MEASUREMENTS OF IN VIVO RATES OF PROTEIN SYNTHESIS IN BRAIN, SPINAL CORD, HEART AND LIVER OF YOUNG VERSUS ADULT RATS, INTACT VERSUS HYPOPHYSECTOMIZED RATS

Abstract— In vivo protein synthesis rates in rats were estimated by single i.p. injections of large quantities of [1-¹⁴C]valine. This method gives reliable estimates of the precursor specific activity and average protein synthesis rates. In the brain, spinal cord, heart and liver, the average rates for adults were 0.65, 0.42, 0.49 and 1.92% replacement of protein-bound amino acid per h. In the brain and liver of 10-day olds the average rates were estimated to be 1.46 and 3.12% per h respectively. Hypophysectomy decreased synthesis rates by 25% or more in all tissues studied. The disadvantages of the method are that applying large amounts of valine i.p. appeared to constitute a stress and that the valine solution required for injection was hypertonic, causing withdrawal of body fluids of the animal.     

A TECHNIQUE FOR MEASURING BRAIN PROTEIN SYNTHESIS

Abstract— Mice were infused intravenously for varying periods of time with L-[U-¹⁴C]- tyrosine. The specific activity of free tyrosine in the blood and the brain, and of protein-bound tyrosine in the brain, was measured and the mean rate of protein synthesis calculated. The half-life of mixed brain proteins was found to be close to 4 days with infusions lasting 0.5–2 h.
The origin of the intracellular tyrosine pool was investigated and it was shown that 60 per cent of this was derived directly from the plasma tyrosine and 40 per cent from protein breakdown within the tissue.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

MEASUREMENTS OF RATES OF PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The use of tracer concentrations of labelled amino acids to measure incorporation in incubated slices of brain results in wide fluctuations with time in the specific activity of the precursor. Using concentrations of about 1 mm of labelled amino acid facilitates the accurate measurement of rates of synthesis. These higher precursor levels in the medium decrease the fluctuations in free amino acid specific activity due to dilution by endogenous amino acid and the production of amino acid by protein degradation, and decrease the lag in incorporation due to transport phenomena. Concentrations of 1 mm amino acid in the medium did not inhibit protein synthesis; with valine, leucine, phenylalanine, lysine and histidine, incorporation rates were similar when measured at trace concentrations and at 1 mm medium levels. The source of amino acid for protein synthesis appears to be intracellular. No evidence could be found for the preferential use of extracellular medium amino acid. The rate of incorporation of amino acids in incubated slices of rat brain was 0.087 per cent of the protein amino acid/h.     

PROTEIN SYNTHESIS IN NORMAL AND SCRAPIE MOUSE BRAIN

Damage to the brain as a result of an intracerebral injection of physiological saline does not appreciably affect the rate of protein synthesis in mouse brain. The in vivo and in vitro incorporation of labelled amino acids into mitochondria and their in vivo incorporation into nuclei, microsomes, nerve ending particles, myelin and cell sap were compared in normal and scrapie-affected mice. No significant differences were found.     

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.     

EFFECTS OF DIFFERENT LEVELS OF DIETARY PROTEIN ON BRAIN GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN YOUNG ALBINO RATS

Abstract— Studies were carried out to identify the minimum levels of protein (casein) needed in the diet in order to prevent or reverse the deficits in brain enzymes previously found with protein deficiency. Groups of weanling albino rats were fed diets containing variable amounts of protein (5, 8, 10, 15 or 20 per cent in experiment I, and 5, 6, 7, 8 or 20 per cent in experiment II) for 5 or 10 weeks. Deficits in brain wt and brain glutamate dehydrogenase and decarboxylase were found to be prevented by a diet containing 8 per cent or more of protein, although for optimum growth 15 per cent protein in the diet was found to be necessary. Groups of rats were fed a 5 or 20% protein diet for 10 weeks after which the 5% protein animals were either continued on the diet for another 10 weeks or changed to one containing 8, 10, 15 or 20% protein. The brain enzyme deficits found with the 5% protein diet were found to be fully reversed by feeding a 10% protein diet during rehabilitation.     

TURNOVER OF PHOSPHATIDYLCHOLINE IN MICROSOMES AND MYELIN IN BRAINS OF YOUNG AND ADULT RATS

We have tested the hypothesis that the turnover of phosphatidylcholine in subcellular fractions of rat brain is a function of the age at which this lipid is deposited. Rats, 60 days of age, were injected intracranially with [2-³H]glycerol and either [methyl-¹⁴C]choline (to label the base moiety) or [U-¹⁴C]glucose (to label acyl moieties). Littermates were killed up to 90 days after injection and brain microsomes and myelin isolated. Lipids were extracted and the phosphatidylcholine was isolated by 2-dimensional TLC and hydrolyzed to its constituent moieties. The ³H in the glycerol backbone and ¹⁴C in the choline or acyl residues was quantitated. The microsomal and myelin ³H/¹⁴C ratios decreased with time with either set of precursors, indicating that labeled choline and acyl moieties were reutilized more efficiently than the glycerol backbone. The various precursors exhibited first order decay curves with half-lives for the glycerol backbone of 6 and 11 days for the microsomal and myelin fractions respectively. These results contrast with those previously obtained with identical experimental procedures when 17-day-old animals were injected. In that study, although much of the phosphatidylcholine turned over rapidly as for the older animals, by 2 weeks after injection most of the remaining phosphatidylcholine was turning over more slowly with a half-life of 13 and 25 days for microsomes and myelin respectively (Miller et al., 1977). The base and acyl moieties also had a corresponding shorter half-life in older animals relative to the slow turnover phase in younger rats.     

HYPOXIC SURVIVAL OF NORMOGLYCAEMIC YOUNG ADULT AND ADULT MICE IN RELATION TO CEREBRAL METABOLIC RATES12

The hypoxic tolerance and the cerebral metabolic rates (CMR) of young adult mice (20 to 25 g, 4 to 5 weeks old) and adult mice (30 g and above, 6 to 7 weeks old), respectively, were determined and their interrelationship was evaluated. CMRs increased from 25 mmol - P/kg.min to 38 mmol/kg.min as the animals grew older from young to full adulthood. Concurrently the tolerance to aerogcnic hypoxia (5% O₂-95%j N₂) declined. The effects of hypoxia on the cerebral energy metabolism were greater in adult than in young adult animals. It is concluded that the full metabolic maturation of the brain is reached in adult animals only. They become more dependent on an adequate oxygen supply as the aerobic activity of the energy metabolism of the brain is further increasing. Hypoxic gasping occurred while the pool of cerebral energy reserves was still far from being depleted. A failure to utilize energy reserves rather than their exhaustion is suggested as the ultimate cause of death from hypoxia. An acid-soluble form of glycogen or related polyglucan was found in addition to the usual amounts of insoluble glycogen. It was utilizcd rapidly during hypoxia and ischaemic anoxia and it may, therefore, constitute an additional source of carbohydrate substrates in thc brain.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

STUDIES ON THE RELATIONSHIP BETWEEN GABA SYNTHESIS AND PROTEIN SYNTHESIS IN BRAIN

Abstract— The synthesis of γ-aminobutyric acid (GABA) in mouse brain was decreased by treatment of the animals with pyridoxal phosphate- γ-glutamylhydrazone, an inhibitor of glutamate decarboxylase in vivo. Under these experimental conditions the following parameters were studied: (1) the incorporation of labeled leucine in vivo , into protein of brain subcellular fractions; (2) the brain polysome profile; (3) the incorporation of labeled leucine into protein in vitro , in ribosomal preparations isolated from brain tissue. In other experiments, GABA synthesis was also decreased in brain cortex slices by preincubation with aminooxyacetic acid. The incorporation of [³H]leucine or [¹⁴C]leucine into protein in these slices was studied, and samples from the proteins were subjected to acrylamide-sodium dodecylsulfate gel electrophoresis. Radioactivity was counted in slices of the gel. The results of the experiments in vivo and in vitro indicate that the previously reported decrease of protein synthesis induced by an inhibition of GABA synthesis affects proteins of all subcellular fractions and all populations of protein as separated by gel electrophoresis. The polysome profile from brains of mice with decreased GABA synthesis was similar to that of control mice. This result differs from that found when brain protein synthesis is inhibited by dopamine and serotonin.     

ENZYMES OF NUCLEIC ACID METABOLISM IN THE BRAINS OF YOUNG AND ADULT RATS

A number of precursors of RNA are incorporated several-fold more readily into the RNA of brain slices from 10-day-old rats than into RNA of slices from adult animals. The brains of the young animals show moderately higher levels of some of the anabolic enzymes of RNA metabolism including RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) and substantially lower levels of the degradative enzymes, the nucleoside phosphorylases. The data suggest that all the enzymes work in a concerted fashion to produce an increased rate of synthesis in young animals rather than that any single controlling enzymic event is responsible.     

A COMPARATIVE STUDY OF BRAIN AND LIVER MITOCHONDRIA FROM NEW-BORN AND ADULT RATS

• 1 A method has been developed for the estimation of organelle number in subcellular fractions and applied to the estimation of the mitochondrial content of brain and liver from new-born and adult rats.
• 2 The respiratory enzyme content per mitochondrion of the adult brain was 3·5 times greater than that of the neonate. This increase in enzyme content was not correlated with an increase in the mean size of the organelle.
• 3 The succinate dehydrogenase activity per mitochondrion of the livers from neonatal and adult rats showed no obvious change. There was, however, an increase in the mean size of the organelle.
• 4 These findings are discussed with reference to the development of the brain and to the development of mitochondria.

     

DIFFERENT TURNOVER RATES OF BRAIN RIBOSOMAL RIBONUCLEIC ACIDS IN MALE AND FEMALE RATS

Abstract— Following a single intracranial injection of [5-³H]orotic acid, the decay dynamics were determined for rRNAs of whole brain in male and female Wistar-inbred albino rats aged 2.5-3.5 months. The turnover rate for male brain rRNAs was significantly lower than in females (mean half-lives being, respectively, 12.2 ± 2.2 (S.D.M.) days, and 7.4 ± 1.3 days in four regression measurements). This difference was apparently not related to the turnover rate of acid-soluble brain nucleotides, which turned over much faster and at a similar rate in rats of both sexes; it also could not be connected with brain levels of rRNAs or DNAs, which were quite similar For males and females. The results are discussed in terms of possible sex hormone determination of brain RNA metabolic patterns especially in males.     

A DIRECT METHOD FOR DETERMINING DOPAMINE SYNTHESIS AND OUTPUT OF DOPAMINE METABOLITES FROM BRAIN IN AWAKE ANIMALS

Abstract— A direct method for measuring the rate of dopamine (DA) synthesis and the DA metabolites by the brain of awake monkeys ( Macaca arctoides ) is described. The method utilizes a coupling of a measure of cerebral blood flow with the mass spectrometrically determined difference in the concentrations of the metabolite under study in plasma obtained from arterial and internal jugular bulb blood. For homovanillic acid (HVA) a consistent and highly significant veno-arterial (V-A) difference of 2.2 ± 0.4 ng/ml of plasma ( P < 0.0005) was found. When this V-A difference was coupled with a measure of cerebral blood flow it was determined that, in the awake monkey, the average output of HVA by brain was 113.4 ± 19.1ng/100g brain min⁻¹. There were large individual variations, however, between animals (range = 38-194 ng/100g brain min⁻¹). In contrast to HVA, no consistent V-A difference for dihydroxyphenylacetic acid (DOPAC) was found; i.e. the concentrations of DOPAC in plasma obtained from arterial and internal jugular bulb venous blood were essentially identical. These data indicate that, in contrast to the rat, in this non-human primate HVA is the major metabolic product of brain DA. Since HVA is the major metabolite of DA, production of HVA under steady state conditions gives a measure of DA synthesis by whole brain; i.e. the rate of DA synthesis by whole brain in the awake monkey is 113.4 ± 19.1ng/100g brain min⁻¹. It is suggested that this technique may be of value in both basic and applied types of studies.     

NUCLEAR PROTEIN PATTERNS IN DEVELOPING AND ADULT BRAIN AND IN ETHYLNITROSOUREA-INDUCED NEUROECTODERMAL TUMOURS OF THE RAT1,2

In a comparative study, the patterns of histones and non-histone proteins were analysed in the chromatin of foetal (18th day of gestation), 10-day-old, and adult BD IX-rat brain, as well as in the chromatin of two ethylnitrosourea-induced neuroectodermal tumours (TV1A1 and GV1A1) and the corresponding malignant cell culture lines TV1C1 and GV1C1. Separation of nuclear proteins at high resolution was obtained by electrophoresis in 15% and 10% polyacrylamide gels containing urea (2·5 m or 6·25 m ). In spite of an overall similarity, significant quantitative and qualitative differences were observed between the respective non-histone proteins banding patterns of normal brain and the neoplastic cells analysed. The non-histone protein banding patterns of brain (∼40 different bands) at different stages of development revealed both quantitative differences and the presence of particular bands characteristic of foetal or adult brain, respectively. Both the‘foetal’and‘adult’non-histone protein bands also appeared in the electrophoretograms of the neoplastic neuroectodermal cells.     

BRAIN SEROTONIN CHANGES IN PHENYLALANINE-FED RATS: SYNTHESIS STORAGE AND DEGRADATION1

The brain serotonin levels of rats maintained on a 5 % phenylalanine diet rose more slowly (0.18 μ g/g brain/hr) after administration of a monoamine oxidase inhibitor than did serotonin levels of controls (0.41 μ g/g brain/hr). The rate of brain serotonin decline following reserpine or dimethylaminobenzoyl methyl reserpate was the same for both groups as was basal monoamine oxidase activity. Brain uptake of monoamine oxidase inhibitor was also the same for both groups. It was concluded that the decrease in brain serotonin levels in phenylalanine-fed animals was due to decreased serotonin formation rather than enhanced degradation. On the basis of available data it was concluded that both hydroxylase inhibition and inhibited precursor transport were involved.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.