EFFECT OF SENSORY STIMULATION ON AMINO ACID INCORPORATION INTO BRAIN PROTEINS IN VIVO

Abstract— Stimulation (AES) of the brachial plexus of anaesthetised rats resulted in an increased incorporation of carbon from [U-¹⁴C]glucose into TCA-insoluble proteins in the contralateral cerebral hemisphere, as compared with the ipsi-lateral hemisphere. The greatest change was observed in the sensori-motor cortex grey matter.
Following intraventricular injections of [U-¹⁴C]glucose, the changes caused by brachial plexus stimulation were variable, depending on which hemisphere received the label. The injection itself severely inhibited the incorporation into protein. Neither the injection, nor stimulation affected the conversion of [U-¹⁴C]glucose into amino acids or its relative distribution between the two hemispheres.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

INCORPORATION OF AMINO ACIDS INTO PROTEINS IN NEURONAL AND NEUROPIL FRACTIONS OF RAT CEREBRAL CORTEX: PRESENCE OF A RAPIDLY LABELLING NEURONAL FRACTION

Abstract— The time course of incorporation of intraperitoneally injected [³H]lysine and [¹⁴C]phenylalanine into neuronal and neuropil proteins has been followed for up to 8 days. At short times after injection (<2 h) the specific activity of the neuronal fraction was higher than that of the neuropil. At longer time intervals, although the total brain specific activity continued to rise, neuronal perikaryal specific activity fell below that of neuropil. Thus the neuronal/neuropil incorporation ratio with [³H]lysine as substrate was 1·5 at 1 h, but by 4 h had fallen to 0·4, a ratio which was maintained for up to 8 days. A similar reversal occurred with phenylalanine as substrate. These changes were interpreted as evidence for the presence of a rapidly-labelling protein fraction in the neurons which is subsequently transported out. Subcellular fractionation showed that over the 4 h period the rapidly labelling fraction was not transported to the synaptosomes. Incubation of prelabelled cortex slices followed by cell fractionation showed that a differential transport of protein of higher than average specific activity from both neurons and neuropil fractions occurred; there is a tendency for preformed highly labelled protein to accumulate during the in vitro incubation in Fraction D, a pellet enriched in red cells, some large neuronal perikarya and cell nuclei. When cell fractions were prepared after in vitro incubation, the distribution of the material down the gradient differed from that when fresh tissue was fractionated, as demonstrated by microscopic examination and the distribution of β-galactosidase, a neuronal marker. Double-label experiments showed that this redistribution could not account for the preferential loss and accumulation of prelabelled protein. It was noted that in vivo incorporation into the rapidly labelling neuronal protein is suppressed under certain changed environmental conditions, such as dark rearing. This is interpreted as lending support to the concept of the state-dependence of neuronal and neuropil protein synthesis and their inter-relations.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

THE EFFECT OF SLEEP DEPRIVATION UPON THE IN VIVO AND IN VITRO INCORPORATION OF TRITIATED AMINO ACIDS INTO BRAIN PROTEINS IN THE RAT AT THREE DIFFERENT AGE LEVELS

Abstract— The effect of sleep deprivation on the in vivo and in vitro tritiated amino acid incorporation into brain proteins was studied in the rat at three age levels. Sleep deprivation was induced either by water tank or handling methods. Three experimental groups of animals were used: control, sleep deprived and post deprivation sleeping rats.
A significant decrease of protein synthesis was found in the cerebellum, telencephalon and in crude subcellular fractions of brainstem of adult rats selectively deprived of paradoxical sleep. However, no alteration of protein synthesis was observed either in vivo or in vitro , in the same brain regions or in the liver after the rebound of paradoxical sleep following deprivation.
In four crude subcellular protein fractions a specific increase of the in vitro labelled amino acid incorporation was observed in the brain stem of 24-day-old rats allowed to recuperate after sleep deprivation as compared with the deprived rats. No significant changes were seen in the telencephalon.
No alteration of incorporation was found in 7-day-old rats deprived of sleep.
The possible functional significance of these results is discussed in relation to stress and to variations in the size of the precursor pool for protein synthesis.     

INCORPORATION OF PHOSPHATE INTO RAT BRAIN DURING SLEEP AND WAKEFULNESS

Abstract— Labelled inorganic phosphate (³²P₁) was administered intraventricularly to unrestrained sleeping and waking adult rats. After about 20 min of sleep or a comparable period of wakefulness, as monitored by EEG and EMG, the animals were frozen in liquid nitrogen and the brains were analysed. One group of animals (A) was not previously acclimatized to the apparatus. A second group (B) was acclimatized. The specific radioactivity of a phosphoprotein fraction was elevated during sleep in group A but not in group B. The specific radioactivity of the phosphatides of group B was depressed in sleeping as compared with waking animals. This effect was not observed in group A. No significant difference was detected between the EEG patterns of sleeping animals in groups A and B, as evaluated by standard criteria. These observations suggest that the physiological conditions attributable to environmental, emotional or other determinants can influence shifts in brain metabolism during the sleep-wakefulness cycle.     

EFFECTS OF SPREADING CORTICAL DEPRESSION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEINS OF RAT BRAIN

Abstract— Incorporation of dl -[1-¹⁴C]leucine into proteins of the cerebral cortex of the rat was measured during spreading cortical depression (CSD) evoked by a single topical application of 25% (w/v) KCI. Maximal inhibition (42 per cent) of the rate of incorporation occurred 1 hr after application of KCI. Spreading depression of 2–3 hr duration was associated with 22 per cent and 13 per cent decreases, respectively, of incorporation of labelled leucine. Specific activity of the free pool leucine was not decreased during CSD but appeared to be higher than controls at 20 min after initiation of CSD. The specific activity of the total free pool amino acids was also increased at 10, 20, 60 and 120 min after application of KCI.
The inhibitory effect of CSD on incorporation of leucine into proteins was uniformly distributed among the crude mitochondrial, microsomal and soluble subcellular fractions from brains of adult animals, while in fractions from 25-day old animals there appeared to be relatively more inhibition in the crude mitochondrial fraction.     

INCORPORATION OF RADIOACTIVE SULPHATE INTO SULPHATIDE DURING MYELINATION IN CULTURES OF RAT CEREBELLUM

Abstract— Explants of rat cerebellum obtained 12-24 h after birth were maintained in culture in Maximow assemblies. About 90 per cent of the cultures myelinated after 10–12 days in vitro . Cultures maintained for varying periods of time were exposed to [³⁵S]Na₂SO₄; labelled sulphatide was recovered from the total homogenate. Preparation of a subcellular fraction with density properties corresponding to those of myelin indicated that labelled sulphatide appeared in this fraction. Cultures which were poorly-myelinated always exhibited a lower rate of inccrpomtion than well-myelmated cultures from littermate animals, but the distribution of labelled sulphatide into the 'myelin' fraction was similar in the two groups. The rate of incorporation of [³⁵S]Na₂SO₄ into total sulphatide increased with the duration of the culture, with a low level of incorporation until the seventh day in vitro , followed by a sharp increase in rate up to the 21st day. This pattern resembles that observed for rat cerebellum in vivo .     

EFFECTS OF ENVIRONMENTAL COMPLEXITY ON AMINO ACID INCORPORATION INTO RAT CORTEX AND HIPPOCAMPUS IN VIVO

Abstract— Amino acid incorporation in vivo was investigated in the cortex and hippocampus of rats raised in enriched and deprived environments for various periods of time following weaning. At early times after weaning (7 days), the incorporation of l -[³H]leucine into all sub-cellular fractions of both cortex and hippocampus was higher in enriched than in deprived rats. At 16 days, incorporation into synaptosomal sub-cellular fractions was higher in enriched than in deprived hippocampus, and lower in enriched than in deprived cortex; incorporation into perikaryal fractions of both brain regions was the same in the two groups of animals. Incorporation into subcortical nuclear protein fractions was higher in enriched rats at this time. At 35 days, the only difference between enriched and deprived rats was a lower incorporation into cortical synaptosomal sub-fractions in the former. Experiments involving double labelling and electrophoresis indicate that there is no stimulation or inhibition of the synthesis of any particular protein in hippocampal nuclear and synaptosomal sub-fractions of enriched rats. Synaptosomal proteins of cortex have a greater half-life in enriched than in deprived rats; proteins of perikaryal fractions of cortex, and of all fractions of hippocampus, are turning over at the same rate in enriched and deprived animals.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

SUBCELLULAR DISTRIBUTION OF RADIOACTIVITY IN NEURONAL AND GLIAL-ENRICHED FRACTIONS AFTER INCORPORATION OF [3H]LEUCINE IN VIVO AND IN VITRO

Abstract— The distribution of protein-bound radioactivity among subcellular organelles of cerebral cortex was followed after intravenous administration of [3H]leucine and after incubation of brain slices in the presence of [3H]leucine. Neuronal and glial cell-enriched fractions were prepared by discontinuous sucroseFicol1 gradient centrifugation of cerebral cortex cell suspensions. Subcellular fractions were obtained from each of the cell prepara- tions and the protein-bound radioactivity determined after in uiuo and in vitro incorporation of [3H]leucine. The unfractionated neuronal material had a considerably higher level of protein-bound radioactivity than the glial material. The most marked neuronal-glial dif- ferences were observed in microsomes and soluble proteins, while the radioactive labelling of the nuclear and mitochondria1 fractions was similar for the two cell types.     

EFFECT OF ELECTROSHOCK ON THYMIDINE INCORPORATION INTO RAT BRAIN DNA

Administration of electroconvulsive shocks in rats induces a marked inhibition of the process of thymidine incorporation into cortical DNA. The inhibitory effect reaches its maximum after 10–15 min. Percentage incorporation returns to normal values within one hour of the treatment. These findings may suggest the participation of a metabolically active fraction of DNA in neural functions.     

BIOSYNTHESIS AND BIODEGRADATION OF RAT BRAIN GANGLIOSIDES STUDIED IN VIVO

Abstract— Metabolic relationships between the four major brain gangliosides, GM1, GD1a, GDlb and GT1 were studied in vivo . Labelled acetate and glucosamine were injected intracerebrally into 6–12-day-old rats and the radioactivities of the cerebral gangliosides were analysed. Radioactivity from [³H]acetate was determined in sialic acid, sphingosine and stearic acid and from [1-¹⁴C]glucosamine in hexosamine and sialic acid. The gangliosides were labelled in proportion to their pool size. In 6 day-old rats the labelling was approx. 30 per cent lower in the sialidase-stable sialyl group than in the labile one. When the brain gangliosides were labelled in 12-day-old rats, however, the specific activities of sialidase-labile and stable sialyl groups were the same at 0.5 months after the injection of precursors and disappeared at the same rate. The results indicate that at the age of 6 days a small pool of monosialogangliosides exists, which is converted to di- and trisialogangliosides. The degradation of gangliosides was studied by following the radioactivities in sphingosine and stearic acid from 2 to 6 months after the injection of labelled acetate. The specific activities of sphingosine and stearic acid decreased simultaneously at the same rate in all the four major gangliosides. The specific activity of stearic acid was the same in total brain lipids as in gangliosides. The half-lives for the degradation of the gangliosides were age-dependent and estimated to 60 days in adult rats. They were much shorter in younger rats but no reliable figures could be determined.     

NUCLEAR PROTEINS IN BRAIN OF 7-DAY-OLD AND ADULT RATS

Abstract— The incorporation of radioactive leucine into proteins of rat brain was considerably higher in the 7-day-old than in the adult rat. The greatest difference in the rate of protein synthesis between these two stages of development occurs in the mitochondrial fraction. Among the nuclear proteins the largest variation was in the histones. The only difference in the relative content of nuclear proteins between 7-day-old and adult brain was in the acidic deoxyribonucleoproteins which was higher in the younger animal. The electrophoretic profile of these proteins changed during brain development.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

AMINO ACID INCORPORATION IN VITRO INTO PROTEIN OF NEURONAL AND GLIAL CELL-ENRICHED FRACTIONS

Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.