FUCOSE INCORPORATION INTO GLYCOPROTEINS OF MOUSE BRAIN

—Radioactive fucose was incorporated into glycoproteins of brain in vivo. After intracerebral administration of this precursor, radioactive glycoproteins were the sole detectable product. The glycoproteins formed appeared to have a slow turnover but this was due, at least in part, to re-utilization of fucose released from degraded glycoproteins. Incorporation of fucose into glycoproteins differed from that of glucosamine, since a much smaller proportion of the radioactive fucose was incorporated into soluble glycoproteins. Fucose was rapidly incorporated into glycoproteins of nerve endings, although there was relatively little incorporation into glycoproteins associated with the soluble component of the nerve-ending fraction. As found in previous studies with glucosamine, soluble glycoproteins of nerve endings turned over relatively rapidly. Pretreatment with acetoxycycloheximide markedly inhibited incorporation of fucose into glycoproteins of brain. In contrast to the results with glucosamine, comparable inhibition was observed for fucose in all subcellular fractions of brain including the particulate and soluble components derived from the nerve-ending fraction.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

THE TURNOVER OF ISOTOPICALLY LABELLED DNA IN VIVO IN DEVELOPING, ADULT AND SCRAPIE-AFFECTED MOUSE BRAIN

Abstract— Tracer experiments using [³H]thymidine have shown that a large proportion of the DNA synthesized in control and scrapie-affected mouse brain is metabolically unstable. Although the turnover of mitochondrial DNA contributed to the loss of radioactivity from whole brain, it has been shown that 70 per cent of the labelled nuclear DNA was removed between 1 and 21 days after injecting the isotopic precursor. Observations on developing mouse brain, where the rate of DNA synthesis is far higher than that in adult brain, also revealed the existence of metabolically unstable DNA. Similar studies on developing and adult brain using [¹⁴C]thymidine indicated that most of the radioactivity lost in vivo was not due to radiation damage to newly labelled DNA molecules. Hydroxyapatite chromatography of heat denatured and renatured DNA from adult brain showed that the rates of turnover of the poorly, moderately and highly reiterated species of nuclear DNA were similar. The results of some dissection experiments have further shown that the observed breakdown of DNA in adult brain was not specifically associated with the turnover of subependymal cells. It is suggested that a metabolically labile fraction of nuclear DNA is present in developing and adult mouse brain and that the amount of tracer incorporated into this fraction is increased in mice infected with scrapie.     

EFFECTS OF OXYGEN INDUCED CONVULSIONS ON THE UPTAKE OF ACETATE INTO RAT BRAIN LIPIDS1,2

Abstract— Rats were exposed to 5 atmospheres absolute of oxygen, and [1-¹⁴C]acetate was injected into the jugular vein either before or at the onset of electroencephalogram-defined convulsions. Levels of ¹⁴C observed 2.2 min after the injection were reduced in the total lipids of brain and elevated in the blood of convulsed rats when compared to the nonconvulsed controls. These differences between convulsed and nonconvulsed animals were less pronounced when measured 15 and 60 min after injection. Convulsions did not change the amount of ¹⁴C incorporated into the total lipids of plasma during the 60 min period studied. Six fractions obtained from total lipid extracts of brain by TEAE-cellulose showed similar ¹⁴C distributions in convulsed and control animals. The results suggest that oxygen-induced convulsions cause an impaired utilization of systemically administered acetate for fatty acid incorporation into the lipids of brain.     

ELEVATED PLASMA PHENYLALANINE CONCENTRATION AND LYSINE INCORPORATION INTO RIBOSOMAL PROTEIN OF DEVELOPING BRAIN

Hyperphenylalaninemia was induced in 7-day-old rabbits over a 6-hr period by intraperitoneal injection of phenylalanine. l -[U-¹⁴C]Lysine was injected intraperitoneally into these rabbits and into a control group. The rate of incorporation of l -[U-¹⁴C]lysme into brain ribosomal protein was decreased during a 5-hr period in the presence of elevated plasma phenylalanine concentrations. Lysine transport from the peritoneum to the plasma was unaffected by the high plasma phenylalanine concentrations.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

THE INCORPORATION OF [1-14C]LINOLENATE INTO LIPIDS OF DEVELOPING RAT BRAIN DURING ESSENTIAL FATTY ACID DEPRIVATION

Abstract— Twenty-one-day old essential fatty acid (EFA) deprived rats incorporated about twice the radioactivity from [1-¹⁴C]linolenate into brain lipid fractions as did controls. At 5 min after injection, 2/3 of the radioactivity was associated with the less polar lipid fraction of both control and EFA deprived animals. By 30 min after injection, 70% of the radioactivity was in the phospholipid fraction. This value increased to 90% at later time points.
The specific activity of brain phospholipids from EFA deprived rats was always greater than that of controls. This held true for the individual phosphatide fractions also. In general, phosphatidylcholine (PC) was labeled most rapidly. With increasing time intervals, radioactivity was transferred to phospha-tidylethanolamine (PE) and phosphatidylserine + phosphatidylinositol (PS + PI).
The transfer of fatty acid radioactivity into phospholipid and the distribution of radioactivity among individual phosphatides did not appear to be affected by the dietary state. However, the total amount of radioactivity incorporated was related to the amount initially retained by brain after injection. Our data suggest that one or more components of the less polar lipid fraction may act as a 'trap' or reservoir for fatty acids which are required for phospholipid synthesis.     

COMPARTMENTATION OF GLUTAMATE METABOLISM IN THE DEVELOPING BRAIN: EXPERIMENTS WITH LABELLED GLUCOSE, ACETATE, PHENYLALANINE, TYROSINE AND PROLINE

—(1) Phenylalanine, proline and presumably tyrosine are precursors of the small glutamate pool in brain. This follows from the finding that with these precursors the specific radioactivity of glutamine is higher than the specific radioactivity of glutamate. (2) Glucose is not as efficient a precursor of glutamate and related amino acids in the brain of 10-day-old mice as it is in the adult brain. (3) Acetate, phenylalanine, tyrosine and proline are incorporated to about the same extent in glutamate, aspartate and glutamine in the brains of 10-day-old and adult mice. (4) The results suggest that the brain of the immature animal uses substrates other than glucose, relative to glucose better than the brain of adult animals.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

INCORPORATION OF AXONALLY TRANSPORTED SUBSTANCES INTO MYELIN LIPIDS

Abstract— The possibility that axonally transported lipids and/or proteins might undergo transaxonal migration and become incorporated into surrounding myelin lamellae was studied by isolating myelin from optic tracts of myelinating rabbits at various times following intraocular injection of [3-¹⁴C]-serine and [2-³H]glycerol. Myelin isolated by a procedure employing ethylene glycol-bis(β-aminoethyl ether)-.N,N'-tetraacetic acid had relatively constant specific radioactivity with respect to both isotopes over a 21 day period. Myelin lipids showed a gradual increase in ¹⁴C specific radioactivity, attributed to reutilization of [¹⁴C]serine from the axon by a compartment of the oligodendrocyte. Free serine is postulated to arise in the axon from catabolism of axonally transported proteins (and possibly lipids) and to migrate transaxonally into the neighboring oligodendroglia. This reutilization mechanism resulted in synthesis of myelin cerebrosides, sphingomyelin, ethanolamine phosphoglycerides and possibly sulfatides, but not gangliosides or serine phosphoglycerides. The data for choline- and inositol-phosphoglycerides are inconclusive. [³H]Glycerol-labeled myelin lipids decreased slowly in ³H specific radioactivity with time, indicating either that [2-³H]glycerol does not participate in the reutilization pathway or that the label is lost in the process. Evidence is presented that ³H- and ¹⁴C-labeled lipids are true myelin constituents. Lipids from the myelin, axolemma- and axon-enriched fractions tended to converge in specific radioactivity over the 21 days, especially the former two fractions. These results together with isotope ratio changes point to an equilibration process whereby lipids are able to transfer. (or exchange) between the 3 compartments. Protein radioactivity in isolated myelin was suggested to arise from residual axon/axolemma contamination, and no evidence was found for transaxonal migration of protein into myelin. The 2 mechanisms elucidated here are believed to account for a quantitatively small portion of myelin lipid and are considered to represent a form of axon-glia interaction.     

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.     

INCORPORATION OF LABELLED PHOSPHATE INTO PHOSPHOLIPIDS IN SQUID GIANT AXONS

Inorganic phosphate labelled with ³²P was applied to giant axons excised from squid (Loligo pealeii) by addition of ³²Pi to the bathing solution, by injection into the axon, or by addition to axoplasm which had been separated from the sheath. The preparations were kept at 10 to 25° for various times up to 4 hr. When ³²P_i was supplied by way of the bathing solution, axoplasm and sheath were usually separated at the end of incubation before extraction of the lipids. Lipids were extracted with chloroform-methanol and resolved by paper chromatography. The lipids which became labelled appeared to be the same in sheath and axoplasm. They were identified by cochromatography with known lipids and by chromatography of products formed from them by mild alkaline hydrolysis. They included phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, and probably somelysophosphatidylethanolamine. Some labelled components remained unidentified. Phosphatidylcholine was apparently present, but did not become significantly labelled either in sheath or in axoplasm, or in a squid's stellate ganglion. There was no evidence that separation from the sheath impaired the capacity of the axoplasm for lipid synthesis.     

STRUCTURE-LINKED ACTIVITY OF LYSOSOMAL ENZYMES IN THE DEVELOPING MOUSE BRAIN

—A longitudinal study of the maturation of mouse cerebral lysosomal enzymes has been completed. Activity of the enzymes, acid phosphatase (I.U.B. 3.1.3.2), β-glucuronidase (I.U.B. 3.2.1.31) and β-acetylglucosaminidase (I.U.B. 3.2.1.30) was assayed spectrofluorimetrically on portions of supernatant from 0.25 M sucrose homogenates spun at 6 x 10³ -min. Activities were obtained in native (free) and Triton X-100 activated samples (total). The neonatal period was characterized by relatively low free and high total acid phosphatase activities. An abrupt rise in free activity occurred during the period 10–20 days. Discontinuous anion exchange DEAE cellulose chromatography (0.01 m -tris–maleate, pH 6.3) with elution by ascending molarities of NaCl of the Triton X-100 activated supernatant revealed three major peaks in the adult. A fourth peak, designated as fraction II (‘maturation fraction’) occurred only during the neonatal period, a time also characterized by increased specific activity of fraction I, with no change in fraction IV. The chromatographic fractions were further characterized by optimal pH, ascorbate, fluoride, Cu²⁺ and Fe²⁺ ions. The maturation profiles of total, β-glucuronidase and total, β-acetylglucosaminidase differed from each other, and from that of total acid phosphatase. Comparable differences existed in the profiles of the free activities, and the ratio of free:total activity differed for each enzyme at any selected time especially during the neonatal period. These findings are are discussed with reference to the maturation of isoenzyme fractions with age, and suggest that the changes in structure-linked organization of individual lysosomal hydrolases are functions of heterogeneity in enzyme complement of individual lysosomes.     

ANTI-WHOLE WHITE MATTER SERUM INHIBITS INCORPORATION OF GLUCOSE AND GALACTOSE INTO THE LIPIDS OF MYELINATING SPINAL CORD CULTURES

Myelin formation was inhibited in fetal mouse spinal cord cultures in the presence of serum from rabbits with experimental allergic encephalomyelitis produced by inoculation of whole bovine spinal cord white matter in complete Freund's adjuvant. Controls were exposed to decomplemented serum. Replacement of serum in inhibited cultures on the 18th day in vitro (DIV) with control serum (disinhibited) resulted in the appearance of visible myelin within 2–3 days. From 20 to 23 DIV, d -[U-¹⁴C]glucose or d -[U-¹⁴C]galactose was present in all media. Total protein, DNA, gangliosides and galactolipids were reduced by 21% in inhibited cultures, and activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase was reduced by 50%. There was little reduction in the incorporation of glucose carbon (21–23 DIV) into several lipid classes examined. Labelling of cerebrosides by galactose carbon in inhibited cultures was only 12% of that of controls while there was no reduction in the labelling of neutral lipid–cholesterol and the glycerophosphatides. Galactolipid labelling by [¹⁴C]galactose in the disinhibited cultures was intermediate between inhibited and control cultures. Differences in the effects of inhibiting medium on the incorporation of glucose and galactose carbon indicate that ceramide synthesis is less affected than is galactose incorporation to form cerebroside.     

THE INCORPORATION OF GLUCOSE INTO BRAIN GLYCOGEN AND THE ACTIVITIES OF CEREBRAL GLYCOGEN PHOSPHORYLASE AND SYNTHETASE: SOME EFFECTS OF AMPHETAMINE

Abstract— The effects of amphetamine sulphate (5 mg/kg intraperitoneally) on the incorporation of radioactive carbon from [U-¹⁴C]glucose into the glycogen of mouse cerebral cortex, midbrain and hind-brain have been investigated. In all brain regions studied amphetamine induced a rapid decrease in glycogen followed by a slower return to control values. No significant alterations were observed in the steady state concentration of cerebral glucose. The initial fall in glycogen was associated with a fall in its specific radioactivity relative to that of cerebral glucose, whereas the resynthesis of the polysaccharide was associated with a marked increase in the relative specific radioactivity of glycogen. Other experiments demonstrated that amphetamine initially stimulates the breakdown of prelabelled glycogen and that the resulting molecule has fewer 1,4 linked glucose side chains.
Studies of the relative forms of the enzymes glycogen phosphorylase and glycogen synthetase suggested that rapid post mortem changes were less likely to occur if cerebral tissue was fixed by means of a freeze-blowing technique. Amphetamine administration resulted in a rapid though transient elevation of phosphorylase a activity in mouse forebrain. The level of glycogen synthetase I activity was unchanged initially but was markedly elevated during the period when there was a large increase in the rate of incorporation of glucose into glycogen. It is suggested that cerebral glycogen metabolism is controlled, at least in part, by the interconversion of the 'active' and 'inactive' forms of glycogen phosphorylase and synthetase.