Phosphorylation of Synaptic Proteins in Chick Forebrain: Changes with Development and Passive Avoidance Training

We have used synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) to study protein phosphorylation at the synapse in the developing chick forebrain and in 1-day-old chick forebrain following training on a passive avoidance task. Endogenous phosphorylation patterns in SPMs and PSDs prepared by extraction with n-octylglucoside isolated from chick forebrain were investigated by labelling with [32P]ATP. The phosphoprotein components of the SPM and PSD fractions were separated using sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis. Autoradiography and densitometry of the Coomassie Blue protein staining pattern revealed phosphate incorporation into several SPM components including those of molecular mass 52, 37, and 29 kilodaltons (kDa). Bands of similar molecular mass were not phosphorylated in PSD fractions. This difference in phosphorylation between SPMs and PSDs was not due to the detergent n-octylglucoside. In a developmental study in which SPM and PSD fractions were prepared from 1-day-old, 14-day-old, and 21-day-old chickens, the phosphorylation patterns of SPMs were similar throughout, but striking differences occurred in PSDs, both in the level of phosphorylation and in the components phosphorylated. A time-course study was carried out in which phosphorylation of SPMs and PSDs from 1-day-old chicks trained on a passive avoidance task was compared with patterns from control chicks trained on a water-coated bead and untrained chicks. In SPMs prepared from forebrains removed 10 mins following training, a consistent but nonsignificant decrease (-21%) in phosphorylation of a 52 kDa band occurred in chicks with passive avoidance training compared with water-trained and untrained chicks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synaptic Vesicle Proteins and Acetylcholine Levels in Chick Forebrain Nuclei Are Altered by Passive Avoidance Training

In a search for biochemical markers of modified synaptic function following training of day-old chicks on a passive avoidance task, we have assayed two monoclonal antibodies to synaptic vesicle proteins (anti-p65 and anti-SV2) and one raised to postsynaptic densities (411B). We have also measured total acetylcholine (ACh) content. Measurements were made on three forebrain regions known to show metabolic and morphological change consequent on training--the lobus parolfactorius (LPO), paleostriatum augmentatum (PA), and medial hyperstriatum ventrale (MHV)--in the right and left hemispheres 2 and 24 h after training chicks on a passive avoidance task, in which they learn to avoid pecking a bead coated with methylanthranilate [methylanthranilate-trained (M-trained)]. Control chicks were trained on a water-coated bead [water-trained (W-trained)]. Twenty-four hours after training, 411B levels showed no differences between W-trained and M-trained chicks in any region. M-training reduced the titre of anti-p65 by 16% in the left PA and 15% in the left MHV and that of anti-SV2 by 19% in the left PA. M-trained chicks showed reduced total ACh content in the LPO by up to 40% and in the PA by up to 48% but had no change in ACh level in the MHV. The decreases in antibody titre were not seen in forebrains analysed 2 h after training, but tendencies toward increases in levels in the right PA and MHV were observed with all three antibodies. Significant differences between right and left hemispheric regions, independent of training, were observed for all the antibodies and for ACh content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cycloheximide inhibits starvation-induced autophagy through mTORC1 activation

Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.     

Effects of the Amnesic Agent 2-Deoxygalactose on Incorporation of Fucose into Chick Brain Glycoproteins

The interaction of the amnesic agent 2-deoxygalactose with fucose incorporation into glycoproteins in day-old chick forebrain has been studied with the aim of identifying glycoproteins whose synthesis is modified during memory formation. 2-Deoxygalactose inhibited total exogenous [14C]fucose incorporation into the forebrain glycoproteins by 26%. Sodium dodecyl sulphate-polyacrylamide gradient gel analysis revealed that intracerebrally injected 2-[3H]deoxygalactose labelled the same eight major glycoprotein bands as were identified using [14C]fucose labelling. Subsequent investigations focussed on these selected components. Subcellular fractionation showed that between 4 and 24 h after administration of the deoxy-sugar, the incorporated radioactivity was found predominantly at the synaptic sites, some glycoproteins being more abundant in synaptic plasma membranes and others in postsynaptic densities. This distribution pattern varied according to the time after injection. The effect of passive avoidance training, using a methylanthranilate-coated bead, on [14C]fucose incorporation into forebrain was to decrease fucose uptake into components of molecular mass 150-180 kilodaltons but to increase significantly labelling of glycoproteins of molecular mass 33 and 28 kilodaltons. The possible implications of these training-induced changes are discussed.     

Passive Avoidance Training Increases Fucokinase Activity in Right Forebrain Base of Day-Old Chicks

Fucokinase (EC 2.7.1.52) activity was estimated in supernatants of homogenate from day-old chick forebrain. Enzyme kinetic studies gave a Km of 4.5 X 10(-6) M and Vmax of 3.72 nmol fucose converted into fucose-1-phosphate/mg prot/h. The pH optimum was 7.5. The enzyme is thus considerably more active than was reported for other species and tissues. There were no differences in enzyme activity between the four forebrain regions studied. One hour after chicks were trained on a one-trial passive avoidance learning paradigm, enzyme activity in the right forebrain base increased 14% over control values (p less than 0.02). The 11.3% increase in activity in the left forebrain base and 10.3% increase in the left roof were not statistically significant. The relationship of this change to the increased fucose incorporation into glycoproteins known to occur over a similar time period and the significance of the lateralization of the increase are discussed.     

Time-Dependent Increase in Release of Arachidonic Acid Following Passive Avoidance Training in the Day-Old Chick

Abstract: We have investigated the role of arachidonic acid, a putative retrograde messenger, in a one-trial aversive learning task in the day-old chick. The left and right intermediate medial hyperstriatum ventrale (IMHV) in the chick forebrain have previously been implicated in the formation of memory for this task. Using an ex vivo technique we have determined the concentrations of various fatty acids liberated from prisms prepared from these brain regions at different time points up to 24 h following passive avoidance training. At 30, 60, and 75 min post-training the concentration of arachidonic acid, but not of other fatty acids, in prisms prepared from the left IMHV, but not the right IMHV, was enhanced compared with that in chicks trained on a nonaversive water-coated bead. To test whether arachidonic acid liberation from the left IMHV was receptor-stimulated we showed that (a) liberation of endogenous arachidonic acid from homogenate prepared from the left and right IMHV of untrained chicks was stimulated by depolarization with KCl (50 m M ) and that (b) glutamate agonists of the NMDA and metabotropic subtypes of glutamate receptor stimulated release of preloaded [¹⁴C]arachidonic acid from prisms prepared from the left IMHV but not the right IMHV. These results indicate that arachidonic acid is liberated from the left IMHV following passive avoidance training in the day-old chick and may play a role as a retrograde messenger in this memory task.     

Induction of Rat Brain Tubulin Following Ammonium Ingestion

The effect of oral administration of ammonium acetate for 2, 15, 30, and 100 days on protein synthesis in rat brain was investigated. Although protein synthesis changes were modest, i.e., maximal increase of 24%, there was induction of synthesis and accumulation of a protein with an Mr of 55,000. We show, on the basis of its position on two-dimensional electrophoresis and its immunological reactivity, that this protein is tubulin. Its content increased by 33% as determined by isolation of tubulin after 15 days of oral administration of ammonium and to 49% after 100 days as determined by quantitative immunoblotting.     

Phencyclidine and Analogues: Effects on Brain Protein Synthesis

Abstract: Phencyclidine and four analogues were tested for their capacity to inhibit total protein synthesis in a brain homogenate. At 1.0 m M they were all found to be potent inhibitors with values ranging from 36% to 96% inhibition. At this high concentration two of the analogues were equal to or more effective than the classic protein synthesis inhibitors cycloheximide and emetine. At lower concentrations the inhibition dropped off sharply to 18% at 1.0 × 10⁻⁴ M and 9% at 1.0 × 10⁻⁵ M for phencyclidine. If the inhibition observed in the brain homogenate occurs in vivo it may account for the high incidence of memory loss reported with phencyclidine use.     

Rapid Nocturnal Increase in Ovine Pineal N-Acetyltransferase Activity and Melatonin Synthesis: Effects of Cycloheximide

Thirty minutes after the onset of darkness, ovine pineal arylalkylamine N-acetyltransferase, N-acetylserotonin, and melatonin increase 5- to 10-fold. No significant changes in hydroxyindole-O-methyltransferase, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxyindoleacetic acid, and 5-methoxytryptophol are detected at this time. Administration of cycloheximide inhibits the rise in N-acetyltransferase and N-acetylserotonin, but not melatonin. Unexpectedly, 5-methoxytryptophol increases after cycloheximide treatment. Taken together, these results, although consistent in part with a role for serotonin N-acetylation in the regulation of melatonin synthesis in sheep, indicate that an N-acetyltransferase-independent mechanism may also be involved.     

Neurofilament Proteins Are Synthesized in Nerve Endings from Squid Brain

Abstract: It is generally believed that the proteins of the nerve endings are synthesized on perikaryal polysomes and are eventually delivered to the presynaptic domain by axoplasmic flow. At variance with this view, we have reported previously that a synaptosomal fraction from squid brain actively synthesizes proteins whose electrophoretic profile differs substantially from that of the proteins made in nerve cell bodies, axons, or glial cells, i.e., by the possible contaminants of the synaptosomal fraction. Using western analyses and immunoabsorption methods, we report now that (a) the translation products of the squid synaptosomal fraction include neurofilament (NF) proteins and (b) the electrophoretic pattern of the synaptosomal newly synthesized NF proteins is drastically different from that of the IMF proteins synthesized by nerve cell bodies. The latter results exclude the possibility that NF proteins synthesized by the synaptosomal fraction originate in fragments of nerve cell bodies possibly contaminating the synaptosomal fraction. They rather indicate that in squid brain, nerve terminals synthesize NF proteins.     

Increased Incorporation of [3H]Fucose into Chick Brain Glycoproteins Following Training on a Passive Avoidance Task

Abstract: Incorporation of [¹H]fucose into the TCA-precipitable material from the particulate and soluble fractions of different regions of the brain in the 1-day-old chick was measured following training on a passive avoidance task. A significant increase in the level of incorporation of [³H]fucose into the particulate fraction of the anterior forebrain roof was observed in the trained birds as compared with untrained controls. The percentage increases in radioactivity in the particulate fraction of the anterior forebrain roof were 29% ( p < 0.01), 16% ( p < 0.01), and 26% ( p < 0.01) in the trained birds as compared with controls, 30 min, 3 h, and 24 h following learning, respectively, but had returned to control levels after 48 h. These results suggest either increased production of glycoproteins or increased fucosylation of preexisting proteins following training, and suggest one of the possible localized neurochemical changes associated with the learning of the passive avoidance response.     

Brain Maturation Following Administration of Phenobarbital, Phenytoin, and Sodium Valproate to Developing Rats or to Their Dams: Effects on Synthesis of Brain Myelin and Other Subcellular Membrane Proteins

Abstract: The anticonvulsant drugs phenobarbital, phenytoin, sodium valproate, and phenytoin-sodium valproate in combination were administered daily to (a) pregnant rats starting on the 5th day after conception, and continued through 17 days postpartum, or (b) to developing rats between 3 and 17 days of age. Each drug was prepared in water and administered at either a therapeutic dose (TD), three times therapeutic dose (³TD), or 9TD. Drug administration had no discernible effect on litter size or sex ratio in the offspring; however, phenobarbital administration to dams caused small but significant reductions in birth weights. Body weights of developing rats treated with anticonvulsant drugs either via dams or directly by intraperitoneal injection lagged behind controls. At 20–24 days of age the brain weights of the offspring of phenobarbital (9TD)-exposed dams lagged control weights by 5% whereas brain weights in the offspring of the other treated groups were indistinguishable from controls. In contrast, administration of phenobarbital directly to developing rats caused no significant brain weight deficits whereas significant deficits were observed with phenytoin (9TD), sodium valproate (9TD), and phenytoin-sodium valproate (9TD) in combination. At 20–24 days of age the relative incorporation of radioactive leucine into purified myelin and crude nuclear proteins of drug-treated rats or the offspring of drug-treated dams was reduced by 10–20% in all cases. Dose-related differences were not observed however, and the effects of phenytoin and sodium valproate in combination approximated those of phenytoin administered alone.     

Effect of Passive Avoidance Training on In Vitro Protein Synthesis in Forebrain Slices of Day-Old Chicks

Slices from the forebrains of day-old chicks represent a highly active in vitro protein-synthesising system. The in vitro incorporation of L-[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell-soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (greater than 40 micron from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04-1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20-50% inhibition of incorporation of radioactive L-leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one-trial passive avoidance paradigm, L-[14C]leucine incorporation was 23% higher (p less than 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.     

Changes in Brain Energy Metabolism and Protein Synthesis Following Transient Bilateral Ischemia in the Gerbil

The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1-2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.     

Effects of luteolin on learning acquisition in rats: involvement of the central cholinergic system

The study was conducted to investigate the ameliorating effects of luteolin on memory acquisition in rats. The effects of luteolin on scopolamine-induced impairment of passive avoidance response were evaluated primarily, as well as the role of the central nervous system through the use of central neurotoxins and central nervous antagonists. Luteolin was not reversed by scopolamine N-methylbromide (M-SCOP) but blocked the impairment of learning acquisition induced by cholinergic neurotoxin (ethylcholine aziridinium, AF64A) and muscarinic (scopolamine hydrobromide, SCOP) and nicotinic (mecamylamine, MECA) receptor antagonists. However, it did not block dopaminergic neurotoxin (6-hydroxydopamine, 6-OHDA)-induced and serotonergic neurotoxin (5,7-dihydroxytryptamine, 5,7-DHT)-induced impairments. From these results, we suggest that the attenuating effect of luteolin (10 mg/kg, i.p.) on the deficits of passive avoidance performance induced by SCOP may be related to the increases in the activities of central muscarinic and nicotinic receptors.     

核糖体失活蛋白研究进展

核糖体失活蛋白是一类毒蛋白,主要存在于植物当中,在真菌和细菌中也有发现.其共同特点是具有N-糖苷酶活性,能水解生物核糖体大亚基rRNA颈环结构上特定位点的腺嘌呤,使核糖体失活,从而抑制了蛋白质合成.本文对核糖体失活蛋白的主要性质、应用以及国内外有关这类蛋白的研究进展加以概述.     

Regional Alterations of Protein Kinase C Activity Following Transient Cerebral Ischemia: Effects of Intraischemic Brain Temperature Modulation

Abstract: It is well established that ischemia-induced release of glutamate and the subsequent activation of postsynaptic glutamate receptors are important processes involved in the development of ischemic neuronal damage. Moderate intraischemic hypothermia attenuates glutamate release and confers protection from ischemic damage, whereas mild intraischemic hyperthermia increases glutamate release and augments ischemic pathology. As protein kinase C (PKC) is implicated in neurotransmitter release and glutamate receptor-mediated events, we evaluated the relationship between intraischemic brain temperature and PKC activity in brain regions known to be vulnerable or nonvulnerable to transient global ischemia. Twenty minutes of bilateral carotid artery occlusion plus hypotension were induced in rats in which intraischemic brain temperature was maintained at 30°C, 37°C, or 39°C. Prior to and following ischemia, brain temperature was 37°C in all groups. Cytosolic, membrane-bound, and total PKC activities were determined in hippocampal, striatal, cortical, and thalamic homogenates at the end of ischemia and at 0.25–24 h of recirculation. PKC activity of control rats varied by region and were affected by altered brain temperature. For both membrane-bound and cytosolic PKC, there was a significant temperature effect, and for membrane-bound PKC there was also a significant effect of region. Rats with normothermic ischemia (37°C) showed extensive depressions of all PKC fractions. Hippocampus and striatum were noteworthy for depressions in PKC activity extending from the earliest (15 min) to the latest (24 h) recirculation times studied, whereas cortex showed PKC depressions chiefly during the first hour of recirculation, and the thalamic pattern was inconsistent. In contrast, in rats with hypothermic ischemia (30°C), significant overall effects were noted only for total PKC in thalamus, which showed depressed levels at both 1 and 24 h of recirculation. Rats with hyperthermic (39°C) ischemia also showed significant overall effects for the time course of membrane-bound, cytosolic, and total PKC activities in the hippocampus, striatum, and cortex. However, no significant reductions in PKC indices were observed in the thalamus. For membrane-bound PKC, significant temperature effects were noted for hippocampus, striatum, and cortex, but not for thalamus. For cytosolic, as well as total PKC, activity, significant temperature effects were noted for all four brain regions. Our results indicate that ischemia, followed by reperfusion, induces a significant reduction in PKC activity and that this process is highly influenced by the brain temperature during ischemia. Furthermore, our data also establish that differences exist in the response of PKC to ischemia/recirculation in vulnerable versus non-vulnerable brain regions. These results suggest that PKC alterations may be an important factor involved in the modulatory effects of temperature on the outcome following transient global ischemia.     

N-Terminal Arginylation of Sciatic Nerve and Brain Proteins Following Injury

N-terminal protein arginylation has been demonstrated in vitro and in situ and has been reported to increase following injury to sciatic nerves of rats. The present study attempts to demonstrate these reactions in vivo by applying [³H]Arg to the cut end of sciatic nerves in anesthetized rats and assaying for N-terminal arginylation using Edman chemistry and acid precipitation of labeled proteins in the proximal nerve segment. No evidence was found for arginylation in an aqueous soluble fraction. However, N-terminal arginylation was detected in a urea soluble fraction at 2 hours after nerve crush. The data show that arginylation of rat sciatic nerve proteins occurs in vivo and suggest that the arginylated proteins formed an aqueous insoluble/urea soluble aggregate after arginylation. In other experiments, rat brains were injured and assayed for arginylation in vitro to test the hypothesis that injury causes an up-regulation of these reactions. Results showed an activation of the reaction at 2 hours post crush and indicate that increases in N-terminal arginylation are likely to be a general response to injury in nervous tissue.