte content, mainly Ca, was increased, NADH₂-TR activity was decreased and membrane ATP-ase was increased.The known increase in hydrolytic enzyme activities in denervated muscles was due to spilling of lysosomal enzymes from degenerating axons at the myoneural junction. This explains the discrepancy between morphological studies indicating paucity of lysosomes in normal muscles and the high hydrolytic enzyme activities in denervation. We propose that denervation changes are at least partly due to the effect of lysosomal spillage from degenerating axons.     

COMPARISON OF THE BEHAVIOUR OF A SOLUBLE AND A MEMBRANE-BOUND ENZYME IN TRANSECTED PERIPHERAL NERVES

Phosphoglucoisomerase (PGI), a soluble enzyme, and AChE, a membrane-bound enzyme were studied in transected peroneal nerves of dog and in isolated segments of these nerves. Although activities of both enzymes increased at the ends of transected nerves, marked differences in their behaviour were observed. The increment in AChE activity was much sharper than that of PGI and continued to grow with time whereas the increase in PGI developed fully within the initial hours after transection and did not change thereafter. In an isolated nerve segment AChE accumulated at both ends with a concomitant decrease in the middle part, whereas changes in PGI activity appeared only in the terminal parts, the rest of the nerve remaining at the normal level. The terminal increase of PGI did not, contrary to that of AChE, depend on the length of the isolated segment. The changes in PGI activity may be features of a local peritraumatic reaction whereas those of AChE indicate involvement of the whole segment along which the enzyme containing organelles are transported.     

Axonal transports of tripeptidyl peptidase II in rat sciatic nerves

Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12-72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Localization of acid phosphatase in the principal cells of the guinea pig epididymis

The activity of acid phosphatase in the principal cells of the guinea pig epididymis was studied histochemically. The enzyme activity was localized in the Golgi and apical regions in segments 1-4. In segments 5-7, the enzyme activity was distributed throughout the entire supranuclear cytoplasm. There was a gradual increase of acid phosphatase activity from segments 1-7. A possible function of acid phosphatase in the epididymis is discussed.     

Anterograde axonal transport of endopeptidase 24.15 in rat sciatic nerves

Axonal transport of endopeptidase 24.15 (EP24.15), a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. At 48h after ligation, a significant amount of the axonal transport of EP24.15 activity was found in the proximal segment, while axonal transport of deamidase activity, a lysosomal enzyme, increased in both proximal and distal segments. Western blot analysis of EP24.15 showed that EP24.15 immunoreactivity in the proximal segment was 1.8-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in the immunoreactive EP24.15 in the proximal segment in comparison with that in the middle segment. In the distal segment, no axonal transport of EP24.15 was found in all methods examined, indicating that EP24.15 is mainly transported by an anterograde axonal flow. These observations suggest that EP24.15 may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Stimulated Release of Phosphatidylinositol Phosphodiesterase in an Isolated Phrenic Nerve-Diaphragm Preparation

Electrical stimulation of the phrenic nerve in an isolated nerve-diaphragm preparation resulted in the release of phosphatidylinositol phosphodiesterase into the organ bath. The released enzyme was Ca2+-dependent and exhibited two pH optima. The enzyme was released in response to nerve stimulation even in the presence of d-tubocurarine in concentrations that block neuromuscular transmission, and was not therefore released from the muscle as a consequence of its contractile activity. Phosphatidylinositol phosphodiesterase activity was determined in the soluble cytosol fractions prepared from different regions of skeletal muscles and from normal peripheral nerves and nerves that were degenerating after transection. The specific activity of the enzyme in the cytosol from the endplate-rich region of the diaphragm was significantly greater than that in cytosol from either the endplate-free region of the diaphragm or from the phrenic nerve. In degenerating nerve the activity of the enzyme was greater in the distal stump than in the proximal stump at 36 h after nerve section. Possible roles for released phosphatidylinositol phosphodiesterase at the neuromuscular junction are discussed.     

Investigations on the Incorporation of a Tritiated Ganglioside, GM1, in the Injured and Intact Sciatic Nerves of the Rat

Following injury of their left sciatic nerves by means of a standardized procedure, male rats received intravenous injections of a tritiated ganglioside. GM1, on different days during the process of regeneration. The rats were killed at two different times after the injection and the concentrations of the total radioactivity, nonvolatile radioactivity, and labelled GM1 were estimated in six segments of the crushed and intact sciatic nerves. The segments of the damaged nerves showed higher concentrations of radioactivity and a higher content of GM1 than the corresponding segments of the contralateral nerves. Within the immediate area of the lesion the highest levels were found on the 3rd and 6th days after the injury; the segments distal from the lesion showed the highest levels of activity on days 9 and 12. The nerve segments proximal to the site of the injury showed a low rate of radioactivity incorporation. The higher concentrations of [3H]GM1 in damaged nerves as well as the rate of incorporation as a function of time indicate that exogenous gangliosides may be involved in the processes of regeneration and have a bearing on the latter.     

Nitric oxide synthase-containing, peptide-containing, and acetylcholinesterase-positive nerves in the cat lower oesophagus

Summary The innervation of the cat lower oesophagus, including the lower oesophageal sphincter, was studied by enzyme histochemistry, immunohistochemistry, and confocal microscopy. In the lower oesophageal sphincter, and at a level 2 cm above it, no apparent differences were seen in the nerve distribution pattern. Among the nerve populations studied, acetylcholinesterase (AChE)-positive nerves were the most abundant in both these regions. The density of AChE-positive nerves was particularly marked in the circular muscle layer. A rich supply of nitric oxide synthase (NOS)-containing nerves was identified by using an antiserum against neuronal NOS, or by enzyme histochemical staining for NADPH diaphorase activity. Vasoactive intestinal peptide (VIP)-immunoreactive nerves had a similar distribution pattern as NOS-immunoreactive nerves, and nerves displaying immunoreactivity for NOS and VIP often showed profiles coinciding with AChE-positive nerves. As judged by confocal microscopy, immunoreactivities for helospectin, pituitary adenylate cyclase-activating peptide (PACAP) and VIP, to a large extent were found in the same nerves. At a level 7 cm above the lower oesophageal sphincter, the total nerve supply was less than in the sphincter itself and 2 cm above it. Immunoreactivity towards VIP, PACAP and helospectin was also found to co-exist with NOS and neuropeptide Y within the same nerve structures. It is concluded that there is an intricate innervation pattern in the feline lower oesophagus reflecting the complexity in the regulation of its motility.     

Increase in NADH-glutamate dehydrogenase activity by mercury in excised bean leaf segments

Application of Hg to excised bean leaf segments increased the glutamate dehydrogenase (NADH-GDH) activity substantially. However, specific activity of the enzyme decreased at lower concentration of Hg, and increased to lesser extent at higher concentration of Hg. Mercury supply increased the glutamate synthase (NADH-GOGAT) activity also. Mercury supply increased the NADH-GDH activity in the presence of NH4NO3, but to a lesser extent than in the absence of NH4NO3. The specific activity of the enzyme decreased considerably at lower concentration of Hg, but increased significantly at higher concentration of Hg. An increase in NADH-GOGAT activity was observed in the presence of NH4NO3, but specific activity of the enzyme decreased marginally. Increase in GDH activity due to Hg remained unaffected by the supply of sucrose, but was reduced by glutamine and glutathione and enhanced by Al. The glutamate dehydrogenase (+Hg enzyme) from mercury treated leaf segments had higher value of S0.5 for NADH than the enzyme (-Hg enzyme) from material not treated with mercury indicating that Hg binding to enzyme prevented NADH binding to the enzyme possibly at thiol groups. However, + Hg enzyme has more reactivity, as apparent Vmax value was higher for it. It has been suggested that Hg activates the NADH-GDH enzyme in the bean leaf segments by binding to thiol groups of protein and pronounced increase in activity by Hg suggests a possible role of enzyme under Hg-stress.     

Morphological alterations induced by sodium valproate on somites and spinal nerves in rat embryos

The antiepileptic drug valproic acid is a well-known teratogenic agent; its main target organ is the neural tube, though skeletal malformations have also been described. In our recent work, respecifications of vertebrae were described in rat fetuses after treatment with 400 mg/kg of sodium valproate at specific somitogenic stages. The observed malformations were stage-dependent. Morphological segmental respecification was observed at the level of segments in formation at the moment of exposure and at the level of more posterior segments. Recently, specific alterations in the development of cranial nerves and ganglia were described in mouse embryos after in vitro exposure to VPA. The aim of the present work was to analyze dysmorphogenetic effects of VPA on embryonic metameric structures: somites, spinal and cranial nerves, and ganglia. Sodium valproate (400 mg/kg) was subcutaneously injected at specific gestational times corresponding to embryonic stages: presomitic or at about 2, 6, 10, 14, 18, or 22 somites. Females were sacrificed on the day 12 post coitum, and embryos were examined. Morphological examination of somites was performed by staining with acridine orange. Morphological examination of nerves and ganglia was performed by immunostaining, using monoclonal antibodies to the 160-kD neurofilament protein. No abnormalities were observed in the cranial nerves and ganglia. Specific and stage-dependent alterations were observed both at the level of the somites and at the level of the spinal nerves. The following characteristic malformations were observed: fusions, duplications, and reductions of somites and corresponding spinal nerves and ganglia. Our morphological data suggest a morphogenetic action of VPA at the level of the axial segments, with a possible respecification of the identity of the interested segments and their derivatives.     

A Rapid Anterograde Axonal Transport of Carboxypeptidase H in Rat Sciatic Nerves

Abstract: Using the highly sensitive HPLC-fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative pro-hormone processing enzyme that removes a basic amino acid from the C-terminus of a precursor peptide, was measured 12–72 h after double ligations of rat sciatic nerves. CPH-like activity in rat sciatic nerves was 60-fold lower than that in the pituitary gland. CPH-like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is ∼50 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.     

Inhibition of 5-amino levulinic acid dehydratase activity by arsenic in excised etiolated maize leaf segments during greening

In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme.     

Anterograde axonal transport of Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme in rat sciatic nerves: cleavage occurs between basic residues

Axonal transport of Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and peaked 72 h after ligation. The optimum pH for Boc-Arg-Val-Arg-Arg-MCA hydrolyzing enzyme activity was 6.5 to 6.9 and did not require Ca(2+) for the activity. Two molecular forms with enzyme activity were identified by size-exclusion chromatography and the molecular masses of the two enzymes were estimated to be 98 and 52 kDa. Two enzyme activities were strongly inhibited by Hg(2+), Cu(2+) and trypsin inhibitors such as TLCK, antipain and leupeptin. It cleaved the substrate, Boc-Arg-Val-Arg-Arg-MCA, between the dibasic sequence Arg-Arg, and needed a support of aminopeptidase B-like enzyme activity for the liberation of 7-amino-4-methylcoumarin. These results suggest that the enzyme is transported in rat sciatic nerves and involved in the post-translational processing of precursor proteins under the anterograde axonal transport. But there is absolutely no evidence for a role in precursor processing and such a putative role is purely speculative.     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.     

Spinal reflexes in the long-tailed stingray, Himantura fai

We have exploited the segregation of motor and sensory axons into peripheral nerve sub-compartments to examine spinal reflex interactions in anaesthetized stingrays. Single, supra-maximal electrical stimuli delivered to segmental sensory nerves elicited compound action potentials in the motor nerves of the stimulated segment and in rostral and caudal segmental motor nerves. Compound action potentials elicited in segmental motor nerves by single stimuli delivered to sensory nerves were increased severalfold by prior stimulation of adjacent sensory nerves. This facilitation of the segmental reflex produced by intense conditioning stimuli decreased as it was applied to more remote segments, to approximately the same degree in up to seven segments in the rostral and caudal direction. In contrast, an asymmetric response was revealed when test and conditioning stimuli were delivered to different nerves, neither of which was of the same segment as the recorded motor nerve: in this configuration, conditioning volleys generally inhibited the responses of motoneurons to stimuli delivered to more caudally located sensory nerves. This suggests that circuitry subserving trans-segmental interactions between spinal afferents is present in stingrays and that interneuronal connections attenuate the influence that subsequent activity in caudal primary afferents can have on the motor elements.     

CYCLOHEXIMIDE ALTERS AXONAL TRANSPORT AND SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE ACTIVITY

—Administration of cycloheximide, 10 mg/kg s.c. led within 4 h to an approx 30% reduction of dopamine-β-hydroxylase (DBH) activity in the abdominal portion of rat sciatic nerves. At least two more hours elapsed before DBH activity in the distal part of these nerves began to fall. This pattern suggests reduced synthesis or delivery of DBH into axons but continued transport of previously delivered enzyme. Coinciding with the time at which DBH activity began to fall in distal segments of sciatic nerve, there was a marked reduction in the accumulation of DBH activity above a ligature in this region. Between 4 and 8 h after administration of cylcoheximide, 10 mg/kg, accumulation above a ligature was 70% less than in untreated nerves (P < 0.001), a reduction significantly greater (P < 0.05) than the accompanying 28% loss of baseline DBH activity. At the same time, the clearance of DBH activity from nerve regions distal to a ligature was greatly reduced. This pattern is consistent with the depletion of a minor but rapidly transported compartment of DBH. Six hours after administration of cylcoheximide, 10 mg/kg, the apparent subcellular distribution of DBH in distal regions of sciatic nerve was altered by a significant 36% loss in sedimentable DBH activity, with non-significant changes in othcr fractions. This suggests that rapidly transported DBH, depleted from the nerve by cycloheximide-induced inhibition of protein synthesis, is more highly associated with intraneuronal particles than is slowly transported or stationary DBH.     

Historical hypotheses regarding segmentation of the vertebrate head

The morphology of the vertebrate head is extremely complex and comprises numerous iterative structures that arise from each of the embryonic germ layers. The search for a fundamental plan uniting all of these serial structures spans ～200 years. The earliest attempt to identify a common plan was J. W. Goethe's vertebral theory of skull organization, in which the skull was interpreted as being formed by a series of trunk vertebrae. This theory was rejected by T. H. Huxley in the 1858 Croonian Lecture and was replaced by the segmented mesodermal model of Francis Balfour, which was elaborated subsequently by A. Marshall, Gavin de Beer, and Edwin Goodrich. This model assumes that the head of the earliest vertebrates consisted of eight segments. It further assumes that each segment contained dorsal muscles arising from the somitic mesoderm, and ventral muscles arising from lateral plate mesoderm, except for the first segment, which lacked ventral muscles derived from the lateral plate mesoderm. The muscles of each head segment were believed to be innervated by two pairs of cranial nerves, homologous to the dorsal and ventral spinal nerves of lampreys. The validity of this theory, known as the Goodrich model, came into question, however, after the discovery that the branchiomeric muscles associated with each pharyngeal arch do not arise from lateral plate mesoderm, as initially proposed by Marshall and subsequently accepted by Goodrich and de Beer, but, rather, arise from paraxial mesoderm. Furthermore, segmentation of the brain into some 14 neuromeres cannot be accommodated by any model involving eight segments. Finally, there is also clear evidence that at least one, if not two, additional series of placodally derived sensory nerves occurs in the head and has no counterpart in the trunk. At present, there is no theory of segmentation that can account for all cephalic iterative structures.