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Methylarginines in free form were identified in bovine brain. Three compounds were isolated from the basic aliphatic amino acid fraction of bovine brain with several ion-exchange chromatographies. They showed the same Rf values in paper and thin-layer chromatographies as those of authentic NG-monomethylarginine, NG,NG-dimethylarginine, and NG,N'G-dimethylarginine. The migration distance of the isolated compounds in high-voltage paper electrophoresis and the retention times in ion-exchange HPLC were also identical to those of the above authentic methylarginines. We concluded that these three compounds are the methyl derivatives of arginine described above. The amount of these three compounds isolated from 1,090 g of bovine brain was 0.3 mumol of NG-monomethylarginine, 0.1 mumol of NG,NG-dimethylarginine, and 0.5 mumol of NG,N'G-dimethylarginine. The occurrence of these free methylarginines may have an important role in regulating the signal transduction through the nitric oxide system.

—3-Methoxy-4-hydroxyphenylglycol (MHPG) formed a sulphate conjugate when incubated with ATP, Mg2+ ions, Na235SO4 and the high-speed supernatant preparations of rabbit or rat brain. The same reactions could be catalysed by similar enzyme preparations from liver. The sulphated product was separated and identified by paper chromatography. On acid hydrolysis, it released both Na235SO4 and the free glycol. The measurement of this labelled sulphate was used as a specific assay procedure for determining the overall sulphoconjugatory process. The pH optimum of the reaction is 7.8. For rabbit brain, the Km for Na2SO4 determined for the activating system is 3.6 × 10−4m, and that for MHPG for the sulphotransferase reaction is 1.05 × 10−4m. The specific enzyme activity, expressed as nmol 35SO4 incorporated/h/mg protein for a 30-min assay is as follows: rat brain, 2.8; rabbit brain, 1.6; rat liver, 33.4and rabbit liver, 15.0. Dithiothreitol at 3 mm concentration had no significant effect on the sulphation of MHPG in all these preparations.

—The uptake of l[14C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm, partially inhibited by dinitrophenol and ouabain and elevated [K+], weakly Na+-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC50= 0·5mm) than by aspartic acid, GABA, glycine or methionine. The [14C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [3H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [3H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (Ki= 1 mm). [14C]glutamine and [3H]GABA were released from brain slices by electrical stimulation or 50 mm K+, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [14C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.

—Effects of acute or chronic administration of ethanol and its withdrawl on the steady-state levels and turnover rates of certain neurotransmitters have been investigated in mice. The influence of long-term administration of ethanol on the activities of enzymes involved in the metabolism of these transmitters has also been studied. Acute administration of ethanol or acetaldehyde or chronic administration of ethanol resulted in a decrease in the cerebral contents of acetylcholine, acetylCoA and CoA. Brain levels of 5-hydroxytryptamine, norepinephrine and choline remained unchanged after acute administration of ethanol. However, chronic administration of ethanol resulted in a decrease in the norepinephrine content without significantly affecting 5-hydroxytryptamine or choline contents. Cerebral levels of γ-aminobutyric acid increased with both acute or chronic administration of ethanol. The total incorporation of [3H]choline into acetylcholine in brain was depressed upon acute administration of ethanol. After withdrawal of ethanol for one day cerebral levels of norepinephrine returned to normal; however, γ-aminobutyric acid and acetylcholine returned to normal levels at 2 and 4 days after ethanol withdrawal, respectively. Pretreatment of mice with pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced decrease in cerebral acetylcholine levels. The activities of cerebral choline acetyltransferase and glutamic decarboxylase were decreased after 2 weeks of chronic ethanol administration. However, the activities of acetyl cholinesterase and GABA-transaminase remained unaffected after 2 weeks of ethanol treatment

—[14C]-Glutamate and [14C]-glutamine were incorporated into calf brain tRNA in the presence of homologous aminoacyl-tRNA synthetases. When the tRNAs were then deaminoacylated and chromatographed, a number of radioactive products were found in addition to the original amino acids. One of the products of glutamate transformation was identified to be glutamine. Formation of the radioactive products of glutamate in the presence and absence of tRNA indicated that glutamine was produced from glutamate at the level of the free amino acid followed by the incorporation of both substances into tRNA. Examination of the products of deaminoacylation of glutaminyl-tRNA showed that glutamine underwent structural alterations at the level of the aminoacyl-tRNAs to give rise to a cyclic derivative of glutarimide. This reaction was specific for glutamine, and constituted approximately 15 per cent of the total radioactivity in the deaminoacylation products of glutaminyl-tRNA.

— 1 Slices of mouse brain were incubated with [U-14C]alanine, valine, leucine, phenylalanine, proline, histidine, lysine, arginine or aspartic acid, and the extent of metabolism was estimated by analyses utilizing paper chromatography of the tissue extracts and with an amino acid analyser. 2 The metabolism of Ala and Asp was high; of Leu and Pro, moderate; and of Lys, Arg and Phe, low; the metabolism of Val and His was not significant. The time-course of metabolism in most cases showed varying rates, indicating heterogeneous metabolic compartments for the amino acids. 3 Production of CO2 was high from Asp, moderate from Ala, and low from Leu; the other amino acids were not oxidized to CO2 to any significant extent. A large portion of the metabolized label was trapped in the form of Glu or Asp. 4 Metabolism increased with increasing concentration of amino acid to some extent and was largely inhibited by omission of glucose, by anaerobic conditions, or by cyanide. Although these conditions also inhibit uptake, the time-course and extent of inhibition uptake and metabolism were different. 5 With Asp, Ala and Phe, metabolism was lowest in slices from pons-medulla; the brain area exhibiting the highest metabolism differed for each amino acid. The metabolism of Asp was lower in brain samples from newborn than in those from adults; the metabolism of Leu was higher in slices from newborn brain. 6 The results indicate that the majority of the amino acids can be metabolized in brain tissue and that the metabolic rates are influenced by a number of factors, among them the level of amino acids and the level of available energy.

Kynurenine formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9) was found to be present in rat brain and was partially purified and characterized. The partially purified enzyme catalysed the hydrolysis of 5-hydroxyformyl-dl-kynurenine to 5-hydroxy-dl-kynurenine and that of formyl-l-kynurenine to l-kynurenine at similar rates. The apparent Km values of the enzyme for 5-hydroxyformyl-dl-kynurenine and formyl-l-kynurenine were 4.0 ± 10−4 and 1.8 ± 10−4m, respectively. The enzyme was active over a wide pH range (5.5–8.5). The activity was inhibited by low concentrations of Ag+ and Hg2+. The physiological significance of the enzyme is discussed.

— A simple, rapid, sensitive and economical assay technique has been developed for any enzyme whose substrate and product differ by charge. It is based on a combination of rapid vacuum filtration with an ion exchange resin. The technique has been successfully applied to brain glutamate decarboxylase.

—The accumulation of [3H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [3H]choline concentrations varying from 0.5 to 100 μm. The accumulation of [3H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with Km. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective Km. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [3H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [3H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [3H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.

— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity. 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids. 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate. 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively. 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates. 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable. 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood. 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

— Putreanine was proven to be formed from spermidine in rat brain by intra-ventricular injection of radioactive spermidine. The ability to form putreanine from spermidine was also found in rat liver. The turnover rate of putreanine was slower in rat brain than in rat liver.

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [3H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11-dehydrocorticosterone, Cortisol and cortisone decreased the amount of [3H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [3H]corticosterone with macromolecules of the cytosol. Further evidence for protein-steroid interaction was obtained by incubating [3H]corticosterone (B), [3H]cortisol (F), or 11-[3H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.

— The de novo biosynthesis of purine nucleotides in rat brain was markedly and rapidly inhibited by intracerebral injections of azaserine. After 48 hr the animals became paraplegic. The biosynthesis of purine and pyrimidine nucleotides, RNA and protein was investigated by radioactive tracer studies over a 4-day period in an attempt to correlate the azaserine-induced, metabolic lesions found in rat brain and the manifested, peripheral, neurological disturbance.

—(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.

— Labelled fucose administered to rats in vivo was rapidly incorporated into brain glycoproteins, but not into any other brain constituents, including glycolipids and acid mucopolysaccharides. Maximum incorporation of tritium-labelled fucose into brain glyco-proteins occurred 3–6 h after intraperitoneal injection in young or adult rats, and the half-time for the turnover of glycoprotein-fucose in young rats was approximately 2 weeks. Within 3 h after the administration of either [1-3H]fucose or fucose generally labelled with tritium, 75 per cent of the total acid-soluble radioactivity in plasma and brain was found to be volatile, and by 24 h after injection more than 90 per cent of the acid-soluble radioactivity was volatile. The tritium in labelled fiicose appears to undergo arapid exchange reaction with hydrogen atoms in body water, although the tritium in fucose glycosidically- linked to glycoproteins is biologically stable. The rapid disappearance of labelled free fucose from the plasma and tissues of the rat precludes the possibility of any significant degree of reutilization of labelled precursor, and provides support for other data indicating that the turnover of fucose in brain glycoproteins is relatively slow in comparison to that of hexosamine and sialic acid. Activities of α-L-fucosidase in rat brain, with pH optima at 40 and 6.0, had essentially the same Km (4 × 10−4 M and 3.2 × 10−4 M, respectively) with p-nitrophenyl-α-L-fucopyranoside as substrate. Activities of both were competitively inhibited by L-fucose. However, the Kt measured at pH 4 (1.9 × 10−2) was almost ten times greater than that measured at pH 6 (1.5 × 10−4).

— Slices from various regions of rat brain, incubated at 25°C, rapidly accumulate [3H]GABA from the surrounding medium until after 60min tissue:medium ratios as high as 300 may be achieved. Kinetic analysis has demonstrated two distinct uptake systems for GABA in all the brain regions examined. One system has a relatively high substrate affinity (Km= 1.2 ± 10-5 M) while the other has a lower affinity (Km= 4 ± 10-4 M). Studies at low GABA concentration (5 ± 10-8 M), as well as estimates of maximum velocities, have shown that the distribution of the high affinity uptake system is heterogeneous. Cortex, hypothala mus, midbrain and hippocampus have relatively high uptake rates while the striatum, cerebellum and pons and medulla have a lower uptake rate. Maximum velocities for the low affinity uptake system show much less regional variation. Lithium, either added to the incubation medium or fed to rats, had no effect on the uptake of GABA by cortical slices.

— Further investigations have been made into ergothioneine in the brains of several mammalian species, and the distribution of ergothioneine in the brain of the ox is described. It has not been possible to confirm many of the findings of earlier workers and the results do not appear to support their conclusion that ergothioneine is identical with the cerebellar factor.

— 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-14C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions. 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered. 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent. 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate. 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour. 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

— Rat brain polysomes were prepared from a deoxycholate-treated postmito-chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation. 1 The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6-day-old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes. 2 The rate of [14C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals. 3 The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals. 4 The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid-soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra-acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals. 5 The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence. 6 Polysomal preparations also contained RNase, phosphomonoesterase, phospho-diesterase and 5′-nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

—Using either tryptophan or 5-hydroxytryptophan as the precursor, and examining the metabolites in whole rat brain and in brain regions of dog, the pattern of metabolites resembled that found under physiological conditions only after tryptophan administration. From these and other observations on the cerebral 5-hydroxyindoles the main conclusions are firstly, that there are regional differences within brain in storage, turnover or metabolic fate of 5-HT. Secondly, that the normal pathway appears to be well localized biochemically with linking of its succeeding steps, and thirdly, that turnover through the system is normally controlled by intracerebral tryptophan 5-hydroxylase in both rats and dogs although there are differences between the species in the cerebral metabolism of 5-HT.

— Some metabolic and biochemical effects of fluorocitrate were studied in vivo in rat brain and cat spinal cord. During the preconvulsant and convulsant phases of fluorocitrate poisoning the contents of free glutamate, glutamine and aspartate declined progressively, while that of alanine increased. Incorporation of 14C from [U-14C]glucose into these amino acids also decreased, although somewhat more gradually. GABA exhibited a biphasic change, its content rising after an initial decrease while its relative specific activity rose initially and subsequently diminished. Incorporation of 14C from [U-14C]glucose and [U-14C]lysine into neural protein declined sharply. The citric acid content rose markedly in rat brain and cat spinal cord. In rat brain the glycogen content declined but ATP and ammonia contents were unchanged. The significance of these results with respect to energy metabolism and the possible mechanism of the convulsions during fluorocitrate poisoning is discussed.

— The formation of N-acetyl-aspartyl-peptides in mouse brain homogenates is described. The formation is completely dependent on histamine and on an ATP-regenerating system, and partially dependent on the addition of N-acetyl-aspartate and certain other amino acids. The N-acetyl-aspartyl-peptide formation is probably non-ribosomal and is apparently not due to hydrolysis of preformed proteins.

— Adenosine metabolism in the homogenate of brain mainly undergoes deamination to inosine and hypoxanthine, while uniformly labelled [14C]adenosine injected into the carotid artery or [8-14C]adenosine incubated with brain slices was mostly phosphorylated to [14C]adenine nucleotides in brain cells. Adenosine kinase has now been partially purified from homogenates of guinea pig brain. The kinase preparation was free of adenosine deaminase, almost free of adenosine triphosphatase and had a Km of the order of 2 × 10-5M for adenosine. Kinetic studies with brain slices showed that adenosine reached the cells by diffusion and that the diffusion was facilitated by subsequent phosphorylation to adenine nucleotides. From the following experimental results, it is concluded that the phosphorylation is catalysed by adenosine kinase quantitatively. (1) During the uptake and phosphorylation of adenosine by brain slices, the nucleoside did not split to adenine and ribose moieties. (2) The rate of formation of adenine nucleotides in the slices was a hyperbolic function of the concentration of adenosine in the medium, showing an apparent Km foradenosine of the order of 2 × 10-5 M. (3) Some analogues of adenosine inhibited both the facilitated diffusion of adenosine and the kinase activity, but ouabain (0.005 mM) did not inhibit either.

SUMMARY A study was conducted on the in vivo incorporation of l-[14C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l-[14C]serine.

— The distribution of tryptophan hydroxylase in the cat brain was investigated and found to parallel roughly the distribution of serotonin. The most active areas are the caudate nucleus, septal area, anterior perforating substance, hypothalamus, amygdala and various areas of the midbrain.

— Synaptosomes from guinea-pig cerebral cortex contain a fetuin: sialyl glyco-protein: glycosyl transferase; evidence is presented which indicates that both a sialyl transferase; evidence is presented which indicates that both a sialyl transferase and endogenous acceptors were located in the synaptosome ‘ghost’ fractions. Following solubilization of synaptosomes with Triton X-100 and the use of fetuin minus NANA as acceptor, 25 per cent of the transferase was recovered after centrifugation and column chromatography on Sephadex G-100 and G-200 with a 64·0-fold purification. The enzyme had a pH optimum of 6·3, required no divalent metal cation for activity, and exhibited high activity with either fetuin minus sialic acid, prothrombin minus sialic acid, Tamm-Horsfall glycoprotein minus sialic acid, or orosomucoid minus sialic acid as acceptor; neither BSM nor PSM minus NANA functioned as an effective acceptor. The fetuin:sialyl transferase using fetuin minus sialic acid and CMP-sialic acid as substrates a and b, respectively, gave the following kinetic constants when using the Cleland bisubstrate model: Ka= 35μM; Kb= 3 μM; Kia, = 25 μM; Kib= 25μM; and V1= 92 pmoles. min−1.mg−1 of protein. The following divalent cations inhibited the reaction: Ba2+ > Hg2+ > Pb2+ > Cu2+.

—The enzymatic decarboxylation of l-DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating 14CO2 evolved from tracer amounts of d l-DOPA[carboxy1-14C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO2 as a reaction product. The enzyme preparation behaved like an aromatic l-amino acid decarboxylase: it also decarboxylated o-tyrosine and when incubated with 5-hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d-DOPA[carboxy1-14C]. Within the human brain, l-DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l-DOPA decarboxylase activity was not significantly above blank values. The activity of l-DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.

— In further experiments on the effects of antibiotic agents on protein synthesis in the goldfish brain, doses of intracranially-injected puromycin or acetoxycycloheximide higher than those previously employed did not hasten the onset of inhibition of incorporation of intraperitoneally-injected [3H]leucine into brain protein. The antibiotic-resistant incorporation was not due to the presence of labelled blood protein in the brain. After the intracranial injection of labelled puromycin, the appearance of radioactivity in the acid-soluble fraction of brain was blocked by acetoxycycloheximide. Repeated daily intracranial or intraperitoneal injections of puromycin were lethal, and acetoxycycloheximide was not protective, indicating that peptidyl-puromycin was present in the brain but did not account for the lethality of puromycin. Behavioural experiments argued against but did not totally exclude the possibility that peptidyl-puromycin was responsible for the amnestic effect of puromycin. Puromycin aminonucleoside, O-methyl tyrosine and 5-guanylyl methylenediphosphonate had little or no effect on protein synthesis in brain, but gougerotin was slightly inhibitory.

— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [3H]5-HT (5 × 10-8 M), for 45 min, when there was apparent equilibrium between [3H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [3H]5-HT in sucrose gradients was separable from the peak for [14C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [3H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.

— After intracerebral administration of [14C]histidine to mice the major labelled substance found in the brain extracts was histidine itself; small amounts of labelled carnosine and homocarnosine were detected. No other labelled substances were detected on radio- autographs of two-dimensional TLC's of the extracts. In the case of the frog, radioactive histidine, N-acetylhistidine, carnosine and homocarnosine were found in the brain extracts at various times after intracerebral injection of the labelled histidine. With time, approximately 90 per cent of the radioactivity in the extracts was found in the N-acetylhistidine. In neither the mouse nor frog could we find unequivocal evidence for the formation either of histamine or imidazoleacetic acid from intracerebrally administered histidine, but our analytical procedures may have lacked sufficient sensitivity to pick up extremely low activities of histamine and imidazoleacetic acid. Experiments with [14C]histamine administered intracerebrally into mice demonstrated the major pathway of metabolism in brain to be histamine methylhistamine methylimidazoleacetic acid. No detectable label appeared in inlidazoleacetic acid. In the frog intracerebral administration of the labelled histamine led to the formation of methylhistamine and imidazoleacetic acid, but at most only traces of methylimidazoleacetic acid were found. The injection of [14C]imidazoleacetic acid intra- cerebrally into mice and frogs resulted in virtually no loss of the label in the form administered in the frog brain over a period of 4 h and in a slow rate of decrease in the mouse brain. No radioactive metabolites of imidazoleacetic acid were found in either species. The limitations of trying to determine natural functions of substances in brain by following the fate of exogenously administered materials is discussed.

– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[14C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.

The first description of Alzheimer's disease (AD) was made in 1907 by Alois Alzheimer (Allgemeine Zeitschrift fur Psyciatrie und Psychisch-Gerichtliche Medizin 64, 3, 1907), although other contemporary physicians had made similar, and rather more complete, assessments of the neuropathological changes present in the AD brain (Fischer, Monatsschr Psychiat Neurol 22, 17, 1907). Our knowledge of AD has increased dramatically and continues to accelerate. This year is 25 years after the publication of a series of papers that, in various ways, articulated the amyloid cascade hypothesis (ACH) for AD (Beyreuther and Masters, Brain Pathol 1, 241–251, 1991; Hardy and Allsop, Trends Pharmacol Sci 12, 383–388, 1991; Selkoe, Neuron 6, 487–498, 1991; Hardy and Higgins, Science 256, 184–185, 1992). This review will cover some familiar territory, but we shall also place the ACH into a wider context, compare it with other hypotheses for AD, explore the evolution of the hypothesis to encompass new findings, and determine, irrespective of the merits of the hypothesis itself, whether it has been useful for the research field, both in academia and in industry. Finally, we shall review how the ACH has led to a number of therapeutic approaches, all of which have, to date, failed to reach their primary efficacy end-points in clinical trials and reflect upon what the future may hold. We review the amyloid cascade hypothesis (ACH) and compare it with other hypotheses that have been posited to explain the initiation and progression Alzheimer's disease. We document the data that support the ACH, and also reflect upon its deficiencies. We list the recent clinical failures of amyloidocentric drugs and anticipate the results that new therapeutic approaches may deliver. This article is part of the 60th Anniversary special issue.

— The activities of tyrosine hydroxylase (T-OH). DOPA decarboxylase (DDC). dopamine-β-hydroxylase (DβH). monoamine oxidase (MAO), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), l-glutamic acid decarboxylase (GAD) and the concentrations of DNA and RNA were measured in 13–20 areas of post-mortem brain tissue from neurologically and psychiatrically normal individuals. Emphasis has been put on regional distribution rather than establishing normal values and detailed comparisons have been made with previously published work on the normal human brain. Despite expressing all results relative to an internal reference point there was substantial inter-brain variability. There was no apparent relation between age, sex, medication, cause of death or time lag between death and dissection and any of the enzyme activities. Enzyme activities were fairly evenly distributed throughout cerebral cortex whereas clear differences existed along the rostro-caudal axis of the brain. It is hoped that this paper, with its companion paper on amine and metabolite concentrations, will be useful as a reference work for investigators of the chemical pathology of the human brain.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting millions of people worldwide. Apart from age, the major risk factor identified so far for the sporadic form of AD is possession of the epsilon4 allele of apolipoprotein E (APOE), which is also a risk factor for coronary artery disease (CAD). Other apolipoproteins known to play an important role in CAD such as apolipoprotein B are now gaining attention for their role in AD as well. AD and CAD share other risk factors, such as altered cholesterol levels, particularly high levels of low density lipoproteins together with low levels of high density lipoproteins. Statins--drugs that have been used to lower cholesterol levels in CAD, have been shown to protect against AD, although the protective mechanism(s) involved are still under debate. Enzymatic production of the beta amyloid peptide, the peptide thought to play a major role in AD pathogenesis, is affected by membrane cholesterol levels. In addition, polymorphisms in several proteins and enzymes involved in cholesterol and lipoprotein transport and metabolism have been linked to risk of AD. Taken together, these findings provide strong evidence that changes in cholesterol metabolism are intimately involved in AD pathogenic processes. This paper reviews cholesterol metabolism and transport, as well as those aspects of cholesterol metabolism that have been linked with AD.

Inorganic phosphate (Pi) is an important polyanion needed for ATP synthesis and bone formation. Since it is found at millimolar levels in plasma it is usually incorporated as a constituent of artificial cerebrospinal fluid (ACSF) formulations for maintaining brain slices. In this paper we show that Pi limits the extracellular zinc concentration by inducing metal precipitation. We present data suggesting that amino acids like histidine may counteract the Pi-induced zinc precipitation by the formation of soluble zinc complexes. We propose that the interplay between Pi and amino acids in the extracellular space may influence the availability of metals for cellular uptake.

—The present paper reports the result of studies undertaken to determine the effects of the in vivo administration of Δ9-THC on the ganglioside and sialoglycoprotein contents of rat brain subcellular fractions. Results indicate that the administration of the drug under both acute and chronic conditions brings about characteristic changes in the sialoglycoproteins and ganglioside content in all the subcellular fractions. Both sialoglycoproteins and ganglioside contents were markedly increased in microsomal and synaptosomal fractions and decreased in the mitochondrial fractions although the increase in the synaptosomal fractions has been found to be most striking. After chronic treatment, both ganglioside and sialoglycoprotein content did not change substantially in all the fractions except for a small increase in case of synaptosomal fractions.

Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C]isopentenyl pyrophosphate [( 1-14C]I-P-P) into long-chain polyisoprenyl pyrophosphates (Poly-P-P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly-P-P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 microM for I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (5) was stimulated by dithiothreitol. The major products were identified as C90 and C95 allylic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product co-chromatographed with authentic Poly-P-P on silica-gel-impregnated paper; (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly-P) by alkaline hydrolysis; (3) treatment of the labeled Poly-P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly-P from lipophilic Sephadex LH-20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (C95) isoprene units when analyzed by reverse-phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long-chain Poly-P-P. The enzymatic reactions by which the Poly-P-P intermediate is converted to dolichyl phosphate remain to be elucidated.

— Myelin has an unusual lipid composition, being particularly rich in sulfatide. This lipid is synthesized by the transfer of sulfate from phosphoadenosine phosphosulfate to galactocerebroside, catalyzed by galactocerebroside sulfotransferase. This paper describes a sensitive assay for the sulfotransferase (capable of measuring activity in as little as 10 μg of extracted rat brain protein) so that this enzyme can be readily investigated in isolated cells, or the small amounts of tissue available in developing animals. Both manganase (20 mm) and thiol reagents were required for optimal activity. This assay was used to monitor the purification of the sulfotransferase from rat brain. Extraction of the enzyme from crude homogenates required the nonionic detergent, Triton X-100, at pH 7–7.5. Removal of Triton X-100 from the extracted enzyme resulted in a soluble but less active enzyme, the activity of which could then be restored with detergents. Stability of the detergent-extracted enzyme was investigated, and even at —40°C there was a 20% loss of activity over 10 days. By standard procedures 500-fold purification of the enzyme has been achieved.

This paper describes a new, sensitive assay for dopamine-beta-hydroxylase (DBH) activity in human cerebrospinal fluid (CSF), serum and brain tissues by high performance liquid chromatography (HPLC) with electrochemical detection (ED). Dopamine (DA) was used as a substrate and was incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by a double-column procedure, the first column of Dowex-50-H+ and the second column of aluminum oxide. NE was adsorbed on the second aluminum oxide column and then eluted with 0.5 M-hydrochloric acid and assayed by HPLC-ED. Epinephrine (EN) was added to each incubation mixture as an internal standard, and this assay was therefore highly reproducible. The peak height in HPLC was linear from 500 fmol to 100 pmol of NE and EN. The lower limit of detection for NE formed enzymatically was about 30 pmol, which indicated that the sensitivity of this procedure was comparable to that of radioassay procedures. We applied the method to measurement of the activity of and examination of some of the characteristics of DBH in human CSF. DBH activity in CSF of Parkinsonian patients was lower than that of control patients. The properties of DBH in human CSF were similar to those in serum and adrenal medulla.

— Pigeons working on a multiple lixed-ratio 50, fixed interval 10 schedule of food reinforcement were injected with l-tryptophan (300mg/kg; I.M.) and killed at various times before, during and after the period of behavioural depression following the administration of this amino acid (0, 25, 50, 90, 170 and 230 min). The levels of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tyrosine, dopamine and norepinephrine were concurrently measured in 4 specific areas of the brain (telencephalon, diencephalon plus mesencephalon, pons plus medulla-oblongata and cerebellum). The course of the increases in the level of 5-hydroxytryptamine in the telencephalon, and subsequent return to pre-injection levels, was temporally related to the onset of the decreased responding and gradual return to normal rates of responding. Changes in dopamine and norepinephrine were not correlated with the onset of and recovery from the decreased response rates. The data in this paper are discussed in terms of (a) the previously reported work with 5-hydroxytryptophan and (b) the importance of the telencephalic serotonergic system in certain types of behavioural depression.

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.

— Abnormalities involving haem biosynthesis have been postulated as underlying mechanisms in the aetiology of the neural manifestations of acute porphyria and of lead poisoning. This paper reports a study of the enzymes of the haem biosynthetic pathway and their control in mammalian brain. The activity of rat brain 6-aminolaevulinate synthetase (ALA synthetase), 6-aminolaevulinate dehydratase (ALA dehydratase), uroporphyrinogen I synthetase, uroporphyrinogen decarboxylase and ferrochelatase were found to be between 12.5 and 0.002% of the corresponding values for liver. This accords with the lower concentrations of total haem and cytochrome P450 found in brain and with the slower rate of incorporation of [4-14C]ALA into brain haem in vivo. The subcellular distribution of radioactivity following intraventricular injection of [4-14C]ALA confirmed that the bulk of brain haemoproteins are intramitochondrial in contrast to liver where the major portion is microsomal. Brain haem biosynthesis was apparently unaffected by factors known to influence this pathway in liver, including starvation and treatment with allylisopropylacetamide or phenobarbitone. These findings suggest that brain haem requirements are considerably less than those of liver and are not subject to significant fluctuations under normal circumstances. Apparent non-inducibility of ALA synthetase suggests that deficient haem and consequently haemoprotein production could result where other enzymes in the pathway become rate-limiting due to genetic defects or inhibition by exogenous agents such as lead.