Cerebral Proteins Research Articles

Different effects of hypothermia on amino acid incorporation and on amino acid uptake in the brain in vivo.

The temperature dependence of the incorporation of amino acids into cerebral proteins and that of the transport of amino acids through the blood-brain barrier were studied. We measured the protein synthesis rate in vivo over a wide temperature range (14 degrees C-38 degrees C) in male Sprague-Dawley rats using a flooding dose of labeled valine. There was a linear dependence of the protein synthesis rate on temperature. The temperature quotient expressed as per cent decrease per 1 degree C was somewhat lower at the lower temperatures, a decrease from 7.8% in the 37.7-32.5 degrees C range to 6.7% in the 25.5-14 degrees C range. The transport of the three amino acids phenylalanine, lysine, and alanine, representing three transport systems, through the blood-brain barrier showed no temperature dependence in vivo. The results show that in hypothermia cerebral metabolic rates are lowered to a great extent, while some aspects of metabolic transport are not affected.

Rates of cerebral protein synthesis are linked to slow wave sleep in the rat

Using L-[1- 14C]leucine autoradiography, rates of cerebral and local cerebral protein synthesis were studied during wakefulness, slow wave sleep (SWS) and REM sleep in the rat. In the cerebrum as a whole, the rate at which labelled leucine was incorporated into tissues was positively correlated with the occurrence of slow wave sleep. We failed to observe a significant correlation of protein synthesis rate with either wakefulness or REM sleep. As in the cerebrum as a whole, most discrete brain regions showed moderate positive correlations between the occurrence of SWS and rates of protein synthesis. There were no brain regions in which rates of protein synthesis showed striking correlations with sleep-wake states. Thus, the occurrence of SWS is associated with higher rates of protein synthesis throughout the brain. These data suggest that SWS sleep favors the restoration of cerebral proteins.

Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats

Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to29 ± 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E-64c ( P < 0.05) to increase MAP2 levels to55 ± 25.7% of control levels. E-64c had no significant effect on the ischemia-induced depletion of myelin-associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia.

Tumor growth-inhibitory glycoprotein secreted from the mouse brain at terminal stage of the ontogeny: molecular homogeneity and requirement of the retained protein conformation for exhibition of the cytotoxic action

The mouse brain at the neonatal stage but not at the adult or fetal stage, secretes a protein which inhibits growth and DNA synthesis of malignant cells preferentially over those of normal cells, and so is termed neonatal brain-derived carcinostatic factor (NBCF). In the present study, NBCF was prepared from conditioned medium cultured from neonatal brain, and was obtained by HPLC as a homogeneous protein (62 kDA, p I 9.1); physico-chemical and biological properties of NBCF differ from those of cerebral proteins known. A 1,2- cis-diol affinity column retained NBCF, which was thereafter eluted with d-sorbitol; NBCF treated with neuraminidase lost part of activity without emergence of new N-terminal amino acids, suggesting glycan moieties required for the activity exhibition. This is supported by repression of NBCF secretion by tunicamycin of doses as low as is not cytotoxic. NBCF did not hydrolyze target proteins; cytotoxic action of NBCF was not counteracted by a diversity of proteinase inhibitors. An ether extract of NBCF was not cytotoxic, whereas the ether-insoluble residuum retained part of the initial activity. NBCF was inactivated with immobilized trypsin or with dithiothreitol combined with guanidine more markedly than with either agent. Thus cytotoxicity exhibition of NBCF, mediated through actions other than proteolysis, is attributed to the proteinic principle but not to protein-bound lipophilic ligands, and requires retention of the protein conformation and intramolecularly buried SS bonds.

Changes in the concentrations of cerebral proteins following occlusion of the middle cerebral artery in rats.

Using an immunoblotting technique, we investigated changes in the concentrations of microtubule-associated protein 2, 200-kDa neurofilament, tubulin, myelin-associated glycoprotein, and 2':3'-cyclic nucleotide 3'-phosphodiesterase in the brains of 40 rats following occlusion of the left middle cerebral artery or sham operation. Compared with those 4 hours after surgery, concentrations of all proteins decreased significantly in the left hemisphere 3 days after surgery (p less than 0.01). Microtubule-associated protein 2 was the most susceptible to ischemia, and its mean +/- SEM concentration decreased to 23 +/- 9.4% of that in concurrent sham-operated controls. Degradation products of microtubule-associated protein 2 and myelin-associated glycoprotein were detected on the blots. Furthermore, in the contralateral hemisphere (where calpain might be activated), concentrations of these two proteins decreased to 57 +/- 12.0% and 83 +/- 4.3% of those in concurrent sham-operated controls, respectively, 3 days after surgery. Changes in the concentrations of cerebral proteins in the contralateral hemisphere are important for understanding clinical symptoms not attributable solely to the ipsilateral lesion following a focal cerebral stroke.

Biochemical and autoradiographic study of cerebral protein synthesis with [18F]-and [14C]fluorophenylalanine.

Fluorine-18-labeled ortho or para isomers of L-fluorophenylalanine were used in double-label experiments together with L-[3H]phenylalanine for amino acid incorporation into cerebral proteins of Mongolian gerbil brain. It was demonstrated by qualitative regional comparison of the 18F and 3H autoradiographic images that L-p-[18F]fluorophenylalanine is incorporated into proteins and exhibits a regional cerebral protein synthesis pattern. To a minor extent, L-p-fluorophenyl[3-14C]alanine and L-o-[18F]fluorophenylalanine are hydroxylated in vivo to form labeled tyrosine or tyrosine analogues that are incorporated into cerebral proteins as well. The advantage and validity of the application of L-p-[18F]fluorophenylalanine with positron emission tomography for noninvasive studies of cerebral protein synthesis in humans are evaluated on the basis of an experimental animal approach.

Extracellular protein phosphorylation in neuronal responsiveness and adaptation.

The significant role of protein phosphorylation systems in the regulation and modulation of multiple neuronal functions has been extensively documented in numerous studies over the last three decades. Beginning with the demonstration by Heald (1957) that brief depolarization of respiring brain slices can cause a significant increase of phosphate incorporation into cerebral proteins, progress in this line of investigation has led to the conclusion that the cyclic process of phosphorylation/dephosphorylation of proteins represents a ubiquitous target for diverse agents which produce rapid and transient changes in neuronal activity (reviewed by Greengard, 1978; Rodnight, 1983). More recent studies, carried out with identified neurons of invertebrates, have begun to provide direct evidence for the role of specific phosphoproteins in certain well defined neuronal functions (reviewed by Nestler and Greengard, 1983). Thus, in the chain of events that occurs intracellularly subsequent to the activation of second-messenger generating systems by neurotransmitters, hormones, growth factors and trophic agents, phosphoproteins constitute a crucial link essential for the process of stimulus-response coupling (for most recent reviews see Nishizuka, 1986 and chapter by Greengard et al., in this volume). In addition, phosphorylative activity has been recognized as a site of molecular adaptation in neurons, since it was shown that modifications in the process of protein phosphorylation are induced by inputs which cause long-lasting alterations in brain function (reviewed by Ehrlich, 1979, 1984, see also chapters by Lovinger and Routtenberg and by Alkon and Naito in this volume).

Protein Synthesis in Postischemic Rat Brain: A Two-Dimensional Electrophoretic Analysis

This study examined the pattern of protein synthesis in the neocortex, caudate-putamen, and the hippocampus following transient forebrain ischemia in rats. The animal model of temporary ischemia used in this study causes permanent damage to vulnerable neurons with a time course of injury that varies from hours (caudate nucleus) to days (hippocampus). To examine the spectrum of proteins synthesized in these regions at 3 and 18 h after recirculation, cerebral proteins were pulse-labeled in vivo by an intravenous injection of [35S]methionine. Newly synthesized (35S-labeled) and constitutive (unlabeled) proteins were analyzed by two-dimensional gel electrophoresis and fluorography. In all three brain regions, specific proteins underwent preferential synthesis (Mr approximately 27,000, approximately 65,000, approximately 70,000, approximately 110,000), while others showed decreased synthesis (neuron-specific enolase, alpha- and beta-tubulin). There was an early (3 h post ischemia) induction of the Mr approximately 70,000 mammalian "stress" protein; at 18 h post ischemia, its synthesis remained high in the hippocampus but was diminished in the neocortex and had largely subsided in the caudate-putamen. All regions at 18 h showed increased synthesis of an Mr approximately 50,000 protein, tentatively identified as glial fibrillary acidic protein. The results show that temporary forebrain ischemia induces changes in protein synthesis that include features similar to those observed in other eukaryotic cells subjected to injurious stress. These postischemic changes in protein synthesis are qualitatively similar in all brain regions examined despite regional differences in the severity of subsequent neuronal damage. The persistent synthesis of the Mr approximately 70,000 stress protein in the hippocampus, however, may reflect continued metabolic injury long after the ischemic episode has passed.

Commentary on “Alzheimer's disease may begin in the nose and may be caused by Aluminosilicates”

The suggestion that the nose is the portal of entry for aluminosilicates is considered to be implausible, but readily testable. The proposed mode of action of aluminosilicates in triggering changes in the structure and function of cerebral proteins is interesting but there are few convincing data supporting the model.

Pattern of labeling of rabbit cerebral cortex soluble proteins 24 hours after a single electroconvulsive shock.

The labeling of proteins in the particulate subcellular fractions and in the cell sap was studied in the cerebral cortex of rabbits 24 hours after a single electroconvulsive shock (ECS). To this end the animals were injected with [3H]valine subarachnoidally and sacrificed 30 minutes later. The incorporation of the labeled amino acid into proteins was slightly increased in all subcellular fractions but the increase was significant only for soluble proteins. Referring to the type of soluble polypeptides found in the cortex at this post ECS phase, no modification was found in the SDS electrophoresis pattern of steady state proteins. The fractionation pattern of labeled soluble proteins showed in one third (3 out of 9) of the experiments a remarkable stimulation of the synthesis of two polypeptides (25,000 and 54,000 Dalton MW), which were not labeled in the controls.

The influence of protein deprivation during development on amino acid incorporation into cerebral proteins.

The incorporation of 3H-valine into protein of cerebrum, liver, and muscle was studied in rats fed a low protein diet. Animals, 15-50 days old, were used to cover the period during and after the most rapid phase of cerebral protein synthesis. The precursor was administered in doses resulting in brain concentrations of free valine in the 0.1-0.3 mM range. Two series of experiments were run and the injected dose varied for comparison of protein synthesis at different concentrations of precursor valine. By concomitant amino-acid analysis it was shown that the concentration of other amino-acids in the brain was not changed by the injected valine. The changes in the amino-acid pool with age in control rats were in agreement with previous findings. Only small alterations were seen in the amino acid pool as an effect of protein deprivation, the results being consistent with those of some previous reports. Uptake of valine into the brain appeared to be decreased only in 15-17-day-old protein-deprived rats. Valine incorporation into cerebral protein was decreased in the youngest age group but no effect was seen in older animals. Liver protein incorporation was slightly decreased by low protein diet but a marked reduction was observed for muscle protein. The changes in brain protein synthesis during normal development were in agreement with previous studies and differed qualitatively from those in liver and muscle.

Regional protein synthesis in the rat brain during bicuculline-induced epileptic seizures.

The incorporation of L-[3,5-3H]tyrosine into cerebral proteins was investigated during the initial phase (30 min) of bicuculline-induced status epilepticus. Autoradiographs of different parts of the cerebral hemispheres, brain stem, and cerebellum were prepared. Marked local reduction of amino acid incorporation was evident in bilaterally symmetrical areas of the cerebral cortex, hippocampus, thalamic nuclei, and the region of the medial geniculate body. No apparent difference of local [3H]tyrosine incorporation was observed in the lower brain stem nuclei and in the cerebellum of control and convulsed animals. The territories showing a decrease of protein synthesis during epileptic seizures coincide largely with the regions of maximal local glucose metabolism and cerebral blood flow. The present investigation demonstrates that autoradiography of regional protein biosynthesis is a suitable method for the visualization of neuronal populations at risk in the very early stages of seizure activity.

Corticotropin-induced changes in protein labelling: lack of molecular specificity.

Previous studies in this and other laboratories have indicated that corticotropin (ACTH) administered to intact or hypophysectomized rats or mice increased the labelling of cerebral proteins by radioactive amino acids, presumably indicative of increased protein synthesis. This study was designed to reveal whether this increased labelling was specific for particular proteins by studying labelling patterns in SDS polyacrylamide gels using a double isotope procedure. Two strains of mice, two different amino acid precursors, leucine and lysine, and both peripheral and central administration of ACTH and amino acids were used. In no case were treatment dependent changes in labelling observed in any region of the gel. This included regions containing S-100 or NSP, two brain specific proteins whose content had been reported to be increased by treatment of rats with an ACTH analog.

Studies of memory: A reevaluation in mice of the effects of inhibitors on the rate of synthesis of cerebral proteins as related to amnesia

Tests were made of the postulates stating that the degree of inhibition of protein synthesis either (a) at training or (b) following training is the critical variable that determines the degree of amnesia. As a first step it was found that the concentrations of numerous cerebral amino acids were substantially increased in 2 strains of mice 0.5 hr after treatment with amnesic doses of the inhibitors of protein synthesis, cycloheximide (CXM) and anisomycin. This observation led, in several different experiments, to a comparison of the apparent degree of inhibition of protein synthesis derived from the acid-soluble radioactivity with that derived from the specific radioactivity of tyrosine tagged with L(1- 14C)-tyrosine. In all instances the apparent degree of inhibition was decreased when based upon tyrosine's specific radioactivity. The effect of several treatments with CXM on memory of a 1-trial passive avoidance task provided data for analysis of the relationship between the degrees of amnesia and those of the more accurate estimates of inhibition of protein synthesis based upon the specific radioactivity of tyrosine. The results failed to support the views that the level of inhibition of protein synthesis at or after training are entirely sufficient to account for the behavioral results. The results also suggest that CXM produces with time some direct or indirect change in the brain that antagonizes the amnesic effects of the antibiotic and that consequently contributes to the survival of memory in mice trained 2 hr after a large amnesic dose of CXM.

Changes in some physicochemical properties of cerebral cortical proteins during clinical death and resuscitation after mechanical asphyxia

During the period of clinical death from mechanical asphyxia in dogs, changes take place in the physicochemical properties of protein of the postimitochondrial supernatant from brain gray matter homogenates. The densitogram obtained by electrophoresis of the proteins in polyacrylamide gel is modified. The total protein concentration after centrifugation and total lactate dehydrogenase (LD) activity are reduced. The combined fraction of unadsorbed β and γ globulins is reduced after adsorption on DEAE-Sephadex A-50 and the activity of LD isozymes (3+4+5) is considerably reduced. The disturbance of the behavior of proteins in sedimentation and electrical fields and changes in their adsorption properties, showing specific features at different times of clinical death, play an important role in the formation of the irreversible changes in the gray matter of the brain tissue. In the early recovery period successive restoration of the adsorption and sedimentation properties of the protein is observed, whereas the electrophoretic spectrum does not return to normal during the first day.

Application of an improved cerebroventricular perfusion technique in the rabbit: Effects of pentobarbital or haloperidol on monoamine metabolites and proteins in the perfusate

An improved procedure for ventriculo-cisternal perfusion of conscious rabbits with artificial cerebrospinal fluid is described. Concentrations of homovanillic acid, a principal metabolite of dopamine, 5-hydroxyindolacetic acid, a major serotonin metabolite, and of endogenous proteins were determined in cerebroventricular perfusates before and after administration of either pentobarbital or haloperidol. Perfusate levels of 5-hydroxyindolacetic acid decreased during pentobarbital anaesthesia but increased following haloperidol whereas concentrations of homovanillic acid increased 50% after pentobarbital and 300% after haloperidol. The rate of cerebrospinal fluid formation was not influenced by either drug. Protein concentrations in the perfusate decreased substantially following either pentobarbital or haloperidol, thus suggesting that the action of these neuroactive drugs may be associated with changes in extracellular cerebral proteins. The perfusion technique is useful for studying experimentally induced metabolic changes in the intact brain.

Variations au cours de la journée de l'incorporation in vivo de la leucine tritiée dans les protéines du cervelet et du cerveau du jeune rat normal et hypothyroïdien. Daily variations of the in vivo [3H] leucine incorporation into the cerebellar and cerebral proteins of the normal and hypothyroid young rat [(author's transl)

In the normal and hypothyroid 6-day-old rat, the specific radioactivity (RSA) and the relative RSA (ratio of the RSA to the [3H] lecine concentration of the acido soluble phase) of the cerebral and cerebellar proteins, changes during the day synchronally. They show a maximum at 15.00 h and a minimum at 0.300 h. At all stages studied, these values are significantly lower in the hyothyroid animals than in normal ones.

Reduced incorporation of [ 3H]leucine into cerebral proteins after long-term ethanol treatment

Reduced incorporation of [ 3H]leucine into cerebral proteins after long-term ethanol treatment

Some cerebral proteins and enzyme systems in Alzheimer's presenile and senile dementia.

The water-soluble proteins of the cerebral gray matter and some enzyme systems (cholinesterase, acetylcholinesterase, lactate dehydrogenase, malate dehydrogenase, acid phosphatase) were studied in 9 autopsy cases of Alzheimer's presenile or senile dementia, 1 case of Pick's disease and 1 case of cerebral arteriosclerosis. The proteins and enzyme patterns were visualized on polyacrylamide gradient gels after electrophoresis. In all patients studied, the profiles of cerebral gray-matter proteins were normal. In the patients with advanced dementia, the enzyme patterns usually were abnormal. Particularly in Alzheimer's disease, the activity of malate dehydrogenase was markedly increased.

The effect of chronic self-administration of d-amphetamine on food intake, locomotor activity, and C14-leucine incorporation into cerebral cortex protein.

Rats had free access to 0.02% d-amphetamine solution instead of water for 23 days. The daily amphetamine consumption was found to increase from 16 mg/kg on day 1 up to 47 mg/kg on day 23. Tolerance to the anorexic effect of the drug was apparent on day 11. The initial depression in body weight persisted throughout the experiment. The hyperactivity of rats remained at the same level despite the daily increase in amphetamine intake. The incorporation of C14-leucine into cerebral cortex proteins was initially increased and returned to control level after 2 weeks of treatment. No direct correlation between hyperactivity and brain cortex protein synthesis was observed.