bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Activity of Substantia Nigra Grafts Reinnervating the Striatum: Neurotransmitter Metabolism and [14C]2‐Deoxy‐d‐glucose Autoradiography
Authors: Schmidt Richard H. , Ingvar Martin , Lindvall Olle , Stenevi Ulf , Björklund Anders
Published: October 2006

Abstract: Dopaminergic innervation of the caudate nucleus in adult rats can be partially restored by the grafting of embryonic substantia nigra into the overlying parietal cortex with concomitant compensation of certain behavioral abnormalities. In this study the function of such grafts was investigated neurochemically by quantification of transmitter metabolism and glucose utilization in the reinnervated target. Rats with unilateral 6‐hydroxydopamine lesions of the nigrostriatal bundle received a single graft to the dorsal caudateputamen and were screened for rotational behavior following 5 mg/kg methamphetamine. The grafts restored dopamine concentrations in the caudateputamen from initially less than 0.5% to an average of 13.6% of normal in rats with behavioral compensation. The ratio of 3,4‐dihydroxyphenylacetic acid to dopamine, which is a measure of the rate of transmitter turnover, were equivalent in transplanted and normal control rats. Moreover, measurements of DOPA accumulation for a 30‐min period after DOPA decarboxylase inhibition indicated similar fractional dopamine turnover rates in normal and transplantreinnervated tissues. Correlations between rotational behavior and dopamine concentrations showed that reinnervation to only 3% of normal was sufficient to counterbalance the motor asymmetry. Measurements of glucose utilization by [14C]deoxyglucose autoradiography indicated equivalent metabolic rates for the grafted tissue and the intact substantia nigra. 6‐Hydroxydopamine denervation of the caudate‐putamen had no significant effect on neuronal metabolism in that region, nor did subsequent reinnervation from a graft. Grafts, however, were associated with a 16% reduction of glucose uptake in the ipsilateral globus pallidus, indicating a significant transsynaptic influence of the nigral transplants on neuronal metabolism in the host brain. Overall the results indicate that behaviorally functional neuronal grafts spontaneously metabolize dopamine and utilize glucose at rates characteristic of the intact nigrostriatal system. This provides further evidence that ectopic intracortical nigral trans‐plants can reinstate dopaminergic neurotransmission in regions of the host brain initially denervated by the 6‐hydroxydopamine lesion.
Preparative Separation and Amino Acid Composition of Neurofilament Triplet Proteins
Authors: Hogue‐Angeletti R. A. , Wu H.‐L. , Schlaepfer W. W.
Published: October 2006

Abstract: Intact neurofilaments were isolated from bovine spinal cord white matter, washed by sedimentation in 0.1 m‐NaCl, and extracted with 8 m‐urea. Solubilized neurofilament triplet proteins of molecular weights approximately 68,000 (P68), 150,000 (P150), and 200,000 (P200) were purified by preparative electrophoresis, using an LKB 7900 Uniphor apparatus. The method provides for an enhanced yield of purified protein and has markedly reduced admixture of electrophoresed protein with acrylamide and associated protein contaminants. Amino acid compositions of the purified neurofilament triplet proteins are reported and compared.
Effects of Kainic Acid on High‐Energy Metabolites in the Mouse Striatum
Authors: Retz Konrad C. , Coyle Joseph T.
Published: October 2006

Abstract: Intrastriatal injection of either kainic acid (0.35 μg) or ibotenic acid (7.0 μg) in the mouse causes a profound and selective degeneration of striatal neurons accompanied by a secondary astrocytic response. The kainate injection (0.35 μg) resulted in significant decrements in the striatal levels of phosphocreatine and ATP by 30 min, a progressive reduction in adenosine phosphates between 30 min and 48 h, and a decrease in energy charge; whereas lactate levels increased by 44% at 2 h, glucose levels fell by 56%. Two hours after intrastriatal injection of ibotenic acid (7.0 μg) similar alternations in striatal high‐energy phosphates and glucose disposition were found. Prior decortication protected against the neurotoxic effects of kainate in the mouse striatum and prevented the alterations in high‐energy phosphates at 2 h although lactate levels increased by 212%. These findings in vivo are consistent with the hypothesis that the neurotoxic effects of acidic excitatory amino acids involve a profound activation of energy consumption by affected neurons.
Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo
Authors: Koeppen Arnulf H. , Papandrea John D. , Mitzen Edward J.
Published: October 2006

Abstract: Labeled palmitic acid ([16‐14C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16‐14C]palmitate to other long‐chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2‐diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16‐14C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.
Activation of Ethanolamine Phospholipase A2 in Brain During Ischemia
Authors: Edgar Alan D. , Strosznajder Joanna , Horrocks Lloyd A.
Published: October 2006

Abstract: Extracts of acetone‐dried powders from ischemic gerbil brain were examined for phospholipase A1 and A2 activities with phosphatidylethanolamine at pH 7.2. Ischemia was induced by bilateral ligation, and the animals were killed by immersion into liquid nitrogen. Bilateral ligation with ketamine as general anesthetic resulted in a rapid, transient increase in phospholipase A2 activity. The activity increased from 0.46 nmolihimg protein at 0 time to 0.82 nmol/h/mg protein at 1 min of ligation. Phospholipase A1 activity also increased from 0.7 to 1.3 nmol/h/mg protein within the 1st min. When Nembutal was used as anesthetic, the phospholipase activation was earlier, within the first 30 s. Similar results were found for ischemia induced by decapitation of Wistar rats without anesthesia. Bilateral ligation of the carotid arteries of the gerbil is known to increase the concentration of free fatty acids, particularly arachidonate. This increase is, at least in part, due to phospholipase A activation. As ethanolamine phospholipase A2 in brain does not require Ca2+ for activity, these results suggest that phospholipase A2 activation in ischemic brain results from a covalent modification of the enzyme.
Nitrogen Metabolism of the Human Brain
Authors: Eriksson Ljusk Siw , Law David H. , Hagenfeldt Lars , Wahren John
Published: October 2006

Abstract: Cerebral nitrogen metabolism was studied in 29 healthy nonobese volunteers by means of a catheterization technique. Arterial levels and arterial‐jugular venous (A‐JV) concentration differences for amino acids, urea, ammonia, 5‐oxoproline, glucose, and oxygen were measured in the basal, postabsorptive state and during an intravenous infusion of a commercial amino acid solution. In the basal state positive A‐JV differences, indicating a net brain uptake, were noted for 12 of 22 amino acids as well as for ammonia. There was no significant net exchange for urea or for 5‐oxoproline. During amino acid infusion, resulting in a 150–300% rise in arterial amino acid levels, the brain uptake of isoleucine, leucine, and tyrosine increased significantly, and a similar tendency was seen for most other amino acids. The infusion was accompanied by a 100% rise in arterial ammonia levels and a 10% increase in urea concentration. For ammonia the small positive A‐JV difference in the basal state became markedly greater during amino acid infusion, whereas no significant alteration was noted for urea exchange across the brain. The A‐JV differences for glucose and oxygen were positive in the basal state and unchanged during the infusion. The present findings demonstrate that in the basal state (a) there is a significant net brain uptake of most amino acids; (b) no single amino acid, urea, or 5‐oxoproline is released from the brain; and (c) ammonia uptake occurs both in this state and during an amino acid infusion. It is concluded that brain uptake of amino acids accounts for a quantitatively significant proportion of the total amino acid turnover in the basal state but the nature of the nitrogenous compound(s) released from the brain to achieve nitrogen balance remains to be determined. Eriksson L. S. et al. Nitrogen metabolism of the human brain.
An Improved Method for In Situ Freezing of Cat Brain for Metabolic Studies
Authors: Yang M. S. , Lutz H. , DeWitt D. S. , Becker D. P. , Hayes R. L.
Published: October 2006

Abstract: This study introduces a new method for rapid freezing of the cat brain. The method employed a Styrofoam box which was fitted around the head of the animal. Liquid nitrogen was poured into the box until the head was submerged. Temperature changes in three brain sites (ventral hypothalamus, the fourth ventricle, and the corpus callosum) and levels of labile carbohydrate metabolites (glycogen, glucose, ATP, P‐creatine, and lactate) in five brain regions (cortex, thalamus, midbrain, cerebellum, and pons) frozen by the box method were compared with those frozen by a conventional cup method in which liquid nitrogen was poured into a hollow Styrofoam cup placed on top of the skull. The box method shortened the time of arrival of the freezing front and improved the freezing rate. The time required to bring the tissue to ‐20°C was shortened, from 20 min at the ventral hypothalamus and 10–12 min at the fourth ventricle with the cup method, to <5 min at both sites with the box technique. Continued perfusion of brainstem prior to freezing was demonstrated. Levels of metabolites frozen by either method were similar. Lactate levels in any of the five brain regions studied by either method were not elevated, indicating no ischemic change. The shorter freezing time resulting from the box method should provide improved temporal resolution in studying dynamic metabolic changes such as those frequently observed in pathological conditions.
Use of Cholate/Sodium Chloride for Solubilisation of Brain D2 Dopamine Receptors
Authors: Hall Jean M. , Frankham Patricia A. , Strange Philip G.
Published: October 2006

Abstract: The use of cholate (in the presence of sodium chloride) is described for solubilising brain D2 dopamine receptors. The method described gives in high yield a preparation of solubilised D2 receptors which when assayed by [3H]spiperone binding show little interference from nonspecific and nonstereospecific binding sites. Characterisation by ultrafiltration, electron microscopy, gel filtration, and sucrose density gradient centrifugation has been achieved, showing the receptors to be truly solubilised. Pharmacological characterisation using [3H]spiperone binding suggested the presence of serotonergic S2 receptors in addition to D2 receptors. The pharmacological properties of the solubilised D2 receptors show a good correlation with those of membrane‐bound receptors, although ligand binding affinities are lower in the solubilised preparation.
Uptake of Acetate and Propionate by Isolated Nerve Endings from the Electric Organ of Torpedo marmorata and Their Incorporation into Choline Esters
Authors: O'Regan S.
Published: October 2006

Abstract: The uptake and incorporation into choline esters of acetate and propionate by electric organ synaptosomes were compared, with the aim of better understanding the basis for the selectivity of choline ester synthesis shown by this tissue for acetate. It was found that propionate uptake, like acetate uptake, was a temperature‐dependent, saturable process. Both uptake mechanisms had similar affinities for their substrates, but the maximal velocity of propionate uptake was considerably lower than that of acetate uptake; and less of the accumulated propionate was used for choline ester synthesis than of the accumulated acetate. While acetate was a good inhibitor of propionate uptake, propionate was a very poor inhibitor of acetate uptake. This finding, in addition to the observation that the two uptakes were not affected in the same way by changes in pH, led to the suggestion that acetate uptake and propionate uptake reflect different processes. In both cases, however, the pH dependence of uptake indicated that these substrates cross the membrane as the charged species. Acetate uptake and acetylcholine synthesis remained closely associated under various experimental conditions, while propionate uptake could be dissociated from the synthesis of propionylcholine. Hence, it appears that acetate is taken up by a specific, high‐velocity mechanism linked to acetylcholine synthesis, whereas propionate uptake may represent a less specific mechanism.
Retinal Hexokinase: Kinetic Properties and the Effect of Cyclic 3′,5′‐Adenosine Monophosphate
Authors: Liu Shu‐Sen , Witkovsky Paul , Yang Chen‐Yu
Published: October 2006

Abstract: We measured hexokinase (EC 2.7.1.1) activity in particulate and soluble fractions isolated from bullfrog (Rana catesbeiana) retinas. Seventy‐three percent of the hexokinase (HK) activity was associated with the particulate fraction, 27% with the soluble fraction. Both HK fractions could phosphorylate fructose, glucose, 2‐deoxy‐d‐glucose, and mannose, but not galactose. The Km for glucose was 0.14 mM, for 2‐deoxy‐d‐glucose. 3.6 mM. With glucose as substrate, the Vmax for particulate HK was 125–148 μM retina−1 min−1, for soluble HK, 37 μM retina−1 min−1. Product inhibition of particulate HK activity by glucose 6‐phosphate was marked, whereas 2‐deoxy‐d‐glucose 6‐phosphate did not inhibit the activity. Cyclic AMP stimulated the HK activity of both retinal fractions nearly twofold at concentrations of 0.2–0.8 mM; AMP was much less effective in this regard.
In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain
Authors: Sajdel‐Sulkowska Elizabeth , Coughlin John F. , Marotta Charles A.
Published: October 2006

Abstract: Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)‐containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)‐cellulose‐purified poly(A)‐containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L‐[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9‐ to 20‐fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two‐dimensional polyacrylamide gel electrophoresis (PAGE): size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)‐cellulose chromatography yielded 1.8 μg/g and 2.0 μg/g, respectively, of poly(A)‐containing mRNA; this represents a two‐ to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two‐dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.
Cerebral Blood Flow and Oxidative Metabolism in Conscious Fischer‐344 Rats of Different Ages
Authors: Takei Hidenori , Fredericks Wendy R. , London Edythe D. , Rapoport Stanley I.
Published: October 2006

Abstract: The cerebral metabolic rates for O2 and for glucose were measured in conscious, fasted male Fischer‐344 rats at the ages of 3, 12, and 24 months, and cerebral blood flow was determined with 14C‐iodoantipyrine. The metabolic rates for oxygen and glucose were obtained by multiplying blood flow by the O2 and glucose concentration differences, respectively, between blood in the femoral artery and in the superior sagittal sinus. Mean cerebral blood flow and the metabolic rates for oxygen and glucose did not differ significantly (p > 0.05) between 3 and 12 or between 12 and 24 months. Nor did the arteriovenous differences for O2 and for glucose change significantly with age. Because the superior sagittal sinus drains blood mainly from the cerebral cortex, the results indicate that average cerebral cortical oxidative metabolism, and the coupling ratios between the cerebral metabolic rate for oxygen and cerebral blood flow and between the cerebral metabolic rate for glucose and cerebral blood flow, do not change significantly with age in the Fischer‐344 rat.
Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies
Authors: Ward H. K. , Thanki C. M. , Bradford H. F.
Published: October 2006

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo. Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30‐150 min by [U‐14C]glucose, [U‐14C]glutamine, or [3H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μM); tetrodotoxin (1 μM) was present in the control medium. Single‐label experiments showed that [U‐14C]‐ glutamine was more effective than [U‐14C]glucose for labelling releasable glutamate and GABA. Double‐label experiments showed that [3H]glutamine and [U‐14C]‐ glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.
Enhancement of Neurite Outgrowth and Aspartate‐Glutamate Uptake Systems in Retinal Explants Cultured with Chick Gizzard Extract
Authors: Kato Satoru , Negishi Koroku , Hayashi Yokichi , Miki Naomasa
Published: October 2006

Chicken gizzard extract promoted a long and radially directed neurite outgrowth from retinal explants of 8‐day‐old chick embryo in cultures of 2–3 days. The neurite outgrowth from retinal explants cultured in the absence of gizzard extract was short and restricted to the explant perimeter. The neurite outgrowth promoted by gizzard extract depended strictly on several factors. (a) Fetal calf serum and polycationic substratum were required in this culture system, (b) Pretreatment of the polyornithine‐coated substratum with gizzard extract allowed the retinal explants to extend neurites even in the absence of gizzard extract in the medium. (c) Maximal neurite outgrowth was observed in retinal explants dissected from 8‐day embryos, but thereafter the explants’response to gizzard extract rapidly declined and was almost lost at the 12th day. As a biochemical parameter of differentiation of cultured neuroretina, uptake systems for neurotransmitter candidates were examined in homogenates of retinal explants cultured in the absence or presence of gizzard extract. After 3 days in culture with gizzard extract, the uptake increased for aspartate and glutamate 1.6‐ to 1.8‐fold and for γ‐aminobutyric acid to a lesser degree when examined at a concentration for high‐affinity uptake (10‐6M). In contrast, the uptake capacity for glycine, choline, and dopamine was not altered in explants cultured with or without gizzard extract. Kinetic analysis showed that the enhanced capacity to accumulate aspartate was not due to an alteration of Km, but to an increase of Vmax. The results suggest that one or several factors in chick gizzard muscle promote not only neurite outgrowth but also the aspartate‐glutamate uptake systems in the developing neuroretina, probably related to ganglion cells.
Differential Changes in Superoxide Dismutase Activity in Brain and Liver of Old Rats and Mice
Authors: Danh Hung Cao , Benedetti Margherita Strolin , Dostert Philippe
Published: October 2006

Superoxide dismutase (SOD) activity was measured in the brain and liver of 24–26‐ and 3‐month‐old rats. No significant age‐related differences in Cu/Zn‐SOD activity were found in any of the tissues studied. A small but significant increase in total SOD activity was observed in the whole brain (10‐20%), cerebral cortex (11%), and hypothalamus (18%) of old rats, whereas a much more important increase in Mn‐SOD activity was found in the whole brain (48%), cerebral cortex (70%), striatum (60%), and hypothalamus (30%). The increase of Mn‐SOD activity in the brain of old rats suggests the enzyme may play an important role in the process of aging. Mn‐SOD is found only in the mitochondrion, which could be an important site of oxygen free radical production, and a significant increase in the enzyme activity was also found in the lung of hypoxic rats. A significant decrease in total SOD and Mn‐SOD activity was observed in the liver of old rats. Preliminary experiments in 23–24‐month‐old mice similarly showed an increase and a decrease in total SOD and Mn‐SOD activity, respectively, in the whole brain and liver. These results suggest that the regulatory mechanisms of Mn‐SOD in the brain and liver vary differentially with age.
In Vivo Elevation of Mouse Brain S‐Adenosyl‐L‐Homocysteine after Treatment with L‐Homocysteine
Authors: Gharib A. , Chabannes B. , Sarda N. , Pachéco H.
Published: October 2006

Intraperitoneal coadministration of adenosine and L‐homocysteine markedly increased S‐adenosyl‐l‐homocysteine in whole mouse brain, but further investigations showed that this elevation could also be produced following administration of l‐homocysteine alone. The noted increase was maximal (+1325%) 10 min after treatment, remaining at about this level for 30‐40 min before returning to control values after 180 min. Cerebral adenosine levels were decreased after treatment with l‐homocysteine, adenosine, or these two substances in combination.
Isolation of PGP 9.5, a New Human Neurone‐Specific Protein Detected by High‐Resolution Two‐Dimensional Electrophoresis
Authors: Doran John F. , Jackson Peter , Kynoch Pamela A. M. , Thompson R. J.
Published: October 2006

Protein gene product (PGP) 9.5 is a new brain‐specific protein originally detected by high‐resolution two‐dimensional electrophoresis of the soluble proteins of human brain and other organs. We have purified this protein from human brain and raised a rabbit antihuman PGP 9.5 antiserum. The protein has a monomer molecular weight of ˜27,000 and is present in brain at concentrations at least 50 times greater than in other organs. Im‐munoperoxidase labelling has localised PGP 9.5 to neurones in the human cerebral cortex with no evidence of staining of glial elements. PGP 9.5 is estimated to be present in brain at concentrations of 200‐500 μg/g wet weight and represents a major protein component of neuronal cytoplasm. This new neurone‐specific cytoplasmic marker may prove useful in studies of neuronal development and in the detection of neuronal damage in disease of the nervous system.
The Enzymes of Acetyl‐CoA Metabolism in Differentiating Cholinergic (S‐20) and Noncholinergic (NIE‐115) Neuroblastoma Cells
Authors: Szutowicz A. , Morrison M. R. , Srere P. A.
Published: October 2006

Dibutyryl cyclic AMP and butyrate inhibited growth of S‐20 (cholinergic) and NIE‐115 (adrenergic) neuroblastoma clones. Both these drugs resulted in a parallel increase of choline acetyltransferase and ATP‐citrate lyase activities in S‐20 neuroblastoma cells. On the other hand, the increase in tyrosine hydroxylase activity in NIE‐115 caused by these drugs was not accompanied by a significant change in ATP‐citrate lyase activity. Both dibutyryl cyclic AMP and butyrate caused a decrease in fatty acid synthetase activity in both cell lines. The activities of pyruvate dehydrogenase, citrate synthase, choline acetyltransferase, and lactate dehydrogenase in both S‐20 and NIE‐115 cells were not significantly influenced by the drugs. ATP‐citrate lyases from S‐20 and NIE‐115 had similar kinetic and immunological properties, and their subunits had the same molecular weight as the rat liver enzyme. These data indicate that the differential regulation of ATP‐citrate lyase activity in cholinergic and adrenergic cells does not result from the existence of different molecular forms of the enzyme in these cell lines. They also provide further evidence to support the hypothesis that ATP‐citrate lyase activity increases during maturation of normal cholinergic neurons and decreases in noncholinergic cells of the brain.
Ca2+– and Calmodulin‐Dependent Phosphorylation of Microtubule‐Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly
Authors: Yamamoto Hideyuki , Fukunaga Kohji , Tanaka Etsuro , Miyamoto Eishichi
Published: October 2006

Abstract: Microtubule‐associated proteins (MAPs) were phosphorylated by a Ca2+– and calmodulin‐dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and t factor were phosphorylated in a Ca2 +‐and calmodulin‐dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+‐ and calmodulin‐dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.
Pharmacological Evidence for the Existence of Multiple Functional Pools of Brain Serotonin: Analysis of Brain Perfusate From Conscious Rats
Authors: Kleven Mark S. , Dwoskin Linda P. , Sparber Sheldon B.
Published: October 2006

Abstract: The serotonin reuptake inhibitor fluoxetine significantly reduced levels of endogenous 5‐hydroxyindoleacetic acid (5‐HIAA) in brain perfusate of rats implanted with push‐pull cannulas. This occurred in conjunction with its suppressant effect upon fixed‐ratio operant behavior. Behavior suppressed with the serotonin agonist lysergic acid diethylamide (LSD) occurred in conjunction with a reduction of 5‐HIAA only after 5‐HIAA was elevated, shortly before, by 5 mg/kg of the serotonin precursor 5‐hydroxytryptophan. Our data demonstrate the likely existence of multiple functional pools of serotonin in brain and support the notion that LSD preferentially affects a newly synthesized pool of this transmitter.
Uptake of L‐[14C]Proline by Isolated Rat Brain Capillaries
Authors: Hwang Shing Mei , Miller Michael , Segal Stanton
Published: October 2006

Abstract: Rat brain capillaries exhibit concentrative uptake of l‐proline. The uptake is mediated by two saturable systems, one with a Km of 0.11 mM and another with a Km of 5.9 mM. Entry also occurred by diffusion, especially at high substrate concentrations. The saturable high‐affinity system is sodium‐dependent, with a Km for sodium of 36 mM. Proline uptake is not inhibited by lysine, but is inhibited by phenylalanine, glycine and leucine. α‐Methylaminoisobutyric acid (MeAIB), a model for sodium‐requiring transport systems, is a competitive inhibitor of the low‐Km system. b‐2‐Aminobicyclo‐[2, 2, 1]‐heptane‐2‐carboxylic acid (BCH), a model for nonsodium‐dependent transport, however, also inhibited proline uptake.
High‐ and Low‐Affinity Binding of [3H]Imipramine in Mouse Cerebral Cortex
Authors: Reith M. E. A. , Sershen H. , Allen D. , Lajtha A.
Published: October 2006

Abstract: Binding of [3H]imipramine in mouse cerebral cortex was found to be nonhomogeneous. Competition experiments, Scatchard analysis and Hill plots are compatible with the existence of binding with high (nanomo‐lar) and low (micromolar) affinity. Low‐affinity binding could be eliminated by the use of low concentrations of imipramine as the competing ligand. In contrast to the high‐affinity binding, the low‐affinity binding was found to be unrelated to the neuronal uptake system for serotonin.
Induction of a Stress Protein in Developing Cell Cultures of the Rat Cerebellum
Authors: Pearce Brian R. , Dutton Gary R. , White Fredric P.
Published: October 2006

Abstract: We examined the ability of developing cere‐bellar cell cultures to synthesize a 71,000 MW stress protein (SP71) in response to heat shock and Cd2+ treatment. The induction of SP71 synthesis appeared to be dependent on both the age of the culture and the stressor used. Heat shock induced SP71 synthesis in freshly prepared cells and in cell cultures at each age examined, whereas Cd2+ was effective only in cultures at 7 days of age and older. These findings are discussed with reference to the development of various cell types in these cultures.
Purification from Brain of Synenkephalin, the N‐Terminal Fragment of Proenkephalin
Authors: Listen Dane , Böhlen Peter , Rossier Jean
Published: October 2006

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin‐containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N‐terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.
Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain
Authors: Michaelis E. K. , Chittenden W. L. , Johnson B. E. , Galton N. , Decedue C.
Published: October 2006

Abstract: A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na‐cholate, followed by solubilization of the binding protein from the membranes by Triton X‐100, and, finally, affinity batch separation of the protein on l‐glutamate‐loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2‐terminal amino acid (tyrosine), and significant absorption at wavelengths >300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound l‐glutamate with high affinity (KD= 0.87 μM), exhibited one class of l‐glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein‐bound l‐glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of l‐glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.
Inhibition of Brain Glycolysis by Aluminum
Authors: Lai James C. K. , Blass John P.
Published: October 2006

Abstract: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain. The IC50 values were between 4 and 9 μM. Aluminum was effective at mildly acidic (pH 6.8) or slightly alkaline (pH 7.2–7.5) pH, in the presence of a physiological level of magnesium (0.5 mM). However, saturating (8 mM) magnesium antagonized the effect of aluminum on both forms of hexokinase activity. Other enzymes examined were considerably less sensitive to inhibition by aluminum. The IC50 of aluminum for phosphofructokinase was 1.8 mM and for lactate dehydrogenase 0.4 mM. At 10–600 μM, aluminum actually stimulated pyruvate kinase. Aluminum also inhibited lactate production by rat brain extracts: this effect was much more marked with glucose as substrate than with glucose‐6‐phosphate. However, the IC50 for inhibiting lactate production using glucose as substrate was 280 μM, higher than that required to inhibit hexokinase. This concentration of aluminum is comparable to those reportedly found in the brains of patients who had died with dialysis dementia and in the brains of some of the patients who had died with Alzheimer disease. Inhibition of carbohydrate utilization may be one of the mechanisms by which aluminum can act as a neurotoxin.
Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy
Authors: Kish Stephen J. , Perry Thomas L. , Hornykiewicz Oleh
Published: October 2006

Abstract: Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus γ‐aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.
Cell‐Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice
Authors: Campagnoni A. T. , Campagnoni C. W. , Bourre J.‐M. , Jacque C. , Baumann N.
Published: October 2006

Abstract: Total polyribosomes were isolated from the brains of 16–20‐day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shi/+, mld, and jp littermates) and translated in a homologous, cell‐free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 ± 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid‐precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close‐to‐normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and < 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an “assembly” or “synthesis” defect.
Further Characterization of an Enkephalin‐Generating Enzyme from Adrenal Medullary Chromaffin Granules
Authors: Lindberg I. , Yang H.‐Y. T. , Costa E.
Published: October 2006

Abstract: An adrenomedullary protease capable of generating Met5‐enkephalin from endogenous precursor(s) has been purified 1,000‐fold using affinity chromatog‐raphy in combination with gel filtration. This trypsin‐like enzyme has an apparent molecular weight of 20,000 dal‐tons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5‐enkephalin‐Arg6‐Phe7 and Met5‐en‐kephalin‐Arg6‐Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1‐Met6‐enkephalin, Met5‐enkephalin, and Met5‐enkephalin‐Lys6, while digestion of Peptide E resulted in the production of Leu5‐enkephalin and Met5‐enkephalin‐Arg6‐Arg7. [3H]‐βm‐Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adrenomedullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin‐converting enzyme.
The Muscarinic Receptor Adenylate Cyclase Complex of Rat Striatum: Desensitization Following Chronic Inhibition of Acetylcholinesterase Activity
Authors: Olianas Maria C. , Onali Pierluigi , Schwartz Joan P. , Neff N. H. , Costa E.
Published: October 2006

Abstract: Chronic inhibition of acetylcholinesterase activity by treatment with diisopropylfluorophosphate (DFP) decreased the capacity of acetylcholine (ACh) acting at a muscarinic receptor to inhibit basal adenylate cyclase activity in homogenates from rat striatum. There was also a loss of the capacity of ACh to inhibit the activation of adenylate cyclase by dopamine. The desensitization of the muscarinic receptor adenylate cyclase complex was associated with a marked attenuation of the capacity of ACh to stimulate a high‐affinity GTPase activity present in striatal membranes. The EC50 value of ACh for inhibiting adenylate cyclase and for stimulating GTPase activity increased following treatment with DFP, while the Hill coefficient for both responses was unaltered.
Developmental Regulation of Phenylethanolamine N‐Methyl Transferase in Avian Sympathetic Nerves
Authors: David J.‐C. , Rush R. A.
Published: October 2006

The presence of the catecholamine synthetic enzyme, phenylethanolamine N‐methyl transferase (PNMT), has been detected in the expansor secundariorum, a smooth muscle of the avian wing. The concentration of the enzyme was estimated over a 10‐week time course from 17 days incubation to 9 weeks posthatch and found to increase rapidly up until hatch in parallel with dopamine β‐hydroxylase activity, but then to fall precipitously to very low levels. The time course of the initial increase in activity corresponds to the presence of ingrowing sympathetic nerve fibres, and denervation of the expansor results in loss of >80% of the PNMT activity.

It is concluded that during the period of innervation the growing nerves contain the enzyme PNMT and therefore have the capacity to synthesize adrenaline, but that shortly after innervation is complete the capacity to synthesize adrenaline is lost. Several alternate mechanisms are proposed to explain the observations.
Autophosphorylation of Calmodulin‐Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity
Authors: Shields Steven M. , Vernon Paula J. , Kelly Paul T.
Published: October 2006

Previous studies have purified from bfain a Ca2+/calmodulin‐dependent protein kinase II (designated CaM‐kinase II) that phosphorylates synapsin I, a synaptic vesicle‐associated phosphoprotein. CaM‐kinase II is composed of a major Mr 50K polypeptide and a minor Mr60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin‐dependent manner. Recent studies have demonstrated that the 50K component of CaM‐kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM‐kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [α‐32P]8‐azido‐ATP was used to demonstrate that the 60K and 50K polypeptides of SJ‐associated CaM‐kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin‐dependent manner. Experiments using 32P‐labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I‐calmoduhn binding sites. These results suggested that the autophosphorylation of CaM‐kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I‐calmodulin overlay techniques and sodium dodecyl Solfate‐polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM‐kinase II polypeptides bound more calmodulin (50–70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM‐kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaMkinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM‐kinase II. The CaM‐kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300–1,000 nM). These findings show that the degree of autophosphorylation of CaM‐kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.
P‐31 Nuclear Magnetic Resonance Analysis of Brain: II. Effects of Oxygen Deprivation on Isolated Perfused and Nonperfused Rat Brain
Authors: Kopp S. J. , Krieglstein J. , Freidank A. , Rachman A. , Seibert A. , Cohen M. M.
Published: October 2006

Phosphatic metabolite (perchloric acid extractable) concentrations of cerebral tissues were analyzed by phosphorus‐31 nuclear magnetic resonance (P‐31 NMR) spectfoscopy following external perfusion of the isolated rat brain (30 min or 60 min) under the following conditions: (a) constant perfusion pressure with either fluorocarbon‐or erythrocyte‐based medium, and (b) constant perfusate flow rate (3 ml/min) with the erythrocyte‐based medium. Metabolite concentrations of control perfused brains were compared with those in nonperfused controls to provide a basis for detecting any qualitative or quantitative changes in cerebral metabolite composition. Metabolic responses of perfused brains to ischemia (incomplete ischemia, 83% reduction in flow for 10 min; transient complete ischemia for 1.5 or 2 min) were evaluated immediately after the ischemic episode and at selected time points during reperfusion (3 and 15 min). Alterations in cerebral metabolite levels induced by hypoxia were analyzed using a nonperfused rat brain model. Irrespective of the perfusion method employed, the phosphatic metabolites of control perfused rat brains were identical quantitatively to those of the nonperfused controls. Cerebral ischemia resulted in significantly increased levels of ADP, AMP + IMP, Pi, fructose 1,6‐diphosphate, and glycerol 3‐phosphate (global ischemia only), whereas ATP and phosphocreatine (PCr) levels declined significantly. The magnitude of these changes varied with the severity of the ischemia; however, following 15 min of control reperfusion metabolite levels had reverted to preischemic values. Significant perturbations in tissue phosphoethanolamine (3.848 resonance) content were evident at various time points during ischemia and postischemic recovery, which varied according to the perfusion conditions. In contrast to the changes observed in response to ischemia, hypoxia affected only cerebral high‐energy phosphate levels. ATP and PCr levels were reduced, while a concomitant, essentially equimolar, increase in Pi and ADP was observed. The present studies indicate that in terms of phosphatic metabolites, the control equilibrated isolated perfused rat brain is quantitatively and qualitatively indistinguishable from the non‐perfused rat brain in vivo regardless of the perfusion conditions (constant flow versus constant pressure). The metabolic responses to ischemia and hypoxia, as measured by P‐31 NMR, were consistent with the pattern of changes reported elsewhere. Overall, P‐31 NMR spectroscopic evaluation of the intact rat brain provides a potential experimental context for dynamic measures of cerebral metabolism under exogenously controlled conditions. The results reported herein from brain PCA extracts contribute a spectroscopic reference for understanding and interpreting the metabolic information contained in the P‐31 profiles of the intact brain.