bluuebunny/crossref_metadata_2025 huggingface dataset:
Astrocyte Leucine Metabolism: Significance of Branched‐Chain Amino Acid Transamination
Authors: Yudkoff Marc , Daikhin Yevgeny , Grunstein Lev , Nissim Ilana , Stern Janet , Pleasure David , Nissim Itzhak
Published: July 2010

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 ± 3.2 nmol/mg of protein/min and a Km of 449.2 ± 94.9 µM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α‐aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α‐ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 µM with a Vmax of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α‐ketoglutarate ? KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the “buffering” of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.
Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes
Authors: McKenna Mary C. , Sonnewald Ursula , Huang Xueli , Stevenson Joseph , Zielke H. Ronald
Published: July 2010

Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U‐13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 mM [U‐13C]glutamate 85% of the 13C metabolized was converted to glutamine. The formation of [1,2,3‐13C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 mM glutamate, 13C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13C]lactate was essentially unchanged within the range of 0.2–0.5 mM glutamate, whereas the amount of [13C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 mM, suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 mM and astrocytes from both rat and mouse brain are consistent with these findings.
Lipopolysaccharide‐Enhanced Expression of Interleukin‐6 in Dibutyryl Cyclic AMP‐Differentiated Rat C6 Glioma
Authors: Slegers Herman , Joniau Marcel
Published: July 2010

Abstract: Rat C6 glioma synthesizes a low basal level of interleukin‐6 (IL‐6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 mM N6,O2′‐dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP‐differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL‐6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 mM dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL‐6 mRNA concentration and IL‐6 immunoreactivity, whereas titration of the biological activity indicated a 100‐fold increase in the secreted IL‐6 activity. The enhanced secretion of IL‐6 is correlated with the induction of differentiation. Chromatography on heparin‐Sepharose and on DEAE‐5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL‐6‐like activities could be neutralized by an anti‐murine IL‐6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.
In Vivo Electrochemical Studies of Dopamine Overflow and Clearance in the Striatum of Normal and MPTP‐Treated Rhesus Monkeys
Authors: Gerhardt Greg A. , Cass Wayne A. , Hudson John , Henson Mike , Zhang Zhiming , Ovadia Aliza , Hoffer Barry J. , Gash Don M.
Published: July 2010

Abstract: Rapid chronoamperometric recordings, using Nafion‐coated carbon‐fiber electrodes (30–90 µm o.d.), were used to investigate overflow and uptake of dopamine (DA) in the striatum of normal and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated rhesus monkeys. The monkeys were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging‐guided sterile stereotaxic procedures were used for implantations of the electrochemical electrodes coupled with single‐barrel micropipettes that were used to apply potassium or DA locally. Potassium evoked a robust overflow of DA‐like electrochemical signals into the brain extracellular space in the unlesioned or normal putamen and caudate nucleus of the rhesus monkeys. In contrast, potassium did not produce any detectable changes (> 97% depletion) of DA in the MPTP‐lesioned striatum. In addition, the diffusion/clearance of locally applied DA was markedly altered in the lesioned caudate nucleus and putamen compared with unlesioned striatum. Cell counts of the number of residual tyrosine hydroxylase‐positive neurons in MPTP‐treated monkeys, in conjunction with whole‐tissue levels of DA and its metabolites, showed that the MPTP lesions produced extensive damage of the nigrostriatal DA system. These data indicate that residual dopaminergic fibers remaining after MPTP lesions are dysfunctional and have a greatly diminished capacity for high‐affinity DA uptake.
The Prefrontal Cortex Regulates the Basal Release of Dopamine in the Limbic Striatum: An Effect Mediated by Ventral Tegmental Area
Authors: Karreman Mignon , Moghaddam Bita
Published: July 2010

Abstract: The present study examined whether the prefrontal cortex (PFC) exerts a tonic control over the basal release of dopamine in the limbic striatum and whether this control is mediated by glutamatergic afferents to the dopamine cell body or terminal regions. Using intracerebral microdialysis in freely moving rats, it was demonstrated that application of tetrodotoxin in the contralateral PFC significantly decreased the release of dopamine in the medial striatum. Conversely, blockade of the tonic inhibitory GABAergic input in the PFC with bicuculline increased the release of dopamine in the medial striatum. Application of excitatory amino acid receptor antagonists into the striatum, while bicuculline was perfused in the PFC, did not affect the bicuculline‐evoked dopamine increase in the striatum. However, infusion of tetrodotoxin or excitatory amino acid receptor antagonists into the ventral tegmental area, a region containing dopamine cell bodies that project to the medial striatum, blocked the stimulation of striatal dopamine release induced by infusion of bicuculline into the PFC. These data demonstrate that the basal output of dopamine terminals in the medial striatum is under a tonic excitatory control of the PFC. Furthermore, this control occurs primarily through glutamatergic projections to the dopamine cell body area rather than the terminal regions.
Metal Selectivity of Exocytosis in α‐Toxin‐Permeabilized Bovine Chromaffin Cells
Authors: Tomsig J. L. , Suszkiw J. B.
Published: July 2010

Abstract: Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca2+, Ba2+, Sr2+, Pb2+, La3+, Cd2+, Co2+, Tb3+, Mn2+, and Zn2+ on the release of norepinephrine (NE) from staphylococcal α‐toxin‐permeabilized bovine chromaffin cells. Pb2+, La3+, Cd2+, Sr2+, and Ba2+ activated NE secretion accompanied by the release of intragranular dopamine β‐hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb2+, La3+, Cd2+, and Sr2+ was nonadditive with Ca2+, indicating a common site of action. In contrast, the Ba2+‐evoked NE release was additive with Ca2+ and the Ca2+ agonists Pb2+, La3+, Cd2+, and Sr2+, suggesting that Ba2+ activates secretion at a site distinct from the Ca2+ receptor. In distinction to the NE release evoked by Pb2+, La3+, Cd2+, and Ba2+, the Sr2+‐evoked NE release was associated with a significant elevation of Ca2+ concentration in the medium and abolished by Ca2+ chelation. This indicates that the secretagogue effect of Sr2+ was indirect and secondary to the displacement of bound Ca2+. Co2+ and Mn2+ inhibited the NE release evoked by Ca2+, Sr2+, Pb2+, La3+, and Cd2+ but had no effect on the Ba2+‐dependent secretion. Tb3+ and Zn2+ were without effect on exocytosis.
Estrogen Alters MPTP‐Induced Neurotoxicity in Female Mice: Effects on Striatal Dopamine Concentrations and Release
Authors: Dluzen Dean E. , McDermott Janet L. , Liu Binjun
Published: July 2010

Abstract: The effects of estrogen on MPTP‐induced neurotoxicity of the nigrostriatal dopaminergic system were examined in C57Bl and CD‐1 mice. Ovariectomized mice with and without estrogen were treated with MPTP or its vehicle. The effects of these treatments on striatal dopamine concentrations and l‐DOPA‐stimulated dopamine and l‐3,4‐dihydroxyphenylacetic acid (DOPAC) release in vitro were determined. Dopamine concentrations of C57Bl mice receiving estrogen before MPTP were significantly greater than those of non‐estrogen‐treated MPTP mice as well as estrogen‐treated mice receiving the MPTP vehicle. Dopamine concentrations of the CD‐1 mice did not differ with these treatments. l‐DOPA‐evoked dopamine release values of estrogen‐treated C57Bl mice were significantly increased compared with non‐estrogen‐treated mice. No such differences were observed in the MPTP‐treated C57Bl mice. DOPAC release rates were similar to that of dopamine in these C57Bl mice. In the CD‐1 mice estrogen also produced a significant increase in l‐DOPA‐evoked dopamine release; however, this response was unaltered by MPTP treatment. A significant increase in l‐DOPA‐evoked DOPAC output was obtained only for estrogen‐treated CD‐1 mice. Both strains show very similar responses to the estrogen treatment, but differential responses of dopamine release to l‐DOPA between the C57Bl and CD‐1 mice were obtained with regard to the interactive effects of estrogen and MPTP. Our results suggest that in addition to its role as modulator, estrogen may also function as a neuroprotectant against MPTP neurotoxicity of the nigrostriatal dopaminergic system in the C57Bl mouse.
Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes
Authors: Hsu Yea‐Nan , Amin Jahanshah , Weiss David S. , Wecker Lynn
Published: July 2010

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µM nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration‐dependent manner with IC50 values in the 10 nM range following incubation for 24 h and in the 1 nM range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 nM nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first‐order process for both subtypes; half‐lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first‐order for both subtypes; half‐lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor‐mediated neurotransmitter release.
Neuroligand‐Evoked Calcium‐Dependent Release of Excitatory Amino Acids from Cultured Astrocytes
Authors: Jeftinija Srdija D. , Jeftinija Ksenija V. , Stefanovic Gordana , Liu Fang
Published: July 2010

Abstract: The release of excitatory amino acids (EAAs) from neuron‐free cultures of neocortical astrocytes was monitored using HPLC. The neuroligand bradykinin caused a dose‐dependent receptor‐mediated increase in release of the EAAs glutamate and aspartate from type 1 astrocyte cell cultures obtained from rat cerebral cortex. Removal of calcium from the extracellular fluid prevented the bradykinin‐induced release of EAAs from astrocytes. The addition of the calcium ionophore ionomycin caused a calcium‐dependent release of EAAs. Inhibitors of the glutamate transporters p‐chloromercuriphenylsulfonic acid, l‐trans‐pyrrolidine‐2,4‐dicarboxylate, and dihydrokainate failed to impair the ability of bradykinin to stimulate glutamate release from astrocytes. α‐Latrotoxin, an active compound of black widow spider venom, caused a significant increase of the release of glutamate in calcium‐containing saline. In calcium‐depleted saline, α‐latrotoxin produced an initial increase in the concentration of glutamate followed by a decline in the concentration of glutamate indicating stimulation of exocytosis coupled with low calcium‐induced inhibition of endocytosis. Taken together, these data suggest that astrocytes may release neurotransmitter through a mechanism that is similar to the neuronal secretory process. Given the important role of glutamate in the induction of long‐term potentiation, learning, memory, and excitotoxicity, it will be important to determine external signals that control both the uptake and release of glutamate by astrocytes.
N‐Methyl‐d‐Aspartate Receptor Proteins NR2A and NR2B Are Differentially Distributed in the Developing Rat Central Nervous System as Revealed by Subunit‐Specific Antibodies
Authors: Portera‐Cailliau Carlos , Price Donald L. , Martin Lee J.
Published: July 2010

Abstract: We have identified the regional distributions and developmental expression of NMDA‐receptor proteins NR2A and NR2B in rat CNS, using two subunit‐specific affinity‐purified polyclonal antibodies that recognize NR2A and NR2B. In western blots of cells transfected with NR2A or NR2B cDNAs, and of brain homogenates, each antibody detects a single predominant 172‐kDa protein corresponding to its homologous subunit. Both subunits are glycoproteins that are enriched in synaptic membranes. In adult rat CNS, NR2A and NR2B are enriched in cortex and hippocampus but are present in other forebrain regions. In hindbrain, NR2A is present at low levels but NR2B is barely detectable. These subunits are differentially expressed in postnatal CNS development. In cortex and striatum, NR2A is absent at birth but expression increases thereafter, whereas NR2B is expressed at nearly adult levels during forebrain development. In hindbrain, low levels of NR2A are present throughout development, whereas NR2B is expressed only transiently in the first postnatal weeks. These results suggest that native NMDA receptors are modulated by NR2A and NR2B in adult forebrain but not appreciably in hindbrain. In contrast, during early postnatal development, NR2B may have a more dominant role than NR2A in modulating NMDA receptors throughout the CNS. Thus, transient changes in NMDA‐receptor function may occur during maturation of certain neuronal and/or glial populations via differential expression of NR2A and NR2B subunits.
Kinetics, Pharmacology, and Autoradiographic Distribution of l‐[3H]Nitroarginine Binding Sites in Rat Cerebellum
Authors: Raghavendra Rao V. L. , Butterworth R. F.
Published: July 2010

Abstract: The kinetics and pharmacology of NG‐nitro‐l‐[2,3,4,5‐3H]arginine (l‐[3H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific l‐[3H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single‐site model. Specific binding was Ca2+ dependent and sensitive to pH (with an optimum of 5.5–7.0). Added calmodulin (1.5–40 µg/ml) had no influence on specific l‐[3H]NOARG binding. However, the calmodulin antagonists W‐5, W‐13, and calmidazolium inhibited l‐[3H]NOARG binding with IC50 values in the micromolar range, and calmodulin (10 µg/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors (NG‐nitro‐l‐arginine methyl ester and NG‐monomethyl‐l‐arginine acetate) and l‐arginine displaced l‐[3H]NOARG binding with IC50 values in the nanomolar range, whereas d‐arginine and basic amino acids (l‐lysine and l‐histidine) displaced l‐[3H]NOARG binding with IC50 values in the millimolar range. A comparison of the NOS functional assay with l‐[3H]NOARG binding in rat cerebellum showed similar profiles of Ca2+ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of l‐[3H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of l‐[3H]NOARG binding to study the regional distribution and functional properties of NOS.
Lipid Metabolism as a Target for Brain Cancer Therapy: Synergistic Activity of Lovastatin and Sodium Phenylacetate Against Human Glioma Cells
Authors: Prasanna Premakala , Thibault Alain , Liu Lei , Samid Dvorit
Published: July 2010

Abstract: Malignant gliomas, the most common form of primary brain tumors, are highly dependent on the mevalonate (MVA) pathway for the synthesis of lipid moieties critical to cell replication. Human glioblastoma cells were found to be uniquely vulnerable to growth arrest by lovastatin, a competitive inhibitor of the enzyme regulating MVA synthesis, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase. The sodium salt of phenylacetic acid (NaPA), an inhibitor of MVA‐pyrophosphate decarboxylase, the enzyme that controls MVA use, acted synergistically with lovastatin to suppress malignant growth. When used at pharmacologically attainable concentrations, the two compounds induced profound cytostasis and loss of malignant properties such as invasiveness and expression of the transforming growth factor‐β2 gene, coding for a potent immunosuppressive cytokine. Supplementation with exogenous ubiquinone, an end product of the MVA pathway, failed to rescue the cells, suggesting that decreased synthesis of intermediary products are responsible for the antitumor effects observed. In addition to blocking the MVA pathway, lovastatin alone and in combination with NaPA increased the expression of the peroxisome proliferator‐activated receptor, a transcription factor implicated in the control of lipid metabolism, cell growth, and differentiation. Our results indicate that targeting lipid metabolism with lovastatin, used alone or in combination with the aromatic fatty acid NaPA, may offer a novel approach to the treatment of malignant gliomas.
Zinc‐Induced Aggregation of Human and Rat β‐Amyloid Peptides In Vitro
Authors: Esler William P. , Stimson Evelyn R. , Jennings Joan M. , Ghilardi Joseph R. , Mantyh Patrick W. , Maggio John E.
Published: July 2010

Abstract: The major pathological feature of Alzheimer's disease is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human β‐amyloid peptide (Aβ), a 39–43‐mer peptide the neurotoxicity of which is related to its aggregation state. Previous work has demonstrated that certain metals that have been implicated as risk factors for Alzheimer's disease (Al, Fe, and Zn) also cause substantial aggregation of Aβ. In particular, we reported that zinc cations at concentrations of >10−4M dramatically accelerate the rate of Aβ aggregation at physiological peptide concentrations at 37°C in vitro. In the present study, we investigate the effect of Zn2+ on aggregation of radiolabeled and unlabeled human and rat Aβ over a wide range of peptide concentrations in the presence and absence of salt and blocking protein. Aggregation was assayed by centrifugation and filtration using amino acid analysis, immunoassay, and γ‐counting for quantification over a wide range of concentrations of Zn2+ and Aβ above and below physiological values. The results of this study demonstrate the following: (a) Radio‐iodinated Aβ accurately tracked unlabeled Aβ, (b) zinc concentrations of at least 10−4M were required to induce significant aggregation of Aβ, and (c) rat and human Aβ species were cleared from aqueous solutions by similar concentrations of zinc. These results stand in significant quantitative disagreement (∼100‐fold in zinc concentration) with one previous study that reported significant aggregation of Aβ by <1 µM Zn2+. Differences between the present study and the latter study from another laboratory appear to result from inappropriate reliance on optical density to measure Aβ concentrations and nonspecific loss of Aβ to plastic in the absence of blocking protein.
Aggregation and Metal‐Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease
Authors: Clements Angela , Allsop David , Walsh Dominic M. , Williams Carvell H.
Published: July 2010

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala21 to Gly or Glu22 to Gln were compared. At pH 7.4 and 37°C the Gln22 peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly21 peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln22 sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly21 peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope 65Zn2+. Of several competing metal ions, tested at 20× the concentration of Zn2+, Cu2+ displaced >95% of the radioactivity from all three peptides and Ni2+ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe2+ and Al3+ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn2+ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly21 mutation is not due to enhanced fibrillogenic or different metal‐binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.
Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine
Authors: Rocha Joao B. T. , Wolosker Herman , Souza Diogo O. , De Meis Leopoldo
Published: July 2010

Abstract: Rat brain microsomes accumulate Ca2+ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K+, Na+, or Li+. Both the Ca2+ uptake and the Ca2+‐dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half‐maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca2+ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca2+ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca2+ influx, and at concentrations >100 µM it inhibits both the Ca2+ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca2+‐ATPase; for the Ca2+‐ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration‐dependent inhibition of Ca2+ uptake. Passive Ca2+ efflux from brain microsomes preloaded with Ca2+ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca2+‐ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.
Phosphorylation and Activation of Tryptophan Hydroxylase by Exogenous Protein Kinase A
Authors: Johansen Patricia A. , Jennings Ian , Cotton Richard G. H. , Kuhn Donald M.
Published: July 2010

Abstract: The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in Vmax without alterations in the Km for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.
Bovine Pituitary Membrane Glycoproteins Contain β‐N‐Acetylgalactosaminylated N‐Linked Sugar Chains
Authors: Taka Junko , Sato Takeshi , Sakiyama Toru , Fujisawa Hirosuke , Furukawa Kiyoshi
Published: July 2010

Abstract: Bovine pituitary glycoprotein hormones contain unique N‐linked sugar chains with GalNAcβ1 → 4GlcNAc structure in their outer chain moieties. In the present study, whether bovine pituitary membrane glycoproteins contain the sugar chains with the disaccharide structure was investigated. Western blot analysis of the membrane glycoproteins using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with β‐N‐acetylgalactosamine residue(s), showed that most protein bands detected with Coomassie Brilliant Blue staining bind to WFA. However, no WFA binding was observed for the bands after treatment of the blotted filter with jack bean β‐N‐acetylhexosaminidase or N‐Glycanase. The WFA‐positive bands were also detected in membrane glycoprotein samples from bovine cerebrum, cerebellum, and medulla oblongata, although their expression levels were low. Structural analysis of the oligosaccharides released by hydrazinolysis from the pituitary membrane glycoproteins by serial lectin column chromatography and sequential exoglycosidase digestion revealed that the major oligosaccharides, which bound to a WFA‐agarose column, are of biantennary complex type with one and two GalNAcβ1 → 4GlcNAc groups in their outer chain moieties. These results indicate that the β‐N‐acetylgalactosaminylation is not unique to the glycoprotein hormones but occurs to most bovine pituitary glycoproteins.
Nitric Oxide Synthase Activity in the Molluscan CNS
Authors: Moroz Leonid L. , Chen Dong , Gillette Martha U. , Gillette Rhanor
Published: July 2010

Abstract: Putative nitric oxide synthase (NOS) activity was assayed in molluscan CNS through histochemical localization of NADPH‐diaphorase and through measurement of l‐arginine/l‐citrulline conversion. Several hundreds of NADPH‐dependent diaphorase‐positive neurons stained consistently darkly in the nervous system of the predatory opisthobranch Pleurobranchaea californica, whereas stained neurons were relatively sparse and/or light in the other opisthobranchs (Philine, Aplysia, Tritonia, Flabellina, Cadlina, Armina, Coriphella, and Doriopsilla sp.) and cephalopods (Sepia and Rossia sp.). l‐Arginine/l‐citrulline conversion was β‐NADPH dependent, insensitive to removal of Ca2+, inhibited by the calmodulin blocker trifluoperazine, and inhibited by the competitive NOS inhibitor N‐nitro‐l‐arginine methyl ester (l‐NAME) but not d‐NAME. Inhibitors of arginase [l‐valine and (+)‐S‐2‐amino‐5‐iodoacetamidopentanoic acid)] did not affect l‐citrulline production in the CNS. NOS activity was largely associated with the particulate fraction and appeared to be a novel, constitutive Ca2+‐independent isoform. Enzymatic conversion of l‐arginine/l‐citrulline in Pleurobranchaea and Aplysia CNS was 4.0 and 9.8%, respectively, of that of rat cerebellum. l‐Citrulline formation in gill and muscle of Pleurobranchaea was not significant. The localization of relatively high NOS activity in neuron somata in the CNS of Pleurobranchaea is markedly different from the other opisthobranchs, all of which are grazers. Potentially, this is related to the animal's opportunistic predatory lifestyle.
Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells
Authors: Sadot Einat , Gurwitz David , Barg Jacob , Behar Leah , Ginzburg Irith , Fisher Abraham
Published: July 2010

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m1 muscarinic acetylcholine receptor. Stimulation of m1 receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation‐dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule‐associated protein.
Expression of the Glucagon‐Like Peptide‐1 Receptor Gene in Rat Brain
Authors: Alvarez Elvira , Roncero Isabel , Chowen Julie A. , Thorens Bernard , Blázquez Enrique
Published: July 2010

Abstract: Evidence that glucagon‐like peptide‐1 (GLP‐1) (7–36) amide functions as a novel neuropeptide prompted us to study the gene expression of its receptor in rat brain. Northern blot analysis showed transcripts of similar size in RINm5F cells, hypothalamus, and brainstem. First‐strand cDNA was prepared by using RNA from hypothalamus, brainstem, and RINm5F cells and subsequently amplified by PCR. Southern blot analysis of the PCR products showed a major 1.4‐kb band in all these preparations. PCR products amplified from hypothalamus were cloned, and the nucleotide sequence of one strand was identical to that described in rat pancreatic islets. In situ hybridization studies showed specific labeling in both neurons and glia of the thalamus, hypothalamus, hippocampus, primary olfatory cortex, choroid plexus, and pituitary gland. In the hypothalamus, ventromedial nuclei cells were highly labeled. These findings indicate that GLP‐1 receptors are actually synthesized in rat brain. In addition, the colocalization of GLP‐1 receptors, glucokinase, and GLUT‐2 in the same areas supports the idea that these cells play an important role in glucose sensing in the brain.
Evidence that Glyceraldehyde‐3‐Phosphate Dehydrogenase Is Involved in Age‐Induced Apoptosis in Mature Cerebellar Neurons in Culture
Authors: Ishitani Ryoichi , Sunaga Katsuyoshi , Hirano Atsushi , Saunders Paul , Katsube Nobuo , Chuang De‐Maw
Published: July 2010

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin‐D and cycloheximide. The level of a 38‐kDa protein in the particulate fraction is markedly increased during age‐induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age‐induced increment of the 38‐kDa particulate protein is suppressed by actinomycin‐D and cycloheximide. N‐terminal microsequencing of the 38‐kDa protein revealed sequence identity with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age‐induced expression of the particulate 38‐kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age‐induced neuronal death and the 38‐kDa protein overexpression. Moreover, the age‐induced expression of the 38‐kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin‐D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age‐induced apoptosis of cerebellar neurons.
Expression of the Neuron‐Specific Enolase Gene by Rat Oligodendroglial Cells During Their Differentiation
Authors: Deloulme J. C. , Lucas M. , Gaber C. , Bouillon P. , Keller A. , Eclancher F. , Sensenbrenner M.
Published: July 2010

Abstract: We have examined the regulation of neuron‐specific γ‐enolase gene (NSE) expression in oligodendrocytes at various steps of their differentiation/maturation. We have demonstrated for the first time that NSE is expressed in oligodendroglial cells in vitro and in vivo, and only at a certain stage of differentiation. A heterogeneity of the γ subunit was observed in cultured oligodendrocytes and the same one was found in adult rat brain. The level of γ mRNA increased when precursor cells differentiated into oligodendrocytes. By contrast, no significant change in α‐enolase gene expression was observed. High NSE (γγ and αγ) enolase activity was detected in cultured oligodendrocytes. Treatment with basic fibroblast growth factor, which stimulates the proliferation of oligodendrocyte precursor cells and reversibly blocks their differentiation, resulted in lower αγ‐ and γγ‐enolase activities in these cells, but it enhanced αα‐enolase activity slightly. These data indicate that γ‐enolase gene expression is associated with the differentiation of the oligodendrocytes and that it is repressed in adult fully mature cells.
Inhibition by Drebrin of the Actin‐Bundling Activity of Brain Fascin, a Protein Localized in Filopodia of Growth Cones
Authors: Sasaki Yo , Hayashi Kensuke , Shirao Tomoaki , Ishikawa Ryoki , Kohama Kazuhiro
Published: July 2010

Abstract: The purification of drebrin, an actin‐binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin‐binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin‐binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low‐speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.
The Desglycinyl Metabolite of Remacemide Hydrochloride Is Neuroprotective in Cultured Rat Cortical Neurons
Authors: Black Marsha A. , Tremblay Roger , Mealing Geoffrey A. R. , Durkin Jon P. , Whitfield James F. , Morley Paul
Published: July 2010

Abstract: The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2‐diphenyl‐2‐propylamine monohydrochloride (desglycinylremacemide; DGR), a low‐affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 µM) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration‐dependent manner by adding DGR (5–20 µM), but not its remacemide precursor (10–100 µM), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5–20 µM) prevented the loss of membrane‐associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 µM NMDA and reduced the NMDA‐triggered increases in intracellular free Ca2+ concentration ([Ca2+]i) by up to 70%. By contrast, remacemide (50 and 100 µM) did not prevent the NMDA‐induced loss of PKC activity or reduce the [Ca2+]i responses. These data suggest that DGR protection against NMDA‐mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA‐triggered [Ca2+]i surges and a prevention of the loss of membrane‐associated PKC activity. In addition, the inhibition of NMDA‐triggered [Ca2+]i responses by DGR was qualitatively different from the inhibition of these responses by the high‐affinity NMDA‐receptor antagonists MK‐801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.
Functional Heterogeneity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin‐Dependent Protein Kinase II
Authors: GuptaRoy Bipasha , Griffith Leslie C.
Published: July 2010

Abstract: The gene for Drosophila calcium/calmodulin‐dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian α, β, γ, and δ isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin‐dependent protein kinase II. Two major isoform‐specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher Kact for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin‐binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower Km for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.
Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment
Authors: Lärkfors Lena , Lindsay Ronald M. , Alderson Ralph F.
Published: July 2010

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron‐specific enolase‐immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT‐4/5, or NT‐3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT‐4/5 (46%), whereas NT‐3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT‐3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT‐4/5, and NT‐3, but not NGF, induced the rapid expression of the immediate early gene c‐fos. Immunocytochemical double‐labeling with antibodies to c‐fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c‐fos. After 4 days in vitro, both BDNF and NT‐3 induced the formation of c‐fos protein in calbindin‐immunopositive neurons, whereas NT‐4/5 did not. The latter results suggest that although BDNF and NT‐4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT‐4/5 are not identical.
Staurosporine Induces Programmed Cell Death in Embryonic Neurons and Activation of the Ceramide Pathway
Authors: Wiesner Douglas A. , Dawson Glyn
Published: July 2010

Abstract: We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1–1.0 µM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N‐Acylsphingosine (C2‐ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [3H]‐palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 µM), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine‐induced and potentially all programmed cell death.
Effect of Hypothyroidism on the Subcellular Distribution of Ca2+/Calmodulin‐Stimulated Protein Kinase II in Chicken Brain During Posthatch Development
Authors: Wang Xin , Rostas John A. P.
Published: July 2010

Abstract: In developing chicken brain Ca2+/calmodulin‐stimulated protein kinase II (CaMPK‐II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T3) at 42 days. The concentrations of α‐ and β‐CaMPK‐II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca2+/calmodulin‐ or Zn2+‐stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK‐II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca2+/calmodulin‐stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP‐stimulated protein phosphorylation. Supplementing the PTU/MMI‐treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.
Differential Cellular Phosphorylation of Neurofilament Heavy Side‐Arms by Glycogen Synthase Kinase‐3 and Cyclin‐Dependent Kinase‐5
Authors: Guidato Sonia , Tsai Li‐Huei , Woodgett Jim , Miller Christopher C. J.
Published: July 2010

Abstract: To investigate the cellular mechanisms regulating neurofilament‐heavy subunit (NF‐H) side‐arm phosphorylation, we studied the ability of three putative neurofilament kinases, glycogen synthase kinase‐3 (GSK‐3)α, GSK‐3β, and cyclin‐dependent kinase‐5 (cdk‐5), to phosphorylate NF‐H in transfected cells. We analysed NF‐H phosphorylation by using a panel of phosphorylation‐dependent antibodies and also by monitoring the electrophoretic mobility of the transfected NF‐H on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis because this is known to be affected by side‐arm phosphorylation. Our results demonstrate that whereas GSK‐3α, GSK‐3β, and cdk‐5 will all phosphorylate NF‐H, they generate different antibody reactivity profiles. GSK‐3α and GSK‐3β induce a partial retardation of a proportion of the transfected NF‐H, but only cdk‐5 alters the rate of electrophoretic migration to that of NF‐H from brain. We conclude that cdk‐5 and GSK‐3 phosphorylate different residues or sets of residues within NF‐H sidearms in cells. We further show that cdk‐5 is active in both the CNS and the PNS but that this activity is not dependent on expression of its activator, p35. This suggests that there are other activators of cdk‐5.