bluuebunny/crossref_metadata_2025 huggingface dataset:
The Functional Significance of the Pentose Phosphate Pathway in Synaptosomes: Protection Against Peroxidative Damage by Catecholamines and Oxidants
Authors: Hothersall J. S. , Greenbaum A. L. , McLean P.
Published: December 2006

Abstract: Catecholamines added in vitro in rat brain synaptosomes activate the decarboxylation of glucose radioactively labelled on carbon 1, suggesting an effective activation of the pentose phosphate pathway. Stimulation also occurred with phenazine methosulphate, reduced glutathione and hydrogen peroxide. The activation of the pentose phosphate pathway by 5‐hydroxytryptamine, noradrenaline and dopamine is ascribed to the activation of monoamine oxidase, producing both the respective biogenic aldehyde and hydrogen peroxide. Evidence is presented that the further metabolism of the aldehyde by aldehyde reductase and the removal of hydrogen peroxide by glutathione peroxidase both release the limitation of N ADP+ availability for the pentose phosphate pathway by leading to the oxidation of NADPH. The relevance of the maintenance of reduced NADP+ on brain is discussed in relation to the metabolism of glutathione and to lipid peroxidation.
Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid‐Sensitive Ca2+‐Dependent Protein Kinase
Authors: Turner R. Scott , Chou C.‐H. Jen , Kibler Robert F. , Kuo J. F.
Published: December 2006

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X‐100 plus 4 mM EGTA, with or without further fractionation, showed that Ca2+‐dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE‐cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid‐sensitive Ca2+‐dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin‐sensitive species of Ca2+‐dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid‐sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulfonamide (W–7).
Differential Expression of Type A and Type B Monoamine Oxidase of Mouse Astrocytes in Primary Cultures
Authors: Yu P. H. , Hertz L.
Published: December 2006

Abstract: Type A and type B monoamine oxidase (MAO) activities were determined in mouse brain and in primary cultures of mouse astrocytes. Thirty‐one‐day‐old astrocyte cultures exhibited predominantly type A MAO activity. In cultures of the same age, treated with 0.25 mM dibutyryl cyclic AMP under the same culturing conditions, 30% type B MAO was expressed, although dibutyryl cyclic AMP up to 1 mM does not affect MAO activity in vitro. The specific activity of type B MAO increased significantly in older cultures, while type A MAO changed only slightly.
Changes in Lipogenic Capacity and Activities of Ketolytic and Lipogenic Enzymes in Brain Regions of Developing Rats
Authors: Yeh Yu‐Yan , Ginsburg Janet R. , Tso T. B.
Published: December 2006

Abstract: Oxidation of ketone bodies (KBs) generates acetyl coenzyme A (AcCoA), which can be further incorporated into fatty acid. We have determined the rates of lipogenesis from ketone bodies in developing rats and their relation to the activities of enzymes involved in the production of cytoplasmic AcCoA via different pathways in brain regions. In the cerebrum (Cbr), rates of fatty acid synthesis from [3‐14C]acetoacetate ([3‐14C]AcAc) were high during the early postnatal period but decreased rapidly thereafter until weaning. Although similar developmental patterns of synthesis characterized the cerebellum (Cbl), midbrain (Mb), brain stem (Bs), and thalamus (Th), maximal rates were highest in the Cbr and lowest in the Th. In all regions, synthetic rates were higher throughout the entire suckling period than in adulthood. There were no appreciable differences in synthetic rates among brain regions of adult rats. The developmental changes in rates of AcAc incorporation into fatty acids were closely related to AcAcCoA synthetase activity, but not to activities of ATP‐citrate lyase or AcCoA synthetase. During the early postnatal stage the enhanced rates of lipogenesis were accompanied by increased activities of AcAcCoA synthetase in all regions, with the highest activity occurring in the Cbr. The sequence of reactions coupling AcAcCoA synthetase and AcAcCoA thiolase in cytoplasm may be an important pathway for generation of AcCoA from KBs for fatty acid synthesis in all regions of the developing brain. This interpretation is strengthened by evidence of concomitant increases in the activities of fatty acid synthetase and AcCoA carboxylase.
Regulation of Glycogen Metabolism in Primary and Transformed Astrocytes In Vitro
Authors: Cummins C. J. , Lust W. David , Passonneau J. V.
Published: December 2006

Abstract: Glycogen metabolism was studied in primary and Herpesvirus‐transformed cultures of neonatal rat brain astrocytes. A small fraction of the glucose consumed was coaserved in glycogen in both the primary and the transformed astrocytic cell cultures. After addition of culture medium containing 5.5 mM glucose, glycogen increased to maximal levels within 2.5 h, the approximate time at which half of the medium glucose was consumed, and rapidly declined thereafter in both the primary and transformed astrocytic cultures. Maximum levels of glycogen were apparently related to the cell density of the Herpesvirus‐transformed cultures, but primary cultures did not show this behavior. At any given cell density, maximal levels of glycogen were dependent on the concentration of extracellular glucose. Administration of glucose caused a transient activation of glycogen synthase a and a rapid inactivation of glycogen phosphorylase a.
The Protein Activator Specific for the Enzymic Hydrolysis of GM2 Ganglioside in Normal Human Brain and Brains of Three Types of GM2 Gangliosidosis
Authors: Hirabayashi Yoshio , Li Yu‐Teh , Li Su‐Chen
Published: December 2006

Abstract: In order to understand the etiology of Type AB GM2 gangliosidosis, we have purified and characterized the activator protein (GM2 activator) specific for the enzymic hydrolysis of GM2 ganglioside from normal human brain. The purified activator from human brain moved as one major protein band in various electrophoretic systems. We have also prepared the antiserum against this activator. The levels and the nature of GM2 activator and β‐hexosaminidase A were examined in the brains of five cases of GM2. gangliosidosis—one Type B, two Type O, and two Type AB. We found that the levels of GM2 activator in both Type B and Type O cases were markedly elevated, and that the two Type AB cases were the results of different causes. One case had a defective β hexosaminidase A and an elevated level of GM2 activator. Although this defective β‐hexosaminidase A could hydrolyze synthetic substrates, it was inactive in the cleavage of natural glycosphingolipids in the presence of the GM2 activator. It could, however, hydrolyze asialo‐GM2 and GbOse4Cer in the presence of sodium taurodeoxycholate. The other case had normal β‐hexosaminidase A, but had a very low level of GM2 activator when analyzed by in vitro assay, suggesting the deficiency of this activator. By immunoelectrophoresis, this case was found to be completely devoid of the protein that cross‐reacts with the antiserum against the GM2 activator.
Effects of Anoxia on the Stimulated Release of Amino Acid Neurotransmitters in the Cerebellum In Vitro
Authors: Bosley T. M. , Woodhams P. L. , Gordon R. D. , Balázs R.
Published: December 2006

Abstract: The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K+] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)‐sensitive and the Ca2+‐dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [14C]GABA and d‐[3H]aspartate (a metabolically more stable anologue of acidic amino acids).[14C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of d‐[3H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of d‐[3H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX‐sensitive veratridine‐induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine‐evoked release of [14C]GABA was already depressed by 10 min and that of d‐[3H]Asp showed a modest elevation only at 5 min. Stimulated release of d‐Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well‐known selective vulnerability of certain neurons during hypoxia.
Distinct Cellular Localization of Membrane‐Bound and Soluble Forms of Catechol‐O‐Methyltransferase in Brain
Authors: Rivett A. Jennifer , Francis Andrew , Roth Jerome A.
Published: December 2006

Abstract: The cellular localization of the two forms of catechol‐O‐methyltransferase (COMT) was investigated by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid, which causes degeneration of striatal neurons followed by proliferation of astroglial cells. Membrane‐bound COMT activity was decreased in the lesioned striatum, while soluble COMT activity was increased. There was a statistically significant correlation between the ratio of lesioned to control activity for membrane‐bound COMT and the neuronal marker enzyme glutamate decarboxylase. Similarly the increase in soluble COMT activity paralleled that of the astroglial marker enzyme, glutamine synthetase. These results indicate that the Km membrane‐bound catechol‐O‐methyltransferase may be localized predominantly in neurons, whereas the high‐Km soluble enzyme is found in glial cells.
Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline‐Induced Status Epilepticus. Effect of Pretreatment with α‐Methyl‐p‐Tyrosine and p‐Chlorophenylalanine
Authors: Turco Elena B. Rodriguez de , de Liberti Susana Morelli , Bazan Nicolas G.
Published: December 2006

Abstract: The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline‐induced seizures were studied. A fourfold increase in cerebral FFA occurred 3–4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic‐clonic convulsion. However, a 13‐ to 17‐fold increase in arachidonic acid was seen approximately 5–6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl‐ and arachidonoyl‐DG were also accumulated. The drug α‐methyl‐p‐tyrosine was found to (a) potentiate the bicuculline‐stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl‐ and arachidonoyl‐DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p‐chlorophenylalanine, but the drug had no effect on the bicuculline‐induced changes. Hydrolysis of phospholipids enriched in stearoyl‐arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.
4‐Aminobutyraldehyde as a Substance Convertible In Vivo to GABA
Authors: Sugahara Masakazu , Asakura Tadashi , Kurioka Susumu , Matsuda Makoto
Published: December 2006

Abstract: [2,3‐3H]4‐Aminobutyraldehyde ([3H]ABAL) was injected subcutaneously into mice, which were sacrificed at various intervals following injection. [3H]γ‐Aminobutyric acid ([3H]GABA) synthesized in vivo from [3H]ABAL was extracted from the brains, separated, and quantitated. The results showed that in the brain, injected [3H]ABAL was rapidly transformed into [3H]GABA. [3H]ABAL may penetrate the blood‐brain barrier into the central nervous system and then be oxidized to [3H]GABA.
Enzymatic Determination of Choline Acetyltransferase by Coenzyme A Cycling and Its Application to Analysis of Single Mammalian Neurons
Authors: Kato Takahiko
Published: December 2006

Abstract: An enzymatic assay for choline acetyltrans‐ferase was developed by measuring acetyl‐coenzyme A (acetyl‐CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl‐CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl‐2.2 μl of assay volume) of freeze‐dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze‐dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500–2,000‐fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000–420,000‐fold amplification, was 40‐fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.
Dihydroxyphenylalanine Production in Rat Brain Striatal Synaptosomes: Stimulation by a Calcium Chelator
Authors: Haycock Dean A. , Patrick Robert L.
Published: December 2006

Abstract: By inhibiting aromatic L‐amino‐acid decarboxylase (EC 4.1.1.28) in rat brain striatal synaptosomes, we have been able to measure dihydroxyphenylalanine production via high performance liquid chromatography‐electrochemical oxidation. This dihydroxyphenylalanine assay was compared to a standard radioisotopic assay of catecholamine synthesis (14CO2 production from L‐[1 ‐ 14C]‐tyrosine) in terms of (1) units of activity, (2) effects of known inhibitory and stimulatory agents, and (3) effects of the calcium chelator, EGTA. The units of activity in the dihydroxyphenylalanine assay were 40% greater than the units in the radioisotopic assay, indicating a mixing of labeled and endogenous tyrosine pools before conversion of the labeled tyrosine to labeled dihydroxyphenylalanine. The inhibition of synthesis produced by either 3‐iodotyrosine or 3, 4‐dihydroxyphenylethylamine was similar in the two assays, as was the stimulation produced by 8‐bromo cyclic AMP. The calcium chelator, EGTA, also activated synthesis to the same extent in the two assays, indicating that the increase observed in the radioisotopic assay is not an artifact of altered precursor specific activity. These data thus indicate the general utility of the synaptosomal dihydroxyphenylalanine synthesis assay, and also demonstrate the specific advantages of this assay for analyzing the effects of agents such as EGTA, which can alter tissue catecholamine precursor levels.
Alterations in Uptake and Release Rates for GABA, Glutamate, and Glutamine During Biochemical Maturation of Highly Purified Cultures of Cerebral Cortical Neurons, a GABAergic Preparation
Authors: Yu A. C. H. , Hertz E. , Hertz L.
Published: December 2006

Abstract: This study demonstrates that virtually homogenous cultures of mouse cerebral neurons, obtained from 15‐day‐old embryos, differentiate at least as well as cultures which in addition contain astrocytes. This was indicated by glutamate decarboxylase activity which within 2 weeks rose from a negligible value to twice the level in the adult mouse cerebral cortex, and by a γ‐aminobutyric acid (GABA) uptake rate which quadrupled during the second week in culture and reached higher values than in brain slices. Within the same period, the GABA content increased four to five times to 75 nmol/mg protein, and a potassium‐induced increase in [14C]GABA efflux became apparent. Although the development was faster than in vivo, optimum differentiation required maintenance of the cultures beyond the age of 1 week. Uptake and release rates for glutamate and glutamine underwent much less developmental alteration. At no time was there any potassium‐induced release of radioactivity after exposure to [14C]glutamate, and the glutamate uptake was only slightly increased during the period of GABAergic development. This indicates that exogenous glutamate is not an important GABA precursor. Similarly, glutamine uptake was unaltered between days 7 and 14, although a small potassium‐induced release of radioactivity after loading with glutamine suggests a partial conversion to GABA.
In Vivo Lipid Peroxidation in Rat Brain Following Intracortical Fe2+ Injection
Authors: Triggs William J. , Willmore L. James
Published: December 2006

Abstract: Cerebral contusion, cortical laceration, intracerebral hematoma formation, and hemorrhagic cortical infarction cause extravasation of red blood cells, followed by hemolysis, decompartmentalization of iron, formation and deposition of hemosiderin, and an increased incidence of epilepsy. In this experiment, 10 μl of an aqueous solution containing 100 mmol/L FeCl2, 100 mmol/L CoCl2, or 0.9% (wt/vol) NaCl were injected at a depth of 1.8 mm into rat isocortex. The rate of formation of fluorescent compounds was measured in chloroform‐methanol extracts of isocortical homogenates. Significant increases in the quantity of fluorescent products of lipid peroxidation were found 120 min after the injection of 100 mmol/L FeCl2. Cobaltous chloride and saline injection had no effect on the levels of fluorescent products found in the cortical homogenates. Although the intracortical deposition of aqueous solutions containing CoCl2 or FeCl2, in rodent cortex causes acute epileptiform discharges, the epileptogenic effect of CoCl2, is transient, while the injection of iron salts causes persistent seizures. Since CoCl2, injection failed to cause formation of lipid peroxidation products while the isocortical injection of iron caused significant increase in fluorescence within the injected hemisphere, we suggest that the occurrence of iron‐induced lipid peroxidation may be of importance in initiation of recurrent seizures in the rat.
The Lipid Composition of Urodele Myelin Which Lacks Hydroxycerebroside and Hydroxysulfatide
Authors: Ki P. F. , Kishimoto Y.
Published: December 2006

Abstract: The concentrations of cerebrosides and sul‐fatides were measured in the nervous systems of urodeles and related orders with a high performance liquid chro‐matographic technique. The peripheral and central nervous systems of all three urodele species, Necturus ma‐culosis (mud puppy, a salamander), Notophthalmus viri‐descens (eastern red spot newt), and Desmognathus ochropheus (mountain salamander), were found to be completely devoid of γ‐hydroxy fatty acid‐containing cerebrosides and sulfatides. All species of reptiles and fish classes close to urodeles contain these galactolipids. The levels of nonhydroxy fatty acid‐containing cerebrosides and sulfatides are essentially similar in both urodeles and reptiles. Myelin isolated from Necturus spinal cord had a specific density of 1.07, lighter than mammalian myelin. Except for the absence of hydroxycerebrosides and hydroxysulfatides, the lipid composition of Necturus spinal cord myelin is essentially similar to that of frog and rat myelin. The fatty acids of nonhydroxycerebrosides are rich in monounsaturated homologs of C22‐C25, and the sphingoid base consists of both sphinganine and sphin‐gosine. Electron microscopic examination of the sciatic nerve showed that the general structure and interlamellar distances of salamander and newt myelin are identical to those of frog, chameleon, and rat. Necturus myelin, therefore, can be used as a model for the study of the functional and structural role of hydroxygalactolipids.
Ethanol and the γ‐Aminobutyric Acid‐Benzodiazepine Receptor Complex
Authors: Greenberg David A. , Cooper Edward C. , Gordon Adrienne , Diamond Ivan
Published: December 2006

Abstract: Ethanol appears to enhance γ‐aminobutyric acid (GABA)‐mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA‐benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 mM failed to alter binding of [3H]flunitrazepam (FNZ), [3H]Ro 15‐1788, or [3H]methyl‐γ‐carboline‐3‐carboxylate (MBCC) to benzodiazepine receptors, or of [3H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4°C and 37°C revealed no significant effects of 100 mM ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [3H]FNZ and/or [3H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA‐benzodiazepine receptor complex.
Quantitative Evaluation of Neurite Outgrowth in Cultures of Human Foetal Brain and Dorsal Root Ganglion Cells Using an Enzyme‐Linked Immunoadsorbent Assay for Human Neurofilament Protein
Authors: Doherty Patrick , Dickson John G. , Flanigan Thomas P. , Walsh Frank S.
Published: December 2006

Abstract: An enzyme‐linked immunoadsorbent assay has been developed to evaluate comparative levels of neurofilament protein in developing primary cultures of human foetal dorsal root ganglion and brain tissue. The quantitative parameters of the assay, relating linearity of response with varying levels of neurofilament protein, were verified by comparing the relative binding of human species‐specific (BF10) and cross‐species‐reactive (RT97) monoclonal antibodies to mixtures of human and baboon spinal cord homogenates that had been passively adsorbed onto microtitre wells. In human neural cultures, the localisation of neurofilament protein to growing neurites was determined by indirect immunofluorescence staining with anti‐neurofilament antibodies and, using the immunoadsorbent assay, a time‐dependent increase in the level of neurofilament protein was detected that correlated with the morphological time course of neurite development. In the case of dorsal root ganglion cells over 6 days in vitro, a seven‐ to ninefold greater increase in neurofilament protein levels was observed in cultures treated with nerve growth factor when compared with control unstimulated preparations. The quantitative responsiveness of dorsal root ganglion neurones to nerve growth factor detected by the neurofilament assay indicates its potential usefulness in the identification and analysis of neurotrophic and neurotoxic factors or cellular interactions operating in vitro.
α‐Ketoglutarate and Malate Uptake and Metabolism by Synaptosomes: Further Evidence for an Astrocyte‐to‐Neuron Metabolic Shuttle
Authors: Shank Richard P. , Campbell Graham Le M.
Published: December 2006

Abstract: This study was undertaken to provide further evidence relevant to the hypothesis that astrocytes supply one or more citric acid cycle intermediates to syn‐aptic terminals, thereby serving an anaplerotic function necessitated by the synthesis and release of amino acid neurotransmitters. In our experiments, two populations of synaptosomes obtained from the brain of rats were separated from myelin and mitochondria by using Percoll to generate continuous density gradients. Both synapto‐somal populations readily accumulated 14C‐labelled a‐ke‐toglutarate and L‐malate by high‐affinity transport systems. Hofstee plots of uptake velocity as a function of substrate concentration were highly nonlinear, indicating that uptake was mediated by two or more carriers, or was subject to negative cooperativity. At least one carrier was selective for a‐ketoglutarate and another for malate, whereas a third carrier appeared to be present which transported both substrates. At low concentrations (1 μM), γ‐ketoglutarate transport was almost totally Na + ‐dependent, whereas malate uptake exhibited little Na+‐dependency. The transport of a‐ketoglutarate was associated with a net influx, and therefore was not due to a homoexchange process. γ‐Ketoglutarate and malate were metabolized rapidly to glutamate and aspartate, respectively, by both synaptosomal preparations; however, in all cases, label accumulated in γ‐aminobutyric acid rather slowly. The incorporation of label into glutamine from γ‐ketoglutarate was much greater in the high‐density synaptosomes than in low‐density synaptosomes, an indication that the former contained a higher proportion of astrogliasomes. The results of our study are consistent with the concept that certain types of synaptic terminals are dependent upon astrocytes for a supply of a‐ketoglutarate and/or malate in order to maintain their pools of citrate cycle intermediates and replenish their neuro‐transmitter pools.
Glutamine, Glutamate, and Other Possible Regulators of α‐Ketoglutarate and Malate Uptake by Synaptic Terminals
Authors: Shank Richard P. , Campbell Graham Le M.
Published: December 2006

Abstract: The uptake of a‐ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of γ‐ketoglutarate by causing an apparent reduction in the substrate‐carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L‐Glutamate and L‐as‐partate were comparatively strong inhibitors of γ‐keto‐glutarate and malate uptake. N‐Acetylaspartate was a weak inhibitor of γ‐ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited γ‐ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8‐ to 14‐day‐old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of γ‐ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of γ‐ketoglutarate by low‐density synaptosomes. By comparison, the uptake of glutamate, glutamine, γ‐aminobutyric acid, and several other amino acids was not affected. The enhancement of γ‐ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U‐‐14C] γ‐ketoglutarate to glutamate, as‐partate, glutamine, and aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.
Determination of Glutathione and ATP in Ganglia and Individual Neurons of Aplysia californica
Authors: Stein Cathy , Weinreich Daniel
Published: December 2006

Abstract: The levels of two γ‐glutamyl cycle substrates, glutathione and ATP, were determined in single identified nerve cell bodies from the CNS of Aplysia californica. The glutathione content of single cells averaged 30 ± 4.9 μmol/g protein. Glutathione levels were similar in identified cholinergic, serotonergic, and histaminergic cells, as well as in neurons whose transmitters are not yet identified. The abdominal rostral white cells, which are enriched in glycine, a component amino acid of glutathione, did not possess distinctively higher glutathione concentrations. The ATP content of single Aplysia nerve cell bodies averaged 15.0 ± 1.5 μmol/g protein. Despite the vast chemical, anatomical, and functional heterogeneity between Aplysia central neurons, no cells were found that contained unusually high or low ATP levels.
Fetal Calf Spinal Cords as Enriched Source of mRNA Encoding Glial Fibrillary Acidic Protein
Authors: Watanabe Sachihiko , Sueoka Noboru
Published: December 2006

Abstract: Total poly(A)+ RNA was isolated from fetal calf spinal cord, adult rat spinal cord, and young rat brain, and was translated using the rabbit reticulocyte lysate system. The amount of glial fibrillary acidic protein in the translation products was measured by immunoprecipitation with antiserum against glial fibrillary acidic protein. RNA from fetal calf spinal cord could direct glial fibrillary acidic protein synthesis such that this protein comprised approximately 1.4% of the total products. RNAs from adult rat spinal cord and brain could direct glial fibrillary acidic protein synthesis much less efficiently, with this protein comprising <0.3% of the total products. These results suggest that the gene for glial fibrillary acidic protein is strongly expressed in fetal calf spinal cord and that this tissue is an enriched source of mRNA encoding glial fibrillary acidic protein.
Alteration of Cerebral Taurine Biosynthesis in Spontaneously Hypertensive Rats
Authors: Kuriyama Kinya , Ida Shuji , Ohkuma Seitaro
Published: December 2006

Abstract: Cerebral taurine biosynthesis in a spontaneously hypertensive rat (SHR) has been studied. Cysteine sulfinic acid (CSA) and cysteic acid (CA), possible key intermediates in taurine biosynthesis, were found in the rat brain, whereas no cysteamine‐cystamine was detected. In the brain of SHR, a statistically significant decrease in the contents of CSA, CA, and taurine was noted in the cerebellum, hypothalamus, and striatum as compared with normotensive Wistar Kyoto rats. Similarly, it was demonstrated that the activity of cysteine dioxygenase, the enzyme catalyzing cysteine to CSA, was attenuated significantly in the same brain areas of SHR. In contrast, no alteration in the activity of CSA decarboxylase, the enzyme converting CSA to hypotaurine or CA to taurine, was observed. A decline in the percent conversion of [14C]cysteine to [14C]taurine was found also in tissue homogenates from the cerebellum, hypothalamus, and striatum of SHR, indicating that the declines in taurine content may be due to an attenuation of taurine biosynthesis, possibly at the step involving cysteine dioxygenase.
Catabolism of N‐Acylethanolamine Phospholipids by Dog Brain Preparations
Authors: Natarajan V. , Schmid P. C. , Reddy P. V. , Schmid H. H. O.
Published: December 2006

Abstract: N‐Acylphosphatidylethanolamine, incubated with dog brain homogenate or microsomes, was hydroyzed to phosphatidic acid and N‐acylethanolamine by a phosphodiesterase of the phospholipase D type. In the absence of F−, phosphatidic acid was further hydrolyzed to diacylglycerol and Pi while N‐acylethanolamine was hydrolyzed by an amidase to fatty acid and ethanolamine. The phosphodiesterase showed an alkaline pH optimum and was also active towards N‐acetylphosphatidyletha‐nolamine, N‐acyl‐lysophosphatidylethanolamine, and glycerophospho(N‐acyl)ethanolamine but showed little activity toward phosphatidylethanolamine and phosphati‐dylcholine. Ca2+ stimulated slightly at low concentrations but inhibited at higher concentrations. Triton X‐100 stim ulated the hydrolysis of N‐acylphosphatidylethanol‐amine, inhibited that of N‐acyl‐lysophosphatidyletha‐nolamine and glycerophospho(N‐acyl)ethanolamine, and had no effect on phosphatidylethanolamine or phospha‐tidylcholine hydrolysis. The N‐acylethanolamine hydrolase (amidase) was also present in the microsomal fraction and exhibited a pH optimum of 10.0. In addition to hydrolysis by the phosphodiesterase, N‐acylphosphati‐dylethanolamine was also catabolized by microsomal phospholipases A1 and/or A2 to N‐acyl‐lysophosphati‐dylethanolamine, some of which was further hydrolyzed to glycerophospho(N‐acyl)ethanolamine.
Anticholinesterase Action of a Bromine Compound Isolated from Human Cerebrospinal Fluid
Authors: Yamane Mototeru , Abe Akihisa , Yanagisawa Isamu
Published: December 2006

Abstract: L‐l‐Methylheptyl‐γ‐bromoacetoacetate was found to be a competitive inhibitor of the acetylcholines‐terases (electric eel, Ki= 17.2 μM; rat brain, Ki= 32.6 μM) and of butyrylcholinesterase (horse serum, Ki= 1.2 μM). The L‐isomer was a more effective inhibitor than the D‐isomer. The bromine atom at the γ‐position of the acidic moiety, the specific length of the carbon chain constituting the secondary alcohol moiety, and the presence of the ketone radical at the acidic moiety of the ester were necessary for the anticholinesterase action. 1‐Methyl‐heptyl‐γ‐bromoacetoacetate formed a complex with acetylcholinesterase or butyrylcholinesterase without hydrolysis of its own molecule.
Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor
Authors: Greene Lloyd A. , Seeley P. John , Rukenstein Adriana , DiPiazza Margaret , Howard Andrew
Published: December 2006

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 nM). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron‐like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF‐induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent Km of the enzyme for tyrosine or for co‐factor (6‐methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.
Effect of Oleate on Neurotransmitter Transport and Other Plasma Membrane Functions in Rat Brain Synaptosomes
Authors: Troeger Melody B. , Rafalowska Urszula , Erecińska Maria
Published: December 2006

Abstract: The effects of fatty acids, oleate and palmitate, on ‐γ‐aminobutyric acid (GABA), aspartate, and 3,4‐di‐hydroxyphenylethylamine (dopamine) transport and a variety of other membrane functions were studied in rat brain synaptosomes at a constant lipid‐to‐protein ratio. Under the conditions utilized oleate, but not palmitate, caused statistically significant changes in synaptosomal functions. Oleic acid inhibited the uptake of the amino acid neurotransmitters and dopamine in a tetrodotoxin‐insensitive manner; it also induced the release of neurotransmitters from synaptosomes. The synaptosomal membrane potential decreased and the maximum GABA accumulation ratio ([GABA]i/GABA]o) declined in parallel. The same depolarizing effect was seen in the presence of 50 μM verapamil or when chloride was replaced by propionate. The rate of respiration was stimulated by the unsaturated fatty acid; neither verapamil (50 μM) nor ouabain (100 μM) was effective in preventing the increase in oxygen consumption. By contrast, ruthenium red substantially decreased the stimulatory effect of oleate. The intrasynaptosomal [Ca2+] was increased by 40%, whereas [Na +]i remained unaltered. It is postulated that under the conditions used the inhibition of neurotransmitter uptake and the decrease in their accumulation caused by oleate result from the depolarization of synaptosomes that arises, at least in part, from increased permeability of the plasma membrane to calcium ions.
Effects of Chronic Ethanol Treatment on the In Vitro Biosynthesis of Pro‐Opiomelanocortin and ItsPosttranslational Processing to (β‐Endorphin in theIntermediate Lobe of the Rat Pituitary
Authors: Seizinger Bernd R. , Höllt Volker , Herz Albert
Published: December 2006

Abstract: Chronic treatment of rats with 15% (vol/vol) ethanol in tap water as their only source of liquid over a period of 3 weeks resulted in a strong decrease by almost 50% in tissue levels and in vitro release of immunoreactive β‐endorphin of the neurointermediate pituitary. Moreover, the in vitro incorporation of [3H]phenylalanine into peptides of the neurointermediate pituitary, immunoprecipitable with β‐endorphin antiserum, was found to be decreased by more than 30%. Analysis of β‐endorphinrelated peptides on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that chronic ethanol treatment reduced the in vitro biosynthesis of the β‐endorphin precursor pro‐opiomelanocortin. This ethanol‐induced effect was combined with a retardation in the time course of the posttranslational processing of the precursor into β‐endorphin. Thus, chronic ethanol treatment may influence the activity of enzymes which process the opioid peptide precursor pro‐opiomelanocortin, leading to a decreased formation of the final secretory product β‐endorphin.
Early Undernutrition and [3H]γ‐Aminobutyric Acid Binding in Rat Brain
Authors: Telang Shaila , Fuller Gregory , Wiggins Richard , Enna S. J.
Published: December 2006

Abstract: The effect of early undernutrition and dietary rehabilitation on [3H]γ‐aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein‐deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high‐ and low‐affinity components in the undernourished group, whereas control animals possessed only a low‐affinity site. The concentration of low‐affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X‐100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA‐activated benzodiazepine binding suggests the possibility that neither the high‐ nor the low‐affinity GABA binding sites are coupled to this receptor component.
Tyrosine Hydroxylase in “Leaky” Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP‐Dependent Systems
Authors: Niggli V. , Knight D. E. , Baker P. F. , Vigny A. , Henry J.‐P.
Published: December 2006

Abstract: The systems responsible for phosphorylating tyrosine hydroxylase, the rate‐limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the other on cyclic AMP. The Ca2+‐dependent system is half‐maximally activated by 1‐2 μM Ca2+ and 0.5 mM ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 mM) does not inhibit significantly Ca2+‐dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP‐dependent system is half‐maximally activated by addition of 0.5 μM cyclic AMP and about 0.3 mM ATP. Ca2+‐dependent and cyclic AMP‐dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10−8M Ca2+ plus 2 × 10−5M cyclic AMP or of 10−5M Ca2+, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca2+‐dependent system, whereas the other site(s) are phosphorylated by both the Ca2+‐ and cyclic AMP‐dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.
Metabolic Stability of Hippocampal Slice Preparations During Prolonged Incubation
Authors: Whittingham Tim S. , Lust W. David , Christakis Dimitri A. , Passonneau Janet V.
Published: December 2006

Abstract: Hippocampal slices were prepared under three conditions: (1) in medium containing glucose and oxygen at 4°C; (2) as in (1), but at 37°C; (3) in medium devoid of glucose and oxygen at 37°C. The rates of recovery to roughly steady‐state levels and through 8 h of incubation were monitored for energy metabolite levels and related parameters. In vitro stable values are compared with in situ hippocampal levels. Regardless of the conditions under which slices were prepared, metabolite levels required up to 3 h to stabilize, and these levels were maintained or improved through 8 h of incubation. Further, the maximal concentrations of metabolites were independent of the conditions of slice preparation. Total adenylates and total creatine levels reached 55% of those in vivo. Lactate decreased from the decapitation‐induced high levels, but stabilized at concentrations about twice those in rapidly frozen brain. Cyclic AMP and cyclic GMP exhibited peak levels at 30 min of incubation, and cyclic GMP remained elevated for 3 h. Although all three methods of slice preparation resulted in similar metabolite profiles on incubation, the initial decreases in high energy phosphates were delayed by chilling. Most striking, the slices prepared in the absence of glucose and oxygen exhibited much smaller orthodromic evoked potentials in the dentate gyrus. The presence of glucose and oxygen during preparation of the slices appears to be critical to the electrophysiological response of the tissue.
Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium
Authors: Pastuszko Anna , Wilson David F. , Erecińska M.
Published: December 2006

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d‐[3H]aspartate, with a Ki of approximately 0.3 mm. Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time‐ and concentration‐dependent manner, but had no effect on the content of γ‐aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem‐brane electrical potential were prevented by the addition of glutamate, whereas the kainate‐induced release of ra‐dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high‐affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.
A Library of Monoclonal Antibodies to Torpedo Cholinergic Synaptosomes
Authors: Kushner P. D.
Published: December 2006

Abstract: A library of monoclonal antibodies was generated to the cholinergic synaptosome. The immunogen was a preparation of highly purified synaptosomes from Torpedo electric organ. One hundred forty‐one hybridoma cell lines were generated from the fusion of a single mouse. Tests reveal these cells produce antibodies with a vast range of neuronal specificities. The initial screen for specificity of antibody production was solid phase radioimmune binding to the original, highly purified synaptosome preparation. Subsequent tissue specificity tests have indicated that most antibodies are synaptosome‐specific amongst the fish tissues tested: brain, liver, and purified synaptic vesicles. Less than 11% cross‐react with liver. Many antibodies cross‐react with frog and rat CNS. Localization within the frog and rat nervous tissue has revealed a vast array of antibody staining patterns. Some antibodies stain in a synaptic fashion. A few stain a restricted set of mammalian CNS neurons. Others define a broader set of CNS neurons. These Torpedo antibodies promise to be valuable probes with which to describe the molecular cell biology of the nervous system, of neurons in general, and of cholinergic neurons in particular.
Orthograde, Retrograde, and Turnaround Axonal Transport of Dopamine‐β‐Hydroxylase: Response to Axonal Injury
Authors: Schmidt Robert E. , Modert Christopher W.
Published: December 2006

Abstract: Reversal of the direction (turnaround) of orthograde axonal transport of dopamine‐β‐hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury‐induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.