bluuebunny/crossref_metadata_2025 huggingface dataset:
ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N‐METHYL‐N‐NITROSOUREA AND METHYL METHANESULPHONATE
Authors: Kleihues P. , Magee P. N.
Published: December 2006

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N‐[14C]methyl‐N‐nitrosourea and [14C]methyl methanesulphonate. The main product with both compounds was 7‐methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7‐methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O6‐methylguanine, accounting for about 12 per cent of the radioactivity present as 7‐methylguanine and cytoplasmic RNA contained about half this amount of O6‐methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O6‐methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1‐methyladenine, 3‐methyladenine and 3‐methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.
THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U‐14C]GLUCOSE AND [U‐14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN
Authors: Gaitonde M. K. , Nixey R. W. K. , Sharman I. M.
Published: December 2006

Abstract— The incorporation of 14C into amino acids of the brain was determined at different times after injection of [U‐14C]glucose and [U‐14C]ribose to rats maintained on thiamine‐supplemented and thiamine‐deficient diets for 22 days.

The 14C‐content of amino acids in the brain of thiamine‐deficient rats decreased at times 2–10 min after injection of [U‐14C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U‐14C]ribose.

The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a‐oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.

Thiamine deficiency in rats showed a 5% decrease in glutamate (P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine (P < 0–01) content of the brain.
ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINO ACIDS: γ‐AMINOBUTYRIC ACID RECEPTOR OF THE SHRIMP MUSCLE
Authors: Robfrtis E. De , DePlazas Sara Fiszer
Published: December 2006

Abstract— The total lipid extract of shrimp muscle (Artemisia longinaris) was precipitated with ether. The supernatant containing 95 per cent of the phospholipids and 50 per cent of the protein showed binding for L‐[14C]glutamatc in the first peak of protein. The sediment, redissolved in chloroform‐methanol was chromatographed on a Sephadex LH‐20 column. A single peak was eluted in the chloroform (20‐40 ml) having no lipid phosphorous and high affinity binding for [14C]GABA. The saturation was achieved at 1 mole per 80.000 g protein and the curve revealed a single type of binding site. The purification achieved was of about 4000‐fold. There was no binding of L‐[14C]glulamate to the ether precipitate. The specificity of the binding of [14C]GABA was further supported by competition experiments with bicuculline. picro‐toxin and muscimol. It is suggested that the hydrophobic protein isolated by us represents the GABA receptor. The findings presented in the two papers of the series suggest that the excitatory and inhibitory receptor from crustacean muscle can be separated as two different proteins.
THE SUBCELLULAR LOCATION OF THE HIGHER ACETYLCHOLINE CONTENT IN RAT BRAIN FOUND AFTER KILLING BY THE NEAR‐FREEZING METHOD AS COMPARED WITH DECAPITATION1
Authors: Richter J. A. , Shea P. A.
Published: December 2006

Abstract— We have confirmed the finding of Takahashi & Aprison (J. Neurochem.11, 887‐898, 1964) that more acetylcholine is found in brains of rats killed by near‐freezing compared to decapitation. The radioenzymic assays for acetylcholine and choline of Shea & Aprison (Analyt. Biochem.56, 165‐177, 1973) and Goldberg & McCaman (J‐ Neurochem.20, 1 8, 1973) were used to measure both compounds and gave very similar results. The larger amount of ACh was observed both in powders of frozen rat brain and in homogenates prepared from animals killed by near‐freezing. When subcellular fractionation of the homogenates was done in the presence of eserine, the larger amount of ACh was found in the soluble fraction (S2). These results indicated that with the near‐freezing method, an extra amount of ACh is preserved in a form that is originally protected from acetylcholinesterase but that becomes esterase‐sensitive on fractionation since no differences were observed in P1. P2 or S2 fractions when no eserine was present. The amounts of choline in homogenates and subcellular fractions were also measured after both methods of killing. Differences in the method of killing and postmortem changes which affect the choline values obtained are described.
THE EFFECT OF CHRONIC DORSAL ROOT SECTION ON THE CONCENTRATION OF FREE AMINO ACIDS IN THE RABBIT SPINAL CORD
Authors: Jones I. M. , Jordan C. C. , Morton I. K. M. , Stagg C. J. , Webster R. A.
Published: December 2006

Abstract— Amino acids may be involved in primary afferent excitatory neurotransmission in the spinal cord. To test this possibility the effect of chronic dorsal root section on amino acid levels of the rabbit spinal cord has been investigated. Dorsal roots L6‐S2 were sectioned under anaesthesia. Control animals were subjected to similar surgical procedures but the dorsal roots were left intact. Electromyogram recordings taken 6 days after surgery confirmed the absence of sensory input to the lower lumbosacral cord of dorsal root sectioned animals although motor function was retained. In contrast to this control animals exhibited normal reflex activity. The spinal cord was removed from each animal and extracted in trichloracetic acid for subsequent analysts of amino acids on an autoanalyser. Sections of cord were retained for histological determination of neuronal degeneration. Comparison of amino acid levels in dorsal root sectioned and control animals revealed that the only excitatory amino acid to be significantly reduced by dorsal root section wasaspartic acid (–50 percent X although glutamic acid was also reduced (– 30 per cent). Two inhibitory amino acids, cystathionine and GABA, were also significantly depleted (– 50 and ‐ 35 per cent). The possible involvement of these amino acids in spinal cord neurotransmission is discussed.
A NEUROCHEMICAL STUDY OF FIELD CASES OF THE DELAYED SPINAL FORM OF SWAYBACK (ENZOOTICATAXIA) IN LAMBS
Authors: Patterson D. S. P. , Foulkes J. A. , Sweasey D. , Glancy E. M. , Tfrlecki S.
Published: December 2006

Abstract— Spinal cords of 5‐8 week‐old lambs affected with the delayed or spinal form of swayback were deficient in myelin lipid components such as cholesterol, cerebrosides and phospholipids and contained less water than cords obtained from clinically and histologically unaffected lambs. The rate of synthesis in vitro of cerebrosides containing hydroxy fatty acids was reduced but the fatty acid composition of this and of the “non‐hydroxy cerebroside’ fractions were quite normal. Isolated myelin was deficient in copper compared with healthy controls and there was a deficiency in one of its basic protein constituents. This suggested an inherent instability of myelin in affected lambs while the presence of some 4 per cent of cholesterol in the esterified form (none normally present) was indicative of degenerative changes. Five out of eight clinically unaffected lambs obtained from the same flocks proved to be sub‐clinical cases when the spinal cord was examined histologically and showed some of the neurochemical abnormalities of the overt clinical disease. When affected lambs were kept at the laboratory until 16 weeks old and fed a copper sufficient diet some of the spinal lesions showed signs of regression and there were fewer neurochemical abnormalities. In particular, cholesterol esters could not be detected in the spinal cord lipids.
METABOLIC COMPARTMENTATION OF GLUTAMATE ASSOCIATED WITH THE FORMATION OF γ‐AMINOBUTYRATE
Authors: Pael. A. J. , Johnson A. L. , Balaazs R.
Published: December 2006

Abstract— The distibution of 14C in the brains of rats that had been given [U‐14C]glucose (10μCi/100g body wt.) at 10 min before death was followed for 20 min post mortem. The results indicated that the input of glucose‐carbon into the tricarboxylic acid cycle stopped instantaneously after death. Although the proportion (more than 40 per cent) of tissue‐14C combined in the amino acids associated with the cycle did not change significantly, there was a characteristic redistribution of 14C within the amino acid fraction after death: significantly, the 14C content of glutamate decreased andthat of GABA increased. The GABA/glutamate specific radioactivity ratio which in vivo was 0‐58, increased progressively in the first 5 min after death, reaching a value of 0‐93. However, by 5 min the rise in the ratio stopped abruptly, although GABA accumulation continued at about half the initial rate beyond that time. These results indicated that GA BA formation is compartmented in the brain andpermitted the evaluation of certain kinetic parameters of the two compartments which could be distinguished under the experimental conditions. One of the compartments was evidently a summation of a number of subcompartments which had certain features in common, such as a low GABA flux relative to the amount of glutamate. The properties of the other compartment were compatible with those of nerve terminals functioning with GABA as the transmitter. This compartment contained about 2 per cent of the total glutamate, but the glutamate pool was labelled about three times more than the average. Further, this compartment accounted for about 50 per cent of the total GABA formation flux andcontained GABA in high concentrations (the probable values were about seven times the mean).
d‐β‐HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN
Authors: DeVivo D. C. , Leckie Mary P. , Agrawal H. C.
Published: December 2006

Abstract— D‐β‐hydroxybutyrate (β‐OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3‐14C]β‐OHB or [2‐14C]glucose was injected subcu‐taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3‐14C]β‐OHB rapidly labelled brain amino acids at all ages whereas [2‐14C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3‐14C]β‐OHB from birth‐15 days of age whereas brain alanine was more heavily labelled by [2‐14C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β‐OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [14C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2‐,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3‐14C]β‐OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [14C]label from [2‐14C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [14C]label into the brain amino acids of the 15‐day‐old [3‐14C]β‐OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?‐OHB at 6 days was 19 8 min and at 15 days was 12‐2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3‐14C]β‐OHB injected rats compared to the [2‐14C]glucose injected rats despite a heavier labelling of brain glutamate in the [3‐14C]β‐OHB injected group. We interpreted these data to mean that β‐OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.
SOME PARAMETERS AFFECTING THE ACTIVITY OF MONOAMINE OXIDASE IN PURIFIED BOVINE BRAIN MITOCHONDRIA 1
Authors: Gabay Sabit , Achee Frances M. , Mentes Gülriz
Published: December 2006

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non‐physiological amines, kynuramine and m‐iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono‐valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non‐competitive but mixed competitive inhibition was found with tyramine and phenylethylamine.

The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non‐protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un‐ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When Km values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected Km was lowest at pH 7.4 for all substrates.

Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.
FURTHER STUDIES ON THE SPECIFIC INTERACTION OF S‐100 PROTEIN WITH SYNAPTOSOMAL PARTICULATES 1
Authors: Donato R.
Published: December 2006

Abstract— The specific interaction of S‐100 protein with disrupted synaptosomes was further investigated. The specific binding is a saturable and reversible process, and is time, temperature, and strictly Ca2+ ‐dependent. Two affinities affect the interaction (Kins= 7.04 × 10−9 M. 1.28 × 1012 binding sites/ mg protein; Kins2= 3.91 × 10−7M, 2.96 × 1013 binding sites/mg protein). The half‐saturation time is about 5.5 min at 37°C. The half‐life of the complex is 17 min at 37°C. At 0°C the binding is 75% slower than at 37° C, and only one‐third of the binding sites are involved. The binding capacity is decreased by high NaCl concentrations and by pretreating membranes at high temperatures. Digestion of membranes with trypsin practically abolishes the specific binding. Treatment of membranes with phospholipase C decreases the specific binding, while phospholipase D enhances it to some extent. Other lipid extractors decrease significantly the extent of the interaction. Synaptic plasma membranes seem to be the synaptosomal component involved in the high affinity binding. The S‐100 binding activity seems to undergo developmental changes, the adult values of kinetic parameters being reached around the 16th postnatal day in the rat. The results are discussed also in relation to the membrane‐bound fraction of S‐100.
ELECTROCONVULSIVE SEIZURE: AN INVESTIGATION INTO THE VALIDITY OF CALCULATING THE CYTOPLASMIC FREE [NAD+]/[NADH] [H+] RATIO FROM SUBSTRATE CONCENTRATIONS OF BRAIN
Authors: Merrill David K. , Guynn Robert W.
Published: December 2006

Abstract— This study is an investigation into the applicability of redox calculations to brain. At six intervals following electroconvulsive scizure, multiple metabolites were measured in freeze‐blown brain from unanesthetized rats. From substrate ratios, the time course of the rapid changes in the cytoplasmic free [NAD+]/ [NADH] [H+] ratio was calculated from the reactions of lactate dehydrogenase [EC 1.1.1.27], malate dehydrogenase [EC 1.1.1.37] and glycerolphosphate dehydrogenase [EC 1.1.1.8], The pattern of the redox ratios in the control animals was also compared with the same ratios determined in freeze‐clamped liver, a relatively homogeneous tissue. Though some evidence for effects of compartmentation are present in the results from brain, these effects are relatively minor. There was found to be very good agreement in the direction and magnitude of change of the redox ratios calculated from lactate dehydrogenase and malate dehydrogenase, and even from glycerolphosphate dehydrogenase at points of low flux. In spite of rapid changes of metabolites, the reaction catalyzed by glutamateoxaloacetate transaminase remained very near its equilibrium position at all time periods. From the results it has been concluded that in spite of the obvious structural heterogeneity of brain, meaningful calculations of the cytoplamic redox state in brain are possible.
CHARACTERIZATION OF HYPOTHALAMIC SUBCELLULAR PARTICLES CONTAINING LUTEINIZING HORMONE RELEASING HORMONE AND THYROTROPIN RELEASING HORMONE
Authors: Barnea Ayalla , Ben‐Jonathan Nira , Porter John C.
Published: December 2006

Abstract— The 900 g supernatant fluid prepared from male rat hypothalamic homogenates was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone and luteinizing hormone releasing hormone in the gradient fractions were quantified by radioimmunoassays. TRH was associated with two populations of particles separable by means of nonequilibrium density centrifugation (100,000 g for 30min). However, after‘equilibrium’centrifugation (100,000 × g for 180 min), a single peak of TRH was observed at 1.07 M‐sucrose. Hypo‐osmotic shock as well as treatment with 0.1% Triton X‐100 or 0.1% deoxycholate (DOC) released TRH from both sets of particles. LRH, as TRH, was associated with two populations of particles which were separable by means of nonequilibrium density gradient centrifugation. After‘equilibrium’centrifugation, both sets of LRH‐containing particles banded at 1.27M‐sucrose as a single symmetrical peak. Although 0.1% Triton X‐100 released LRH from both populations of particles, hypo‐osmotic shock or 0.1% DOC released LRH only from the large LRH‐containing particles. The small LRH‐containing particles were resistant to hypo‐osmotic shock and to 0.1% DOC. Based on these criteria, it is concluded that in hypothalamic homogenates the TRH‐containing particles and the large LRH‐containing particles are synaptosomes. The small LRH‐containing particles may be of different cellular and/or subcellular origin.
EFFECTS OF Mn2+ AND OTHER DIVALENT CATIONS ON ADENYLATE CYCLASE ACTIVITY IN RAT BRAIN 1
Authors: Walton K. G. , Baldessarini R. J.
Published: December 2006

Abstract— Mn2+ caused an 8‐to 16‐fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn2+. with a doubling of activity at 50‐60μM. and was unaffected by a 60‐fold excess of Mg2+. Whether or not Mg2+ was added, inclusion of a low concentration of Mn2+ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca2+. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn2+ in brain homogenates was potentiated by F−. Other ions, notably Hg2+. Pb2+. Cu2+ and Zn2+. in order of decreasing potency, inhibited both basal and Mn2‐‐stimulated cyclase activity. It is proposed that the effect of Mn2+ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F− stimulates the enzyme. These results suggest that the effects of low concentrations of Mn2+ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides.
EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF HUMAN BRAIN
Authors: Yusuf H. K. M. , Merat A. , Dickerson J. W. T.
Published: December 2006

Abstract— The ganglioside content and composition of brains from twenty‐five human fetuses, three new‐born babies and ten children, were studied. The ages ranged from 13 weeks gestation to 26 months postpartum. Each brain was divided into forebrain. cerebellum and brain stem. The concentration of total gangliosides rose to a plateau at different stages of development in the different parts, whereas the total amount reached a constant value at 9 months of age in each part. The developmental profile of individual gangliosides differed in the different parts of the brain. Thus, in the forebrain GD1a. and in the cerebellum GD1a rose to become the major gangliosides. The brain stem showed little change in its ganglioside pattern during the developmental period studied. The possible significance of these charges in the gangliosides during development is discussed.
A COMPARISON OF METHODS FOR STOPPING INTERMEDIARY METABOLISM OF DEVELOPING RAT BRAIN1
Authors: Miller A. L. , Shamban Ava
Published: December 2006

Abstract— Freeze‐blowing (Veechet al. 1973), focussed microwave irradiation (Stavinohaet al. 1973) and immersion in liquid nitrogen were compared as methods for stopping metabolism in order to assay in vivo levels of intermediary metabolites in developing rat brain. Freeze‐blowing was superior at all ages (5. 10, 15 and 20 days post‐natal). The differences between this method and immersion in liquid nitrogen were quite small in the youngest rats and increased with age. reflecting the increased time needed to freeze larger brains. Brains frozen by immersion in liquid nitrogen showed evidence of increased anaerobic metabolism, with increased fructose 1.6‐diphosphate. dihydroxyacetone phosphate and lactate and decreased glucose 6‐phosphate and creatine phosphate concentrations. When brain metabolism was stopped by microwave irradiation there were many differences from freeze‐blown brain. Increases in fructose 1.6‐diphosphate. dihydroxyacetone phosphate, ADP and AMP, and decreased in ATP and creatine phosphate were especially striking. The differences between microwave irradiation and freeze‐blowing were not attributable simply to anoxia. Rather, the changes produced by this method seem to reflect the different thermal characteristics of the various enzymes which must be denatured to stop metabolism of the substrates measured. Unlike freezing in liquid nitrogen, the efficacy of microwave irradiation was not a simple function of head size, in that better results were achieved with 15‐ and 20‐day‐old than 5‐ or 10‐day‐old rats.

Many glycolytic and Krebs cycle intermediates, as well as glutamate and aspartate, progressively increased over the course of development. The reasons for these increases are uncertain but are probably‐related to the concomitant rises in rates of glycolysis and oxidative phosphorylation in brain.
A COMPARATIVE STUDY OF L‐GLUTAMATE DECARBOXYLASE FROM MOUSE BRAIN AND BOVINE HEART WITH PURIFIED PREPARATIONS1
Authors: Wu Jang‐Yen
Published: December 2006

Abstract— L‐Glutamate decarboxylase (EC 4.1.1.15) (GAD), the enzyme responsible for the formation of GABA, has been purified to homogeneity from mouse brain (Wu et at., 1973) and antibodies specific for neuronal GAD have been obtained (SAITO et al., 1974a). The present report describes the purification of GAD from bovine heart more than 2000‐fold over the homogenate by initial solubilization with Triton X‐100. subsequent fractionation with ammonium sulfate, column chromatography on DEAE cellulose, calcium phosphate gel, and DEAE‐Sephadex, and gel filtration. At least two forms of GAD have been observed in bovine heart preparations; one of them appears as a high molecular weight form (Peak I, MW 360,000) and the other one as a low molecular weight form (Peak II, MW 105,000). Cysteine sulfinic acid and cysteic acid, both precursors of taurine, had no effect on the purified heart enzyme or on neuronal GAD at 10 mM, suggesting that cysteine sulfinic acid and cysteic acid probably are not substrates for any species of GAD described above. The heart enzyme and neuronal GAD differ in several respects. First, they are different immunochemically as judged by the lack of cross reactivity between the purified heart enzyme and the antibody against purified neuronal GAD. Second, they are different biochemically. 5,5′‐Dithiobis[2‐nitrobenzoic acid] (DTNB). one of the most potent inhibitors of neuronal GAD [Ki= 1.0 × 10−8M] inhibits the heart enzyme only to a small extent at 1 mM. On the other hand, pyruvic acid, which inhibits the heart enzyme to an extent of 90% at 10 mM, only inhibits the neuronal enzyme slightly. Third, they are different in their substrate specificity. The neuronal enzyme can catalyze α‐decarboxylation of both L‐glutamate and L‐aspartate while the heart enzyme can use only L‐glutamate as substrate. Moreover, an unidentified product probably derived from L‐glutamate is obtained in the reaction mixture of the heart enzyme but is not observed with the brain enzyme, suggesting that the heart enzyme may catalyze a reaction converting L‐glutamate to products other than GABA. It is therefore concluded that heart GAD and neuronal GAD are two different entities. Work is in progress to determine whether the heart enzyme is related to the glial enzyme. Should the antibody against the heart enzyme cross‐react with the glial enzyme, the role of the glial enzyme in GABA function can then be studied by immunochemical and immunocytochemical methods.
A ROLE FOR THIAMINE IN THE REGULATION OF FATTY ACID AND CHOLESTEROL BIOSYNTHESIS IN CULTURED CELLS OF NEURAL ORIGIN1
Authors: Volpe J. J. , Marasa J. C.
Published: December 2006

Abstract— Cultured glial (C‐6) and neuronal (neuroblastoma) cells were utilized to define the role of thiamine in the regulation of fatty acid and cholesterol biosynthesis. Glial cells subjected to thiamine deficiency exhibited rates of fatty acid synthesis that were only 13% of the rates in thiamine‐supple‐mented cells. The decrease in fatty acid synthetic rate was accompanied by a comparable decrease in the activities of fatty acid synthetase and acetyl‐CoA carboxylase, the two critical enzymes in the pathway. Immunochemical techniques demonstrated that the decrease in activity of fatty acid synthetase reflected a decrease in enzyme content and that this change in content was caused by a decrease in enzyme synthesis. The disturbance of fatty acid synthesis was exquisitely sensitive to thiamine–i.e. marked improvement was evident within hours of replenishment with only 0.01 μ/ml of thiamine. Total recovery occurred in 1–2 days. Thiamine‐deficient glia also exhibited reduced rates of cholesterol biosynthesis, i.e. 60% of the rates in thiamine‐supplemented cells. This effect was accompanied by a comparable reduction in activity of 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase, the rate‐limiting step in cholesterol biosynthesis. Unlike the glial cells, the neuronal cells exhibited either no or only a slight reduction in lipid synthesis under similar conditions of thiamine deficiency.

The data have important implications for the genesis of the neuropathology in states of altered thiamine homeostasis and for the mechanisms of regulation of lipid synthesis.
OCTOPAMINE DISTRIBUTION IN THE INSECT NERVOUS SYSTEM
Authors: Evans P. D.
Published: December 2006

Abstract— Octopamine distribution has been surveyed in the nervous systems of two insect species, the locust, Schistocerca americana gregaria, and the cockroach Periplaneta americana. It is essentially similar for both species, being highly localised in the ganglia of the ventral nerve cord. Large amounts of octopamine are also found in the optic lobes especially, in the locust where it is concentrated in the medulla of the optic lobe. Octopamine can also be shown to be associated with insect neurohae‐mal structures such as the corpora cardiaca and the neurohaemal organs of the medial nervous system. The significance of the distribution is discussed.
EFFECTS OF ELEVATED [K+]0 ON THE RELEASE OF NEUROTRANSMITTERS FROM CORTICAL SYNAPTOSOMES: EFFLUX OR SECRETION?
Authors: Haycock John W. , Levy William B. , Denner Larry A. , Cotman Carl W.
Published: December 2006

Abstract— Elevated K+0 elicited a substantial Ca‐independent efflux of accumulated GABA from cortical synaptosomal fractions. Efflux from tissue labelled with either NE or choline was affected considerably less by elevated K+ pulses in the absence of calcium. K‐facilitated Ca‐dependent efflux was large for all three of the accumulated substances. K‐dependent (Ca‐independent) efflux of accumulated GABA was associated with all subcellular fractions exhibiting GABA accumulation whereas K‐facilitated Ca‐dependent efflux was restricted to fractions containing synaptosomes. Eighty per cent of both GABA accumulation and K‐dependent efflux was, however, recovered in a purified synaptosomal fraction. Alanine slightly decreased GABA accumulation, but % K‐dependent efflux was not affected.

Elevated K+, in the absence of calcium, released GABA from accumulated pools in preference to endogenous pools, whereas the Ca‐dependent efflux, facilitated by K+, was similar for both accumulated and endogenous GABA.

The Ca‐independent efflux of accumulated GABA increased linearly with log [K+]0 between 10 and 70 mM‐K+ in sodium‐containing media. Prior treatment with veratridine or Na‐free medium substantially decreased the Ca‐independent but not the Ca‐dependent GABA efflux produced by elevated K+ pulses.

The Ca‐dependent and Ca‐independent efflux of accumulated GABA in response to elevated K+ pulses is suggested to arise not only via different flux mechanisms but also from different GABA pools. The Ca‐dependent efflux is interpreted to reflect stimulus‐secretion coupling processes whereas the Ca‐independent efflux may reflect membrane transport processes.
PURIFICATION OF CHOLINE ACETYLTRANSFERASE FROM DROSOPHILA MELANOGASTER
Authors: Driskell W. J. , Weber B. H. , Roberts E.
Published: December 2006

Abstract— Purification of choline acetyltransferase (ChAc) from heads of Drosophila melanogaster, the richest known source of ChAc, has been accomplished. The stability of the enzyme was preserved by working with a concentration of protein above 0.1 mg/ml. The purification was carried out with ammonium sulfate fractionation and column chromatography on QAE‐Sephadex, CoA‐Sepharose, G‐200 Sephadex, and PCMB‐Sepharose. In a procedure using 100 g of Drosophila heads, the specific activity of the crude homogenate was 0.028 μmol/min/mg protein and that of the final product was 43 μmol/min/mg protein, representing a 1500 fold purification. A single protein band, containing all of the ChAc activity, was seen by polyacrylamide gel electrophoresis. A sharp pH optimum at 7.2 was observed. Apparent Km's for acetyl CoA and choline were 90 μM and 47 μM, respectively. The molecular weight was determined to be 69,000. Isoelectric focusing of extracts of Drosophila heads showed only one peak of choline acetyltransferase activity with an apparent pi of 5.1.
A SENSITIVE ENZYMATIC‐RADIOISOTOPIC ASSAY FOR APOMORPHINE
Authors: Kebabian John W.
Published: December 2006

Abstract— An assay for the dopaminergic agonist, apomorphine, is described. The assay is capable of detecting as little as 3 pmol of apomorphine. The basis of the assay is the O‐methylation of apomorphine utilizing S‐[methyl‐3H]adenosyl‐L‐methionine and a partially purified catechol‐O‐methyl transferase to form [methyl‐3H]apocodeine. The radiolabeled apocodeine is purified by solvent partition. Following systemic administration of apomorphine, the compound is uniformly distributed throughout the brain of the rat: there is no selective accumulation of apomorphine in dopaminergic regions of the brain. The level of apomorphine found in the substantia nigra is as great as the level found in the caudate‐putamen. This latter observation, in addition to a variety of other evidence, raises the possibility that some of the in vivo effects of apomorphine may occur via an action of the drug within the substantia nigra, rather than by an action within the caudate‐putamen.
DOPAMINE‐CONTAINING VASOMOTOR NERVES IN THE DOG KIDNEY
Authors: Bell C. , Lang W. J. , Laska F.
Published: December 2006

Abstract— The dopamine (DA) content of the canine renal cortex is greater than can be attributed to its presence in noradrenergic axons only. Most of the excess DA is in the outer part of the cortex. By fluorescence histochemistry numerous catecholamine‐containing axons are seen to be associated with renal cortical arteries and arterioles. The fluorescence is abolished following treatment of animals with 6‐hydroxydopamine or with reserpine, but is restored to some axons if reserpine is followed by systemic administration of 1‐DOPA. This procedure does not restore fluorescence to atrial noradrenergic axons after reserpine‐induced depletion. Pretreatment of animals with guanethidine abolishes axonal fluorescence and depletes tissue NA and DA from atrium, but in renal cortex the tissue DA level is little affected and some fluorescent axons remain. These results are discussed in the light of previous functional evidence for dopaminergic autonomic axons in the canine kidney.
GLIAL VERSUS NEURONAL ORIGIN OF MYELIN PROTEINS AND GLYCOPROTEINS STUDIED BY COMBINED INTRAOCULAR AND INTRACRANIAL LABELLING
Authors: Matthieu J.‐M. , Webster H. deF. , De Vries G. H. , Corthay Sylvia , Koellreutter Brigitte
Published: December 2006

Abstract— —The contribution of axonal transport to the production of myelin proteins and glycoproteins was investigated using the double labelling technique of combined intraocular and intracerebral injections in the same animal. Myelin and an axolemma‐enriched fraction were isolated from pooled optic nerves, chiasma and optic tracts. Separation by gel electrophoresis showed that typical myelin proteins and glycoproteins were only significantly labelled by intracerebral injection. Intraocular injection labelled high molecular weight proteins other than the major Wolfgram protein and the major myelin glycoprotein. Fifteen days after intraocular injection the label was concentrated in a high molecular weight protein which migrated slightly more slowly than the major Wolfgram protein. The pattern of proteins and glycoproteins in myelin labelled by intraocular injection was very similar to that obtained in the axolemma‐enriched fraction by the same route. These results indicate that neuronal metabolism and axonal transport do not contribute significantly to the synthesis of specific myelin proteins and glycoproteins, but suggest that the components of myelin fractions which are labelled by intraocular injection are contaminants of axolemmal origin. One of these glycoproteins may prove a useful marker of axolemma membranes.
A COMPARISON OF SOME ASPECTS OF THE PHYSIOLOGY AND METABOLISM OF THE NERVOUS SYSTEM OF THE LOCUST SCHISTOCERCA GREGARIA IN VITRO WITH THOSE IN VIVO
Authors: Clement E. M. , Strang R. H. C.
Published: December 2006

Abstract— Studies were made on isolated ganglia of the locust in a modified O2 electrode. The basic requirements of a suitable incubation medium were determined and found to include saturation of the medium with 100% O2, and 10mm‐glucose as energy source. The effects of incubation in vitro were determined for a number of biochemical characteristics including O2 uptake and the concentrations of Arg P, ATP, ADP, glycogen, glucose, lactate and pyruvate. Action potentials were recorded from a major nerve both in vivo and in vitro. On the basis of these biochemical and physiological studies, it was concluded that the tissue functions well in vitro. Some comparisons were made between the metabolic characteristics found in the locust nervous system, and those of mammalian tissue. The locust ganglia were found to have a rate of O2 uptake much higher than comparable mammalian tissue, and to contain a considerable reserve of glycogen. The low ratio of lactate/pyruvate suggests a more aerobic metabolism than is the case in mammalian brain.
REGIONAL LEVELS OF GABA IN THE BRAIN: RAPID SEMIAUTOMATED ASSAY AND PREVENTION OF POSTMORTEM INCREASE BY 3‐MERCAPTO‐PROPIONIC ACID
Authors: van der Heyden J. A. M. , Korf J.
Published: December 2006

Abstract— A simple and rapid semiautomated assay for GABA in central nervous tissue is described. The method is based on a simple manual procedure of isolating GABA from tissue extracts on small CM Sepharose Cl 6B columns, followed by an automated fluorimetric detection (continuous flow system) with o‐phthalaldehyde (OPA) and β‐mercapto‐ethanol (β‐ME) at an alkaline pH. GABA is separated from other compounds that fluoresce in our detection system. By using low concentration of OPA and β‐ME and allowing only a short reaction time with these reagents, the detection is specific towards GABA. The detection limit of the assay is 1 nmol.

A procedure is described for the prevention of postmortem GABA increase in rat and mouse brain by intravenous injection of 3‐mercapto‐propionic acid (1.2 nmol/kg) 2min before decapitating the animal. This treatment and microwave irradiation result in similar GABA levels in mouse brain and substantia nigra tissue from rat brain. We found a great conformity in regional GABA levels in the rat and the mouse brain.
TRYPTOPHAN LOADING: CONSEQUENT EFFECTS ON THE SYNTHESIS OF KYNURENINE AND 5‐HYDROXYINDOLES IN RAT BRAIN
Authors: Gaal E. M. , Young R. B. , Sherman A. D.
Published: December 2006

Abstract— Tryptophan loading of rats resulted in a continuous non‐linear uptake of l‐tryptophan from plasma into the brain. The optimum tryptophan load for increasing cerebral 5‐hydroxytryptamine (5‐HT) level was 25 mg/kg. Above this, there was a gradual decrease both in the levels and synthesis of 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) as assessed from simultaneous intraperitoneal or intraventricular injections of l[14C]tryptophan. A 5–10 fold increase in cerebral tryptophan produced a limited stimulation of 5‐HT synthesis. When the cerebral tryptophan level reached 1 ± 10‐4, substrate inhibition in vivo of the tryptophan monooxygenase (tryptophan‐5‐hydroxylase) but not of the indoleamine‐2,3‐dioxygenase occurred. Cerebral synthesis of kynurenine increased linearly with increasing tryptophan load. At a plasma ratio of 50:1 tryptophan to kynurenine, tryptophan loading interfered with the entry of peripheral kynurenine. Tryptophan loading also increased the efflux of 5‐hydroxyindoles from the brain. One hour after intraperitoneal injection of l‐kynurenine sulfate (5 mg/kg) into rats, there was a shift in the plasma ratio of l‐tryptophan to l‐kynurenine to 4:1. In these rats, a 20% reduction of cerebral tryptophan was noted.
SYNAPTOSOMAL TRANSPORT: A CHLORIDE DEPENDENCE FOR CHOLINE, GABA, GLYCINE AND SEVERAL OTHER COMPOUNDS
Authors: Kuhar Michael J. , Zarbin Marco A.
Published: December 2006

Abstract— The synaptosomal transport of fourteen different neurotransmitters, neurotransmitter precursors and other amino acids was tested for a chloride dependence. Those compounds having sodium‐dependent‘high‐affinity’transport systems exhibited a chloride dependence which was maintained over time and which was concentration dependent. Low chloride concentrations altered the transport kinetics of the compounds examined. The reduced accumulation of these compounds in chloride deficient media was not due to increased efflux or alterations in metabolism. The significance of these results for models of transport, methods in transport studies and for other factors are discussed.
Synthesis of Retinal Gangliosides During Chick Embryonic Development
Authors: Panzetta P. , Macdoni H. J. F. , Caputto R.
Published: December 2006

Abstract: Embryonic retina cells incorporated radioactivity from D‐[6‐3H]glucosamine into gangliosides in vitro. The incorporation was higher in retinas from younger embryos. The pattern of labeling of individual gangliosides of the retina changed gradually from a predominant labeling of gangliosides running chromatographically as GD3 (nomenclature of Svennerholm) and GM3 in retinas from 8‐day‐old embryos to a predominant labeling of those running as GDIa and GT, in retinas from 13–18‐day‐old embryos and newly hatched chicks. The shift in the pattern of labeling correlated with a temporary increase of about sixfold of the activity of UDP‐N‐acetyl‐galactosamine:GM3 N‐acetylgalactosaminyltransferase occurring between days 8 and 14 of embryonic development and with a regular increase of the activity of the UDP‐galactose:GM2 galactosyltransferase occurring from day 8 until hatching. The activities of the CMP‐NeuAc:lactosylceramide‐, CMP‐NeuAc:GM3‐, and CMP‐NeuAc:GM1‐sialosyltransferases in the retinas of newly hatched chicks were 40, 20, and 40%(in comparison with the corresponding activities determined in retinas of the 8‐day‐old embryo.
Effect of Oxotremorine on Ornithine Decarboxylase Activity of the Adrenal Gland in Rat
Authors: Ramirez‐Gonzalez Maria Dolores , Widy‐Tyszkiewicz Ewa , Sourkes Theodore L. , Almazan Guillermina
Published: December 2006

Abstract: In this work we have studied the mechanism for the increase of adrenal ODC (ornithine decarboxylase, EC 4.1.1.17) activity provoked by oxotremorine, a muscarinic agonist. 1. Oxotremorine increased medullary ODC activity maximally at 2 h. Cortical enzyme responded much more slowly. 2. Blockade of peripheral muscarinic receptors with methylatropine partially reduced the response to oxotremorine in the medulla, but not cortex. 3. Hy‐pophysectomy abolished the cortical, but not the medullary, responses to oxotremorine. Methylatropine reduced the effect of oxotremorine on medullary ODC in hypophysectomized rats. 4. In unilaterally splanchnicotomized rats oxotremorine caused an increase of ODC activity of the denervated adrenal gland relative to control value; activities in both medulla and cortex were significantly lower than those observed in the innervated gland. Evidence was obtained for a compensatory increase of ODC activity of the adrenal cortex (but not medulla) on the intact side of unilaterally operated rats. 5. Surgical intervention, in the form of a sham operation for transection of the spinal cord, leads to an increase of ODC activity in both parts of the adrenal gland. Transection of the cord attenuates these increases. 6. The additional increase of medullary ODC activity owing to the administration of oxotremorine to sham‐operated rats is partially reduced in the adrenal medulla by muscarinic blockade, and completely in the cortex. This effect of methylatropine in regard to cortical ODC activity was not apparent in the other experiments with intact or unilaterally splanchnicotomized (unoperated side) rats. The results with unilaterally splanchnicotomized rats and those with transected spinal cord suggest that oxotremorine‐induced modifications of adrenal ODC activity are centrally mediated, above the level of origin of the splanchnic nerves in the spinal cord (T8–10). Experiments with hypophysectomized rats show that the response of the adrenal cortex to oxotremorine is entirely mediated by the hypophysis.
Investigation of the Axonal Transport of Three Acidic, Soluble Proteins (14‐3‐2, 14‐3‐3, and S‐100) in the Rabbit Visual System
Authors: Erickson Paul F. , Moore Blake W.
Published: December 2006

Abstract: The question of whether three acidic, water‐soluble proteins (14‐3‐2, 14‐3‐3, and S‐100, the first and last known to be brain‐specific) are axonally transported was investigated in the rabbit visual system. The water‐soluble proteins were obtained from individual optic nerves, combined optic tracts and lateral geniculate bodies, superior colliculi, and, in some instances, retinas at various times (1–56 days) after monocular injections of [3H]leucine. These proteins were separated by a two‐step polyacrylamide gel electrophore‐sis procedure that isolated 14‐3‐2, 14‐3‐3, and S‐100 almost uncontaminated by other radioactivity. The isolated 14‐3‐2 and S‐100 were demonstrated to be approx. 90% pure by a new method based on retarding the migration of these proteins by immunoadsorption during the first step of electrophoresis. An analysis of the radioactive labeling of the total soluble proteins (TSP) and the isolated acidic proteins revealed that: (1) S‐100 was not axonally transported; (2) both 14‐3‐2 and 14‐3‐3 were part of one of the slow components of axonal transport (2‐4 mm/day); (3) the radioactivity of 14‐3‐2 and 14‐3‐3 represented about 2.7% and 3.2%, respectively, of the radioactivity incorporated into the axonally transported TSP; (4) the ultimate distributions of the radioactively labeled 14‐3‐2 and 14‐3‐3 were the same (about 70% of each destined for the superior colliculus) and differed from that of the TSP; and (5) the rates of catabolism of the axonally transported 14‐3‐2 and 14‐3‐3 were slightly greater than that of the TSP, with half‐lives for 14‐3‐2 and 14‐3‐3 estimated to be 11 and 10 days, respectively.