EFFECT OF HYPERPHENYLALANINEMIA ON VITAMIN B6, METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The turnover of the different forms of B₆ vitamers in the brains of normal and hyperphenylalaninemic preweanling rats was compared after administration of a load of [¹⁴C]pyridoxol. Metabolic transformations occurred in the following sequence: oxidation of pyridoxol to pyridoxal, which was in turn phosphorylated to the 5'-phosphate ester. No significant amount of pyridoxamine was formed during the 8-h experimental period. Pyridoxamine 5'-phosphate was derived from pyridoxal 5'-phosphate. The specific radioactivity of pyridoxal phosphate in the hyperphenylalaninemic brain was significantly lower and increased at a slower rate than in control brains. This difference could not be accounted for by either a deficient supply or inhibited activity of the enzyme, pyridoxal kinase. The synthesis of pyridoxamine 5'-phosphate in the experimental animals also lagged behind the controls. Decreased activity of enzymes dependent on pyridoxal phosphate as cofactor would explain the slower turnover of this B₆-coenzyme.     

EFFECTS OF LEUCINE ON BRAIN SEROTONIN

Abstract— The effect of excess leucine in the diet on serotonin metabolism in the brain was investigated in experimental animals. It was found that:
(1) Animals receiving diets containing 3 % and 8 % leucine and those receiving jowar diets had significantly lower levels of serotonin in the brain.
(2) Intraperitoneal administration of the precursor amino acid 5-HTP increased the serotonin concentration in brain in both control and leucine-fed animals. However, the serotonin concentration in leucine-fed animals was significantly lower than that of pairfed controls. Larger amounts of the synthesized serotonin were found to be catabolized in 3 hr in leucine-fed animals than in control animals.
(3) The in vitro uptake of [¹⁴C]5-HTP by brain slices of animals fed leucine was found to be similar to that of control animals.
(4) The basal concentration of 5-HIAA in brain was higher in leucine-fed animals, suggesting a higher rate of catabolism of serotonin.
(5) Administration of nicotinic acid resulted in a further fall of serotonin concentration in the brains of leucine-fed animals but not in control animals.     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

Abstract— The fatty acid composition of cerebrosides, sulphatides and ceramides has been determined at 20 days postpartum in the brains of Quaking mutant mice and of littermate controls. There was a significant deficit in the proportion of long-chain fatty acids (C₂₂-C₂₄) affecting both normal and a-hydroxy fatty acids of the cerebrosides. The proportion of normal but not the a-hydroxy long-chain fatty acids of the sulphatides was also decreased. Striking and disproportionate deficits of the C_24:1 and C_{24 h:1} fatty acids of cerebrosides, sulphatides and ceramides characterized the brain of the Quaking mutant, and an increased proportion of C_{23 h:O} fatty acid was found in the cerebrosides and sulphatides of the brain of this mutant. We compared these data with findings on the Jimpy mutant which has been examined by the same techniques. The deficiency of long-chain fatty acids which was found in the cerebrosides and sulphatides of both mutants was less extensive but more selective in the Quaking mutant.     

THE BINDING OF 5-HYDROXYTRYPTAMINE TO BUTANOL EXTRACTS OF RAT BRAIN STEM

Abstract— When butanol-water extracts of rat brain stem were incubated with [³H]5-HT, (5 × 10⁻⁷ m ), and the components resolved by chromatography on LH₂₀ Sephadex, a peak representing approximately 70% of the eluted radioactivity was found in chloroform-methanol 4:1. The peak was not found in identically prepared extracts from rat diaphragm, neither was a similar peak found when brain extracts were incubated with [¹⁴C]ACh (10⁻⁶ m ), suggesting a degree of selectivity. Binding was not saturated at concentrations of 5 × 10⁻⁵ m -5-HT. The binding was highly sensitive to the presence of water, requiring about 15% (v/v) for optimum binding. The implications of these findings are discussed in terms of a possible '5-HT receptor'.     

COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal 'ghost', and microsomal fractions of white and grey matter. Ester-type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine- and serine-containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C₁₈ and C₂₀ homologues.
The possibility is discussed that microsomes furnish characteristic lipids for the synthesis or renewal of specific membranes, and that these lipids are accumulated somewhat before being released.     

THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—II

Abstract— The levels of tritiated catecholamines and metabolites were measured in regions of the rat brain at intervals after the intraventricular injection of [³H]dopamine, [³H]nor-adrenaline and [³H]normetanephrine. The disappearance of catecholamines and appearance of metabolites with time and the regional turnover rates of these amines indicate that the major pathway of the metabolism of noradrenaline and dopamine actively released from physiological storage sites is to the neutral alcoholic metabolites. The acid metabolites, homovanillic acid and 3,4-dihydroxyphenylacetic acid appear to be only minor products of normal dopamine metabolism in rat brain regions including the striate, but are the main end products of the metabolism of excess exogenous dopamine.
The active metabolism of stored noradrenaline to alcohol metabolites is also indicated by the increase in neutral alcohol metabolites accompanying the increased noradrenaline turnover when rats were subjected to electroshock stress. Therefore in the rat brain, neutral alcohol metabolites of dopamine and noradrenaline have great significance in the study of physiological catecholamine turnover in any region.     

THE UPTAKE OF PURINES BY RAT BRAIN IN VIVO AND IN VITRO

Abstract— The uptake of [¹⁴C]guanine and some of its [¹⁴C]-labelled derivatives into rat brain was studied in vivo and in vitro. In vivo guanine, guanosine, and hypoxanthine penetrated the brain of adult rats to a very small extent. Inosine was taken up somewhat better. In young animals, also, guanosine was taken up poorly, but guanine was taken up fairly well. When guanine was administered to adult animals, only guanine was found in the brain. In young animals, by contrast, radioactivity from guanine appeared in guanosine and in guanine nucleotides, but no free guanine was found. In vitro guanine was taken up much better and, in fact, remained mostly as guanine in slices from 10-day-old rats. The in vitro conversion of guanine to GMP and its incorporation into RNA was unimpaired by the addition of unlabelled guanosine, an indication that guanine was converted directly to GMP. The uptake of guanine in vitro was not subject to competitive inhibition or influenced by the presence of dinitrophenol. This finding suggested that guanine entered the slice by simple diffusion.     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.     

INFLUENCE OF ANAESTHETICS ON THE BALANCE BETWEEN PRODUCTION AND UTILIZATION OF ENERGY IN THE BRAIN

Abstract— The influence of general anaesthesia upon the metabolic state of the brain was evaluated from the tissue concentrations of ATP, ADP and AMP, and from the concentrations of glycolytic and citric acid cycle intermediates, in immobilized and artificially ventilated rats anaesthetized either with 70% N₂O, 1% halothane or 60 mg/kg of pentobarbitone. The results were compared to the results obtained on awake animals in fentanyl-analgesia. The adenylate energy charge was identical in all groups studied and there were no H⁺-independent changes in the phosphocreatine/creatine ratios. In pentobarbitone anaesthesia there was an accumulation of glucose 6-phosphate and a fall in fructose 1,6-diphosphate, indicating inhibition of phosphofructokinase. No significant changes in these metabolites were observed with halothane or nitrous oxide anaesthesia and the substrate patterns differed from that obtained with pentobarbitone.
The blood glucose concentrations were higher in the unanaesthetized, immobilized rats given fentanyl than in those anaesthetized. There was a direct relationship between the glucose concentrations in blood and in tissue. The glucose concentration ratios intracellular water to blood were higher in the anaesthetized than in the unanaesthetized animals, increasing with increasing depth of anaesthesia. The intracellular lactate concentrations were lowest in the groups given pentobarbitone and fentanyl citrate, and there was thus no direct relationship between lactate concentration and depth of anaesthesia.     

RAPIDLY LABELED AND SECRETED PROTEINS OF THE CHICK BRAIN

Abstract— The extracellular and cerebrospinal fluids (ECF) of the chick brain were found to contain a distinctive group of rapidly labeled proteins. Gel staining patterns suggest that most ECF protein bands correspond with components also found in either the homogenized whole brain cytoplasmic fraction or the blood serum. The valine-incorporation profiles of these three fractions, however, were entirely distinctive. Comparisons were carried out using a sensitive double-labeling method, in which ECF proteins from chicks labeled for 1 h with [³H]valine were comigrated on SDS-polyacrylamide gels with the cytoplasmic or serum proteins from a ¹⁴C-labeled animal. Analyses of the ³H- and ¹⁴C-labeling profiles from these gels showed that certain newly-synthesized proteins are heavily enriched in the ECF relative to the other two fractions. Most prominently, material with an apparent molecular weight of # 17,000 was found to incorporate nearly one-third of all the radioactivity appearing in the ECF proteins, but was not heavily labeled in either the cytoplasmic or serum fractions. The effects of a simple training experience on the pattern of chicks' brain protein synthesis were also examined.     

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.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

EFFECTS OF ACETYLCHOLINE ON THE INCORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF NERVE-ENDING PARTICLES FROM GUINEA PIG BRAIN

Abstract— Guinea pig brain nerve-ending particles (synaptosomes) were incubated with [³²P]orthophosphate in a medium with or without 10⁻⁴M-acetylcholine and 10⁻⁴ M-eserine. Phospholipids were then extracted and separated by chromatography. About 60 per cent of the ³²P was found in phosphatidic acid and about 20 per cent in triphosphoinositide. Acetylcholine significantly increased the specific radioactivity of phosphatidic acid but had no effect on that of phosphatidylinositol or the nucleotide fraction. Labelling of the other phospholipids, including diphosphoinositide and triphosphoinositide, was not altered significantly by acetylcholine. Labelling of the nucleotide fraction and the polyphosphoinositides reached a peak at 40 min, that of phosphatidic acid at 80 min, while that of phosphatidylinositol was still rising at 160 min.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]labelled material into subcellular fractions of 30-day-old rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C] mevalonic acid. As in the adult and 15-day-old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15-day-old animal, the myelin fraction from the 30-day-old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [¹⁴C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non-myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non-myelin to myelin fractions at about 37–44 days of age.     

CONTRIBUTION OF THE PENTOSE CYCLE TO THE METABOLISM OF GLUCOSE IN THE ISOLATED, PERFUSED BRAIN OF THE MONKEY

Abstract— Isolated brains from three adult monkeys were perfused for 1 hr with [2-¹⁴C]glucose. Glycogen was isolated from the brain stem, cerebral hemispheres, cerebellum and the hypothalamic area at completion of the perfusions. The distribution of ¹⁴C in carbons of the glucose unit of glycogen was determined and from this the contribution of the pentose cycle to metabolism of glucose was calculated. The data indicate a maximum contribution by the pentose cycle of 5–8 per cent in brain. No significant difference was observed in the various portions of brain. Oxygen consumption was noted to be low in relation to the amount of glucose utilized, as measured in these experiments.     

THE SULPHATION OF VANILYLMANDELIC ACID AND HOMOVANILLIC ACID BY LIVER, SMALL INTESTINE AND BRAIN

Abstract— The sulpho-conjugates of vanilylmandelic acid (VMA) and homovanillic acid (HVA) were biosynthesized in vitro from ATP and sodium sulphate and from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) obtained commercially or generated in vitro . Hydrolysis with sulphatase indicated the sulphate nature of these conjugates. In decreasing order of activity, the liver, small intestine and brain of various animals exhibited this sulpho-conjugatory ability. The K _m, values of VMA and HVA for the sulphotransferase of mouse liver were, respectively, 1740 and 93 μM. Competition studies suggested that the same enzyme may be involved in the sulphation of these two acids.     

INHIBITION PATTERN BY ANALOGS INDICATES THE PRESENCE OF TEN OR MORE TRANSPORT SYSTEMS FOR AMINO ACIDS IN BRAIN CELLS

Abstract— We surveyed the transport systems present in the brain for amino acids. Cellular transport was measured in brain slices, and capillary transport was estimated by measuring in vivo the short-term (15 s) extraction by brain from the blood. Specific analog inhibition of uptake was used to distinguish the classes. Amino acid levels (close to physiological) were such that primarily the 'low-affinity' transport was measured.
In brain tissue we could distinguish 10 overlapping amino acid transport classes. Five of these, described in a number of tissues, were characterized by their substrates: alanine (A system), leucine (L system), alanine-serine-cysteine (ASC system), glutamic acid (Glu system), and arginine (Ly⁺ system), respectively. The others distinguished were each fairly specific for one of the following five amino acids: glycine, proline, γ-aminobutyric acid (GABA), taurine, and lysine. Of these 10 systems only 4 could be clearly found in capillary transport: L, ASC, Ly ⁺, and Glu.
The properties and the distribution of the transport systems are different. Examples are that at least one of the systems is present primarily only in neurons (GABA), and one primarily in glia (taurine). The specificity of some of the systems, e.g. A, is altered during development. In contrast to the properties of most other systems, there is little Na⁺, energy, or temperature dependence of the L system. This is reflected in the properties of capillary neutral amino acid transport when the L system is the predominant one.     

ELECTROSHOCK-INDUCED SEIZURES AND THE TURNOVER OF BRAIN PROTEIN IN THE RAT

Abstract— A total of ten electroshock seizures (two seizures per day) were induced in rats beginning 3 days after an injection of [U-¹⁴C]glucose. Despite the intense stimulation, the labelling of the protein and nucleic acid fractions in the brains of convulsed animals decreased only slightly and not significantly. During the first 2 days after administration of [¹⁴C]glucose to untreated animals, there was a slight decrease in the specific activity of protein-bound glutamic acid relative to that of aspartic acid and the total protein fraction, suggesting the presence of a protein with a high content of glutamic acid and a rapid turnover.     

SUBCELLULAR DISTRIBUTION OF B6 VITAMERS IN CEREBRAL CORTEX

Abstract— The distribution of B₆ vitamers in subcellular fractions of cerebral cortex was examined. No pyridoxine and only traces of pyridoxamine could be detected in cerebral cortex. Significant quantities of pyridoxal were found in the cytoplasmic fraction. No vitamin B₆ was detected in the subcellular fractions carrying microsomes, myelin, vesicles, and small membranes settling above the 1-0 M-sucrose gradient. Pyridoxamine phosphate was the predominant form of vitamin B₆ in cerebral cortex of mature rats and guinea pigs, being present in almost twice the concentration of pyridoxal phosphate. More of the latter compound was present in the cytoplasm than in the mitochondria. In contrast, pyridoxamine phosphate was compartmentalized in extraterminal and intraterminal mitochondria. Of especial interest was the finding that there was a significant amount of pyridoxamine phosphate attached to the presynaptic membrane and a small but detectable amount in the fraction containing postsynaptic membranes.