Experimental alcoholism in rats. Effect of acute ethanol intoxication on the in vitro incorporation of ( 3 H)leucine into neuronal and glial proteins

Abstract— Ethanol administered in vivo or in vitro during incubation of brain slices was studied with respect to its effect on brain protein synthesis. In the in vivo series the rats were given a single intraperitoneal injection of ethanol 3 h before death. Slices of cerebral cortex and liver were incubated in isotonic saline media containing [³H]leucine. Amounts of free and protein-bound radioactivity were determined. Subcellular fractions and fractions enriched in neuronal perikarya and in glial cells were prepared from cortical slices subsequent to incubation, and the specific radioactivity determined for each cell type. The incorporation of [³H]leucine into brain proteins was inhibited while incorporation into liver proteins was stimulated in ethanol-treated rats. The levels of TCA-soluble radio-activity, however, did not differ between the ethanol group and the controls. In the fractionated material from cerebral cortex, the specific radioactivity in the neuronal fraction was unaffected by ethanol, while the radioactivity in the glial fraction was significantly depressed. In vitro administration of ethanol induced a non-linear response in both brain and liver, with depression of leucine incorporation into proteins of cerebral cortex at all concentrations used. When brain slices were exposed to ethanol in vitro, in concentrations corresponding to the in vivo experiments, a similar reduction of the leucine incorporation into the glial fraction was obtained. Incorporation of leucine into subcellular fractions from whole brain cortex was also investigated. The specific sensitivity of the glial fraction to ethanol is discussed in relation to the involvement of the different cell types with transport processes in the brain.     

CEREBRAL ANOXIA: EFFECT ON TRANSCRIPTION AND TRANSLATION1

Abstract— The activity of DNA-dependent RNA polymerase and the synthesis of microsomal protein were investigated after various periods of anoxic condition produced with rabbit brain in an in vitro experimental model. There was prompt inhibition of protein synthesis even after an anoxic period of 5 min, and inhibition was more than 80 per cent after an anoxic period of 30 min. However, RNA polymerase activity was retained during the early stage of anoxia, but definite inhibition appeared after an anoxic period of 15 min. Comparisons with other available information suggest that the inhibition of protein synthesis observed with brain slices is closely related to their polysomal function, that irreversibility of inhibition of protein synthesis might be related to the involvement of nuclear RNA synthesizing mechanism, and that these can occur both in the neuronal and glial elements.     

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.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

FURTHER EVALUATION OF POLYPEPTIDE SYNTHESIS IN CEREBRAL ANOXIA, HYPOXIA AND ISCHEMIA

The alteration of polypeptide synthesis was evaluated with microsomes isolated from anoxic rabbit, hypoxic rat and ischemic gerbil brains to estimate the extent of functional or structural changes in polyribosomes in situ and the extent of artifact during tissue preparation. By using two-stage experimentation with combination of control and pathological microsomes and supernatant, it was found that the previously observed effects on microsomal or polyribosomal polypeptide synthesis in the above pathophysiological conditions were mainly the reflection of the alteration of polyribosomes in situ rather than the artifact during tissue preparation by degradative processes. In support of this finding. the use of inhibitors of degradative enzymes did not significantly protect microsomes either in normal or in pathological conditions. It was noted that the decline of tissue pH, to a certain extent, could be correlated with dysfunction of polyribosomes both in situ and during tissue preparation in cerebral hypoxia and anoxia. Since there is little change in ATP level, it was postulated that the alteration of pH in situ is responsible for the observed suppression of polypeptide synthesis in vitro at least in cerebral hypoxia. This hypothesis was supported by the subsequent experiments with incubation of brain slices and homogenization of brain tissue under various pH. It was emphasized that the environmental biochemical elements surrounding polyribosomes in cytoplasm should be evaluated as possible contributing factors for polyribosomal dysfunction in such pathological conditions as cerebral anoxia, hypoxia or ischemia if the alteration of energy state does not explain the phenomenon entirely.     

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.     

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.     

Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage

Summary.  Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Effect of Passive Avoidance Training on In Vitro Protein Synthesis in Forebrain Slices of Day-Old Chicks

Slices from the forebrains of day-old chicks represent a highly active in vitro protein-synthesising system. The in vitro incorporation of L-[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell-soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (greater than 40 micron from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04-1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20-50% inhibition of incorporation of radioactive L-leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one-trial passive avoidance paradigm, L-[14C]leucine incorporation was 23% higher (p less than 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism.

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [14C]leucine into protein and had a decreased uptake of [14C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would be consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [14C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Leucine-nitrogen metabolism in the brain of conscious rats: its role as a nitrogen carrier in glutamate synthesis in glial and neuronal metabolic compartments

The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched-chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N-precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U-13C] and/or [15N]leucine as additions to the diet were killed at 0-9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine-N turnover was more than 100%/min. The isotopic dilution of [U-13C]leucine (brain/plasma ratio 0.61 +/- 0.06) and [15N]leucine (0.23 +/- 0.06) differed markedly, suggesting that 15% of cerebral leucine-N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 micro mol/g/h and at least 50% of glutamate-N originally derived from leucine. The enrichment of [5-15N]glutamine was higher than [15N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [15N]GABA, [15N]aspartate, [15N]glutamate greater than [2-15N]glutamine suggests direct incorporation of leucine-N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N-carrier from the plasma pool and within the cerebral compartments.     

TURNOVER OF PROTEIN PHOSPHORUS IN RESPIRING SLICES OF GUINEA PIG CEREBRAL CORTEX: CELLULAR LOCALIZATION OF PHOSPHOPROTEIN SENSITIVE TO ELECTRICAL STIMULATION

Abstract— In experiments designed to localize the increased turnover of phosphoprotein-P which occurs in respiring brain slices as a result of electrical stimulation, a cell separation procedure was used to prepare a fraction enriched in neuronal cell bodies from incubated slices labelled with [³²P]phosphate. Labelled phosphoprotein was found to be twice as concentrated in the neuron-enriched fraction as in other fractions. Electrical stimulation for 10 s increased the rate of incorporation of [³²P]phosphate into phosphoproteins in the neuron-enriched fraction by 25 per cent ( P < 0.05), but had no effect on incorporation into a partially purified glial fraction contaminated with neuropil and cell debris.     

Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study

Abstract— A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [¹⁴C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the ¹⁴C-labelled proteins in both cell types were greater, the younger the brain. The ¹⁴C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [¹⁴C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [¹⁴C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of ¹⁴C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [¹⁴C]phenylalanine, the specific radioactivities of all ¹⁴C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.     

On the mechanism of the inhibitory effect of potasium ions on amino acid incorporation into rat cerebral cortex proteins in vivo.

Earlier results on potassium ion inhibition of amino acid incorporation into the brain proteins in vivo (spreading cortical depression) led to the hypothesis that inhibition of protein synthesis is based on ATP deficiency. In the present study we tested various aspects of the aminocylation of tRNA, an ATP-dependent process, during spreading cortical depression produced by the topical application of 25% KCl. On using a 7-min interval between the subcutaneous injection of L-[U-14C] leucine and killing the rat, incorporation into the tRNA fraction was found to be reduced by 25%. Total amino acid radioactivity in the soluble fraction was unaltered. The acceptor capacity of tRNA, measured in vitro, and the proportion of non-acylated tRNA in vivo were likewise unchanged.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

A comparative study in vivo and in vitro of the ability of ribosomes from Xenopus liver and ovary to incorporate L-[U-14C]leucine

1. A system for the incorporation in vitro of amino acids into protein is described for the South African clawed toad (Xenopus laevis laevis Daudin). 2. The incorporation of l-[U-(14)C]leucine by Xenopus-liver microsomes is very much greater per mg. of microsomal RNA than the incorporation by ovary microsomes. 3. The incorporation by Xenopus-liver and -ovary polysomes is approximately the same when expressed per mg. of polysomal RNA. 4. It was predicted from the above results that ovary microsomes should contain a ribosomal fraction inactive in protein synthesis. This was shown to be the case by a labelling experiment in vivo with l-[U-(14)C]leucine. 5. The labelling experiment in vivo also showed that the active polysomal fraction in ovary is associated with membranes and is liberated by treatment with deoxycholate; this is also true of liver microsomes in vivo. 6. The results are discussed in relation to previous work on the synthesis of proteins by amphibian ovarian tissue, and on the role of bound and free ribosomes in protein synthesis.     

Some effects of aluminium on rat brain protein synthesis

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

1H-NMR study of the metabolome of an exceptionally anoxia tolerant vertebrate, the crucian carp (Carassius carassius)

The crucian carp (Carassius carassius) can tolerate anoxia for days to months, depending on the temperature. In this study, we applied ¹H-NMR-based metabolomics to polar extracts of crucian carp brain, heart, muscle and liver samples obtained from fish exposed to either control normoxic conditions, acute anoxia (24 h), chronic anoxia (1 week) or reoxygenation (for 1 week following chronic anoxia) at 5 °C. Spectra of the examined tissues revealed changes in several energy-related compounds. In particular, anoxic stress resulted in decreased concentrations of phosphocreatine (muscle, liver) and glycogen (liver) and ATP/ADP (liver, heart and muscle) and increased concentrations of lactate (brain, heart, muscle) and beta-hydroxybutyric acid (all tissues). Likewise, increased concentrations of inhibitory compounds (glycine, gamma-amino butyric acid or GABA) and decreased concentrations of excitatory metabolites (glutamate, glutamine) were confirmed in the anoxic brain extracts. Additionally, a decrease of N-acetylaspartate (NAA), an important neuronal marker, was also observed in anoxic brains. The branched-chain amino acids (BCAA) valine/isoleucine/leucine increased in all anoxic tissues. Possibly, this general tissue increase can be due to an inhibited mitochondrial function or due to protein degradation/protein synthesis inhibition. In this study, the potential and strength of the ¹H-NMR is highlighted by the detection of previously unrecognized changes in metabolites. Specifically, myo-inositol substantially decreased in the heart of anoxic crucian carp and anoxic muscle tissue displayed a decreased concentration of taurine, providing novel insights into the anoxia responses of the crucian carp.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.