Brain Protein Synthesis Research Articles

Changes in regional protein synthesis in rat brain and pituitary after systemic interleukin-1 beta administration.

A convenient and sensitive method has been developed for measuring changes in protein synthesis in discrete areas of the brain and pituitary of conscious freely moving rats. A single injection of high-concentration low-specific activity L-[35S]methionine is given to flood amino acid precursor pools, thereby equalizing the specific activity of the L-[35S]methionine throughout the tissue. Unincorporated L-[35S]methionine is removed from cryostat sections by treatment with perchloric acid (2%) before quantitative autoradiography. The sensitivity of this technique is demonstrated by the detection of changes in protein synthesis in regions of the brain and pituitary after systemic administration of interleukin-1 beta, a cytokine that has centrally mediated effects but which is not thought to cross the blood-brain barrier. Areas of the brain found to exhibit significant increases in protein synthesis were the subfornical organ, the choroid plexus, the medial habenular, the dentate gyrus, and the anterior and posterior lobes of the pituitary. In the brain, the cingulate cortex and the pineal gland showed significant decreases in the rate of protein synthesis.

Triiodothyronine does not affect the average incorporation of l-[ 35S]methionine in rat brain structures

The autoradiographic method with l-[ 35S]methionine was used to examine the effect of acute administration of l-triiodothyronine on local rates of brain protein synthesis in free-moving adult rats. Triiodothyronine was given intraperitoneally at doses of 12.5 or 25 μg kg −1. It did not modify the rate of plasma methionine incorporation in the 40 brain regions examined, despite a 4- to 8-fold increase of plasma free triiodothyronine levels. Biochemical analysis confirmed that triiodothyronine (25 μg kg −1) had no apparent effect on the overall rate of protein synthesis in the brain as a whole. These results suggest that changes in the circulating levels of thyroid hormones do not exert a general and direct metabolic effect in brain of intact adult rats.

N-3 fatty acid deficiency increases brain protein synthesis in the free-moving adult rat.

The autoradiographic method with L-[35S]methionine was used to determine the effects of an n-3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n-3 fatty acid-deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole.

The effect of elevated plasma phenylalanine levels on protein synthesis rates in adult rat brain

Increasing the plasma phenylalanine concentration to levels as high as 0.560-0.870 mM (over ten times normal levels) had no detectable effect on the rate of brain protein synthesis in adult rats. The average rates for 7-week-old rats were: valine, 0.58 +/- 0.05%/h, phenylalanine, 0.59 +/- 0.06%/h, and tyrosine, 0.60 +/- 0.09%/h, or 0.59 +/- 0.06%/h overall. Synthesis rates calculated on the basis of the specific activity of the tRNA-bound amino acid were slightly lower (4% lower for phenylalanine) than those based on the brain free amino acid pool. Similarly, the specific activities of valine and phenylalanine in microdialysis fluid from striatum were practically the same as those in the brain free amino acid pool. Thus the specific activities of the valine and phenylalanine brain free pools are good measures of the precursor specific activity for protein synthesis. In any event, synthesis rates, whether based on the specific activities of the amino acids in the brain free pool or those bound to tRNA, were unaffected by elevated levels of plasma phenylalanine. Brain protein synthesis rates measured after the administration of quite large doses of phenylalanine (> 1.5 mumol/g) or valine (15 mumol/g) were in agreement (0.62 +/- 0.01 and 0.65 +/- 0.01%/h respectively) with the rates determined with infusions of trace amounts of amino acids. Thus the technique of stabilizing precursor-specific activity, and pushing values in the brain close to those of the plasma, by the administration of large quantities of precursor, appears to be valid.

Age Affects Brain Protein Synthesis in Rats

The purpose of the present study was to determine whether age affected the rate of brain protein synthesis in rats. Experiments were conducted on three groups of rats, ages 4, 7 or 24 wk. The fractional rates of protein synthesis in brain, liver and kidney decreased and the RNA concentration (mg RNA/g protein) declined with age. In brain, liver and kidney, the RNA concentrations were significantly correlated with the fractional rates of protein synthesis. The RNA activity [g protein synthesized/(g RNA.d)] was not related to the fractional rate of protein synthesis in any organs except cerebral cortex. The results suggest that the rate of protein synthesis in brain declines with age after weaning, and that RNA concentration is at least partly related to the fractional rate of brain protein synthesis.

Adrenalectomy-induced increase of brain protein synthesis is antagonized by corticosterone replacements in free-moving rats.

The autoradiographic method with L-[35S]-methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of methionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy-induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham-operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long-term neurochemical processes.

Visualization of neurotransmitter receptors in the living human brain by position emission tomography (PET)

Various brain imaging techniques have become available in the past decade. These include techniques to evaluate brain structure using X-ray computerized tomography or magnetic resonance imaging and techniques to assess brain functional activities (cerebral blood flow, brain energy metabolism and brain protein synthesis) using positron emission tomography (PET). PET also makes it possible for the first time to evaluate the states of various types of neurotransmitter receptors, such as dopamine D2 and D1, serotonin 5-HT2, muscarinic cholinergic, opiate, benzodiazepine and histamine H1, and to determine the state of monoamine oxidase (A and B) under in vivo conditions. These techniques cannot only be used to map "brain neurochemistry" in normal human beings, but they will also increase our knowledge by demonstrating neurochemical abnormalities in a wide range of neurological and psychiatric disorders or those that happen during normal aging. The PET techniques may be applicable to the development of new drugs in the pharmaceutical industry. We have been exploring the methodology of using 11C-labeled antagonists for mapping functional neurotransmitter receptors in human brain directly and noninvasively by PET. The present review article provides an outline of the conceptual and methodological progress over the past several years that has made it possible to visualize neurotransmitter receptors in the living human brain by PET on the basis of our original work.

Perspectives on lead toxicity

Lead toxicity causes hematological, gastrointestinal, and neurological dysfunction in adults and children. Symptoms are usually noted with blood lead greater than 1.93 μmol/L. Severe or prolonged exposure may also cause chronic nephropathy, hypertension, and reproductive impairment. Lead inhibits enzymes; alters cellular calcium metabolism; stimulates synthesis of binding proteins in kidney, brain, and bone; and slows nerve conduction. Less severe exposure to lead, designated by blood lead levels fo 0.48–0.96 μmol/L, has been implicated in poor pregnancy outcome, impaired neurobehavioral development, reduced stature in young children, and higher blood pressure in adults. Biochemical and systemic effects of high and low level lead toxicity are described. Dust, water, and paint chips are still major sources of lead but lead from folk remedies, cosmetics, food supplements, food preparation utensils, and improperly prepared infant formula has caused epidemic and sporadic severe lead toxicity. Screening for pediatric low level lead exposure requires measurement of blood lead.

Comparison of protein synthesis in brain and peripheral tissue during aging. Relationship to insulin-like growth factor-1 and type 1 IGF receptors.

Comparison of protein synthesis in brain and peripheral tissue during aging. Relationship to insulin-like growth factor-1 and type 1 IGF receptors.

Initiation of protein synthesis and heat-shock protein-72 expression in the rat brain following severe insulin-induced hypoglycemia.

Following stress such as heat shock or transient cerebral ischemia, global brain protein synthesis initiation is depressed through modulation of eucaryotic initiation factor (eIF) activities, and modification of ribosomal subunits. Concomitantly, expression of a certain class of mRNA, heat-shock protein (HSP) mRNA, is induced. Here we report that the activity of eucaryotic initiation factor-2 (eIF-2), a protein that participates in the regulation of a rate-limiting initiation step of protein synthesis, transiently decreases following insulin-induced severe hypoglycemia in the rat brain neocortex. Expression of HSP 72, a 72-kDa HSP, in surviving neurons was seen at 1-7 days of recovery following 30 min of hypoglycemic coma, but not at 1 h and 6 h of recovery. In the neocortex, HSP 72 was first seen in layer IV, and later also in surviving neurons in layer II. In the CA1 region and in the crest of dentate gyrus, HSP 72 expression was evident in cells adjacent to irreversibly damaged neurons. In the CA3 region and the hilus of dentate gyrus, HSP 72 was expressed in a few scattered neurons. In septal nucleus, HSP 72 was expressed in a lateral to medial fashion over a period of 1-3 days of recovery. We conclude that severe insulin-induced hypoglycemia induces a stress response in neurons in the recovery phase, including inhibition of protein synthesis initiation, depression of eIF-2 activity, and a delayed and prolonged expression of HSP 72 in surviving neurons. The HSP 72 expression may be a protective response to injurious stress.

The effect of an AMPA antagonist (NBQX) on postischemic neuron loss and protein synthesis in the rat brain.

Two groups of rats were subjected to 12 min of global cerebral ischemia and 6 days recirculation using the four-vessel occlusion model with hypotension and then treated with the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxalinedione (Honoré et al. 1988]. One group was used for routine and quantitative histology and immunostaining for glial fibrillary acidic protein (GFAP). The second group was subjected to autoradiographic studies of regional cerebral protein synthesis, with special emphasis on the hippocampus, the frontal cortex, and the thalamus. It was found that neuroprotective treatment with NBQX normalized cerebral protein synthesis rate (CPSR) in all investigated regions 6 days after ischemia. In untreated ischemic animals CPSR was normalized in all regions except for the CA3 and thalamus, where it had increased by 29% and 41%, respectively. Treatment of controls with NBQX had no effect on CPSR after 6 days. The histological investigations revealed that NBQX did not protect vulnerable cells in the dentate hilus and the reticular thalamic nucleus (RTN). In these regions reactive astrocytosis visualized by GFAP immunostaining was equally pronounced in both ischemic and NBQX-treated animals, and most neurons in the RTN were eosinophilic. The 80-100% pyramidal neuron loss in CA1 was accompanied by a high degree of reactive astrocytosis, whereas the NBQX-treated animals showed no signs of astrocytosis in this region. The ischemic CA1 pyramidal layer was also massively invaded by microglia.(ABSTRACT TRUNCATED AT 250 WORDS)

Postischaemic changes in protein synthesis in the rat brain: effects of hypothermia.

Protein synthesis, measured as [14C]-leucine incorporation into proteins, was studied in the normothermic rat brain following 15 min of transient cerebral ischaemia and 1 h, 24 h and 48 h of recirculation, and in the hypothermic (33 degrees C) brain following 1 h and 48 h of recirculation. Ischaemia was induced by bilateral common carotid occlusion combined with hypotension. Following normothermic ischaemia, incorporation of [14C]-leucine was depressed by 40-80% at 1 h of recirculation in all brain regions studied. At 48 h postischaemia, incorporation returned to normal or above normal levels in the inner layers of neocortex, the CA3 region, the striatum and the dentate gyrus, while in the outer layers of neocortex and in the hippocampal CA1 region the incorporation was persistently decreased by 26% and 40% respectively. At 24 and 48 h postischaemia, protein synthesis in the CA1 region and the striatum could be attributed to proliferating microglia. Intra-ischaemic hypothermia ameliorated the persistent depression of protein synthesis in the CA1 region at 48 h postischaemia, and a two-fold increase compared to the normothermic group was observed both in the CA1 region and the striatum. In the cortex, eucaryotic initiation factor 2 activity transiently decreased at 30 min postischaemia. In animals subjected to intra-ischaemic hypothermia, the eucaryotic initiation factor 2 activity was reduced by 50% of control at 30 min of recirculation compared with 77% in normothermic animals. We conclude that the postischaemic depression of protein synthesis is in part caused by a decrease in eucaryotic initiation factor 2 activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of brain protein synthesis by cycloheximide does not affect formation of long-term memory in honeybees after olfactory conditioning

The honeybee forms a long-term memory in different training situations that lasts for a lifetime, but the cellular mechanisms of long-term memory formation are not known. We analyzed the dependency of long-term memory on the de novo brain protein synthesis. The protein synthesis inhibitor cycloheximide was injected via the median ocellus directly into the brain. 3H-leucine incorporation into brain proteins was inhibited by > 95% for > 3 hr. The time of protein synthesis inhibition was prolonged by a second injection of the same dose. Worker honeybees were conditioned to an olfactory stimulus at different times before and after injection. The proboscis extension response (PER) of bees restrained in tubes was classically conditioned with sugar water applied first to the antennae followed by feeding (unconditioned stimulus) paired with odor presentation (conditioned stimulus). The bees were tested by presenting the odor alone at different times up to 24 hr after injection. No significant reduction in the probability of the conditioned response in cycloheximide-treated bees was found when compared to the Ringer-injected controls in 4 series of experiments. Since protein synthesis was inhibited between 7 hr pre- and 7 hr postconditioning without affecting the formation of long-term memory, a possible role of de novo protein synthesis in the formation of long-term memory after olfactory conditioning of the PER is not supported by these experiments.

Studies of the protein synthesis system in the brain cortex during global ischemia and reperfusion

Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase ( ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein ( hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.

Pathophysiological mechanisms of brain damage from status epilepticus.

Human status epilepticus (SE) is consistently associated with cognitive problems, and with widespread neuronal necrosis in hippocampus and other brain regions. In animal models, convulsive SE causes extensive neuronal necrosis. Nonconvulsive SE in adult animals also leads to widespread neuronal necrosis in vulnerable regions, although lesions develop more slowly than they would in the presence of convulsions or anoxia. In very young rats, nonconvulsive normoxic SE spares hippocampal pyramidal cells, but other types of neurons may not show the same resistance, and inhibition of brain growth, DNA and protein synthesis, and of myelin formation and of synaptogenesis may lead to altered brain development. Lesions induced by SE may be epileptogenic by leading to misdirected regeneration. In SE, glutamate, aspartate, and acetylcholine play major roles as excitatory neurotransmitters, and GABA is the dominant inhibitory neurotransmitter. GABA metabolism in substantia nigra (SN) plays a key role in seizure arrest. When seizures stop, a major increase in GABA synthesis is seen in SN postictally. GABA synthesis in SN may fail in SE. Extrasynaptic factors may also play an important role in seizure spread and in maintaining SE. Glial immaturity, increased electronic coupling, and SN immaturity facilitate SE development in the immature brain. Major increases in cerebral blood flow (CBF) protect the brain in early SE, but CBF falls in late SE as blood pressure falters. At the same time, large increases in cerebral metabolic rate for glucose and oxygen continue throughout SE. Adenosine triphosphate (ATP) depletion and lactate accumulation are associated with hypermetabolic neuronal necrosis. Excitotoxic mechanisms mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors open ionic channels permeable to calcium and play a major role in neuronal injury from SE. Hypoxia, systemic lactic acidosis, CO2 narcosis, hyperkalemia, hypoglycemia, shock, cardiac arrhythmias, pulmonary edema, acute renal tubular necrosis, high output failure, aspiration pneumonia, hyperpyrexia, blood leukocytosis and CSF pleocytosis are common and potentially serious complications of SE. Our improved understanding of the pathophysiology of brain damage in SE should lead to further improvement in treatment and outcome.

811 The effect of seizures on translation processes of brain protein synthesis in seizure-susceptible E1 mice

811 The effect of seizures on translation processes of brain protein synthesis in seizure-susceptible E1 mice

The Quality and Quantity of Dietary Protein Affect Brain Protein Synthesis in Rats

The influence of the amino acid supply in diets with different quality and quantity of protein on the rate of protein synthesis in the brain was investigated. Amino acid concentrations in serum and brain altered in accordance with the amino acid levels of the diets, with the exception of some amino acids such as aspartic acid, glutamic acid, glycine and threonine. When rats were fed various levels of dietary casein (0, 5 and 20%), the aggregation of ribosomes increased and the fractional rate of protein synthesis tended to increase with the increase in dietary protein. When rats were fed a 20% casein diet, greater aggregation of brain ribosomes and protein synthesis rate were observed compared with those in rats fed 20% wheat gluten or gelatin diets. The RNA activity was related to the degree of the aggregation of brain ribosomes and the fractional rate of protein synthesis.

Some effects of aluminium on rat brain protein synthesis

1. Infant rats and rabbits received intraperitonal aluminium (Al) chloride (5, 10 or 20 mg Al/kg body weight) every third day from one to four weeks of age. 2. When the polysomal fraction was tested in a protein synthesizing system, a significant increase in the incorporation of [ 14C] leucine, [ 14C] phenylalanine, or [ 35S] methionine into proteins in vitro was observed at the higher doses in rats but not rabbits. 3. The incorporation of [ 35S]methionine into brain ferritin was measured using polysomal mRNA or mRNA “stored” in the ribonucleoprotein (RNP) particle fraction. 4. The results suggest that Al exposure causes the mobilization of ferritin mRNA from the latter fraction to the polysomal fraction for increased ferritin synthesis.

Brain protein synthesis in the conscious rat using L-[35S]methionine: relationship of methionine specific activity between plasma and precursor compartment and evaluation of methionine metabolic pathways.

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.