bluuebunny/crossref_metadata_2025 huggingface dataset:
A Reevaluation of the Role of Mitochondria in Neuronal Ca2+ Homeostasis
Authors: Budd S. L. , Nicholls D. G.
Published: July 2010

Abstract: The ability of mitochondrial Ca2+ transport to limit the elevation in free cytoplasmic Ca2+ concentration in neurones following an imposed Ca2+ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura‐2 imaging. Following KCI depolarization, addition of the protonophore carbonylcyanide m‐chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca2+ into the cytoplasm in both somata and neurites. No CCCP‐releasable pool was found in nondepolarized cells. Although the KCI‐evoked somatic and neurite Ca2+ concentration elevations were enhanced when CCCP was present during KCI depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine‐123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP‐releasable pool was found subsequent to KCI depolarization, consistent with the abolition of mitochondrial Ca2+ transport; however, paradoxically the KCI‐evoked Ca2+ elevation is decreased. It is concluded that the CCCP‐induced increase in cytoplasmic Ca2+ response to KCI is due to inhibition of nonmitochondrial ATP‐dependent transport and that mitochondrial Ca2+ transport enhances entry of Ca2+, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage‐activated Ca2+ channel activity.
Brain μ‐Calpain Autolysis During Global Cerebral Ischemia
Authors: Neumar Robert W. , Hagle Scott M. , DeGracia Donald J. , Krause Gary S. , White Blaine C.
Published: July 2010

Abstract: Proteolytic degradation of numerous calpain substrates, including cytoskeletal and regulatory proteins, has been observed during brain ischemia and reperfusion. In addition, calpain inhibitors have been shown to decrease degradation of these proteins and decrease postischemic neuronal death. Although these observations support the inference of a role for μ‐calpain in the pathophysiology of ischemic neuronal injury, the evidence is indirect. A direct indicator of μ‐calpain proteolytic activity is autolysis of its 80‐kDa catalytic subunit, and therefore we examined the μ‐calpain catalytic subunit for evidence of autolysis during cerebral ischemia. Rabbit brain homogenates obtained after 0, 5, 10, and 20 min of cardiac arrest were electrophoresed and immunoblotted with a monoclonal antibody specific to the μ‐calpain catalytic subunit. In nonischemic brain homogenates the antibody identified an 80‐kDa band, which migrated identically with purified μ‐calpain, and faint 78‐ and 76‐kDa bands, which represent autolyzed forms of the 80‐kDa subunit. The average density of the 80‐kDa band decreased by 25 ± 4 (p = 0.008) and 28 ± 9% (p = 0.004) after 10 and 20 min of cardiac arrest, respectively, whereas the average density of the 78‐kDa band increased by 111 ± 50% (p = 0.02) after 20 min of cardiac arrest. No significant change in the density of the 76‐kDa band was detected. These results provide direct evidence for autolysis of brain μ‐calpain during cerebral ischemia. Further work is needed to characterize the extent, duration, and localization of μ‐calpain activity during brain ischemia and reperfusion as well as its role in the causal pathway of postischemic neuronal injury.
Neuronal Loss and Cytoskeletal Disruption Following Intrahippocampal Administration of the Metabolic Inhibitor Malonate: Lack of Protection by MK‐801
Authors: Pang Zhen , Umberger Gloria H. , Geddes James W.
Published: July 2010

Abstract: Impaired energy metabolism may contribute to the pathogenesis of late‐onset neurodegenerative disorders such as Alzheimer's disease by increasing neuronal vulnerability to excitotoxic damage through the NMDA receptor. The effects of metabolic impairment on the striatum have been extensively examined, but relatively little is known regarding the vulnerability of the hippocampus. To examine the effect of metabolic impairment on the hippocampal formation, malonate (0.25–2.5 µmol), a reversible inhibitor of succinate dehydrogenase, was administered by stereotaxic injection into the hippocampus of male Sprague‐Dawley rats. Neuronal loss was assessed by Nissl stain, and immunocytochemistry was used to examine cytoskeletal disruption. Malonate produced a dose‐dependent lesion in which CA1 pyramidal neurons were most vulnerable, followed by CA3 and dentate gyrus. Cytoskeletal alterations included the loss of microtubule‐associated protein 2 (MAP2) and dendritic MAP1B immunoreactivity, whereas axonal MAP1B and τ proteins were relatively spared. Spatially and temporally correlated with the loss of MAP2 was an increase in the immunoreactivity of calpain‐cleaved spectrin. A similar pattern of neuronal damage and cytoskeletal disruption was produced by intrahippocampal injection of quinolinate (0.1 µmol), an NMDA agonist. Although these results are consistent with the hypothesis that metabolic impairment results in excitotoxic death, MK‐801 (dizocilpine maleate), a noncompetitive NMDA receptor antagonist, did not attenuate the lesions produced by malonate but was effective against quinolinate. The results suggest that NMDA receptor activation is not required for malonate‐induced damage in the hippocampal formation.
Involvement of Na+,K+‐ATPase in the Mitogenic Effect of Insulin‐Like Growth Factor‐I on Cultured Rat Astrocytes
Authors: Matsuda Toshio , Murata Yosuke , Tanaka Koh‐ichi , Hosoi Rie , Hayashi Masako , Tamada Kazuko , Takuma Kazuhiro , Baba Akemichi
Published: July 2010

Abstract: We have previously reported that insulin/insulin‐like growth factor (IGF)‐I induced the α1 isoform of Na+,K+‐ATPase in cultured astrocytes. In this study the effects of insulin/IGF‐I on Na+,K+‐ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF‐I on astrocytes. Insulin increased Na+,K+‐ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [3H]thymidine incorporation in astrocytes preincubated in fetal calf serum‐free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na+,K+‐ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. 125I‐IGF‐I binding experiment showed that all the cells used here had similar binding characteristics. The insulin‐induced increase in enzyme activity was not affected by 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), and it was observed even in Ca2+‐free medium. The stimulation by IGF‐I of [3H]thymidine incorporation was attenuated by ouabain and a low external K+ level. These findings suggest that stimulation of Na+,K+‐ATPase activity is involved in the mitogenic action of IGF‐I on cultured astrocytes.
Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C‐Dependent Mechanism
Authors: Kimball Beth C. , Mulholland Michael W.
Published: July 2010

Abstract: Calcium signaling in fura‐2 acetoxymethyl ester‐loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 mM), glutamate (100 µM), norepinephrine (10 µM), and substance P (1 µM) did not increase the intracellular calcium concentration ([Ca2+]i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µM), bradykinin (11%; 10 µM), and histamine (31%; 100 µM), whereas 100% of glia responded to ATP (100 µM). ATP‐evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca2+]i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca2+]i evoked, ATP = UTP > ADP > β,γ‐methyleneadenosine 5′‐triphosphate ≫ 2‐methylthioadenosine 5′‐triphosphate = α,β‐methyleneadenosine 5′‐triphosphate = AMP = adenosine, suggesting a glial P2U receptor. Depletion of d‐myo‐inositol 1,4,5‐trisphosphate‐sensitive calcium stores by thapsigargin (10 µM) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U‐73122 (10 µM), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12‐myristate 13‐acetate (100 nM), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca2+]i. Our data suggest that glial responses to ATP are mediated by a P2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.
Activation of Adenosine A2 Receptors Stimulates Phosphoinositide Metabolism in Rat Peripheral Nerve
Authors: Sheldon Rhonda L. , Bonasera Lara K. , Eichberg Joseph
Published: July 2010

Abstract: The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [3H]‐cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [3H]cytidine monophosphate phosphatidic acid ([3H]‐CMP‐PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5′‐N‐ethylcarboxamidoadenosine (NECA), a nonselective analogue, and two different A2‐selective analogues, N6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl)ethyl]adenosine and 2‐p‐(2‐carboxyethyl)phenethylamino‐NECA (CGS 21680), but not by N6‐cyclopentyladenosine, an A1‐selective analogue. The stimulatory action of CGS 21680 was blocked by the A2‐selective adenosine receptor antagonists 3,7‐dimethyl‐1‐propargylxanthine (DMPX) and 1,3‐dipropyl‐7‐methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP‐PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A2 subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine‐receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.
The α1 and α6 Subunits Can Coexist in the Same Cerebellar GABAA Receptor Maintaining Their Individual Benzodiazepine‐Binding Specificities
Authors: Khan Zafar U. , Gutiérrez Antonia , De Blas Angel L.
Published: July 2010

Abstract: Two GABAA receptor subunit‐specific antibodies anti‐α6 and anti‐α1 have been used for elucidating the relationship between the presence of α1 and/or α6 subunits in the cerebellar GABAA receptors and the benzodiazepine‐binding specificity. Receptor immunoprecipitation with the subunit‐specific antibodies shows that 39% of the cerebellar GABAA receptors have α6, whereas 76% of the receptors have α1 as determined by [3H]muscimol binding. Results show that 42–45% of the receptors having α6 also have α1, whereas 13–15% of the receptors that contain α1 also have α6. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABAA receptors; i.e., one has both α1 and α6 subunits, a second type has α1 but not α6, and a third type has α6 but not α1 subunits. The results also show that receptors where α1 and α6 subunits coexist have two pharmacologically different benzodiazepine‐binding properties, each associated with a different α subunit. The α1 subunit contributes the high‐affinity binding of [3H]Ro 15‐1788 (flumazenil) and the diazepam‐sensitive binding of [3H]Ro 15‐4513. The α6 subunit contributes the diazepam‐insensitive binding of [3H]Ro 15‐4513, but it does not bind [3H]Ro 15‐1788 with high affinity. Thus, in the cerebellar α1–α6 GABAA receptors, there is no dominance of the pharmacology of one α subunit over the other.
Antibody Recognition of Calcium‐Binding Proteins Depends on Their Calcium‐Binding Status
Authors: Winsky L. , Kuźnicki J.
Published: July 2010

Abstract: Previous studies have revealed changes in immunohistochemical stains for calcium‐binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium‐induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium‐free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium‐dependent conformational changes. A calcium‐insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium‐binding proteins preferentially recognize particular calcium‐induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium‐binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.
Differential Effects of Nerve Growth Factor on Expression of Choline Acetyltransferase and Sodium‐Coupled Choline Transport in Basal Forebrain Cholinergic Neurons in Culture
Authors: Pongrac Julie L. , Rylett R. Jane
Published: July 2010

Abstract: Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high‐affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high‐affinity choline transport activity was not changed relative to control. Dose‐response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC50 of 7 ng/ml, with no enhancement of high‐affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high‐affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high‐affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.
Inhibition of Neuroblastoma Cell Phosphatidylinositol 3‐Kinase by CDP‐Diacylglycerol and Phosphatidate
Authors: Lavie Yaakov , Agranoff Bernard W.
Published: July 2010

Abstract: Phosphatidylinositol (PI) 3‐kinase is activated by a variety of agents, including various growth factors, and has been proposed to play a role in initiation of cell growth, proliferation, and differentiation. We here investigate the effect of various membrane lipids on PI 3‐kinase immunopurified from human SH‐SY5Y neuroblastoma cells. CDP‐diacylglycerol (CDP‐DAG) inhibited PI 3‐kinase activity with an IC50 of 6 µM. Phosphatidate (PA) was also inhibitory (IC50 = 38 µM) as was lysophosphatidate. Neither DAG nor any of the other phospholipids examined affected PI 3‐kinase activity. The results offer the possibility that CDP‐DAG or PA at critical membrane sites may exert functionally significant metabolic regulation at the point of convergence of the PI 3‐kinase‐directed and the PI 4‐kinase‐directed phosphoinositide signal transduction pathways.
The Lex Carbohydrate Sequence Is Recognized by Antibody to L5, a Functional Antigen in Early Neural Development
Authors: Streit Andrea , Yuen Chun‐Ting , Loveless R. Wendy , Lawson Alexander M. , Finne Jukka , Schmitz Brigitte , Feizi Ten , Stern Claudio D.
Published: July 2010

Abstract: The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex‐type N‐glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N‐linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5‐positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N‐glycans containing a 3‐fucosyl N‐acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid‐linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3‐fucosyl N‐acetyllactosamine sequence [Lewisx (Lex) or X‐hapten] also recognized by anti‐SSEA‐1 and anti‐CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti‐SSEA‐1 and anti‐CD15.
Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells
Authors: Grant Nancy J. , Claudepierre Thomas , Aunis Dominique , Langley Keith
Published: July 2010

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one‐half, whereas NGF increased L1 protein levels ∼2.3‐fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10−7M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down‐regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.
α‐Linolenic Acid Dietary Deficiency Alters Age‐Related Changes of Dopaminergic and Serotoninergic Neurotransmission in the Rat Frontal Cortex
Authors: Delion Sylvie , Chalon Sylvie , Guilloteau Denis , Besnard Jean‐Claude , Durand Georges
Published: July 2010

Abstract: The effects of α‐linolenic acid diet deficiency on rat dopaminergic and serotoninergic neurotransmission systems were investigated in the frontal cortex, striatum, and cerebellum of male rats 2, 6, 12, and 24 months of age. The diet deficiency induced a severe decrease in the 22:6n‐3 fatty acid levels in all regions and a compensatory increase in n‐6 fatty acid levels. A recovery in the levels of 22:6n‐3 was observed in deficient rats between 2 and 12 months of age; however, this recovery was lower in frontal cortex than in striatum and cerebellum. In the striatum and the cerebellum, dopaminergic and serotoninergic receptor densities and endogenous dopamine and serotonin levels were affected by aging regardless of the diet. In contrast, a 40–75% lower level of endogenous dopamine in the frontal cortex occurred in deficient rats according to age. The deficiency also induced an 18–46% increase in serotonin 5‐HT2 receptor density in the frontal cortex during aging, without variation in endogenous serotonin level, and a 10% reduction in density of dopaminergic D2 receptors. Monoamine oxidase‐A and ‐B activities showed specific age‐related variations but regardless of the diet. Our results suggest that a chronically α‐linolenic‐deficient diet specifically affects the monoaminergic systems in the frontal cortex.
Polyamine Modification Increases the Permeability of Proteins at the Blood‐Nerve and Blood‐Brain Barriers
Authors: Poduslo Joseph F. , Curran Geoffry L.
Published: July 2010

Abstract: The permeability of the blood‐nerve barrier (BNB) and the blood‐brain barrier (BBB) to superoxide dismutase (SOD), insulin, albumin, and IgG in normal adult rats was quantified by measuring the permeability coefficient‐surface area product (PS) with the intravenous bolus injection technique before and after covalent protein modification with the naturally occurring polyamines—putrescine (PUT), spermidine (SPD), and spermine (SPM). The PS value of the BNB for PUT‐SOD was 21.1‐fold greater than the native SOD, and the PS values of the BBB for PUT‐SOD ranged from 17.6‐fold greater for the thalamus to 23.6‐fold greater for the caudate‐putamen compared with native SOD. In a similar manner, polyamine‐modified insulin showed a 1.7–2.0‐fold increase in PS of the BNB and BBB compared with the high values of native insulin. Polyamine‐modified albumin showed a remarkable 54–165‐fold increase in PS of the BNB and BBB compared with native albumin, whereas PUT‐IgG resulted in an even higher increase in the PS that ranged from 111‐ to 349‐fold for nerve and different brain regions compared with native IgG. Polyamine modification of proteins, therefore, can dramatically increase the permeability at the BNB and BBB of a variety of proteins with widely differing Mr and function. It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT > SPD > SPM). Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density. It is suggested that the polyamine transporter may be responsible for the transport of these polyamine‐modified proteins. Systemic administration of polyamine‐modified peptides and proteins might prove to be an efficient approach to deliver therapeutic agents into the CNS and PNS for the treatment of a variety of neurological diseases.
Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria
Authors: Davey Gavin P. , Clark John B.
Published: July 2010

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.
Isolation and Characterization of Two Fatty Acid Binding Proteins from Mouse Brain
Authors: Myers‐Payne Sean C. , Hubbell Timothy , Pu Lixia , Schnütgen Frank , Börchers Torsten , Wood W. Gibson , Spener Friedrich , Schroeder Friedhelm
Published: July 2010

Abstract: Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross‐reacted with antisera to recombinant liver L‐FABP. One protein, designated brain H‐FABP, migrated on tricine sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H‐FABP bound NBD‐stearic acid and cis‐parinaric acid with KD values near 0.02 and 0.5 µM, respectively. Brain H‐FABP cross‐reacted with affinity‐purified antisera to recombinant heart H‐FABP. The second protein, mouse brain B‐FABP, migrated on tricine SDS‐PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B‐FABP bound NBD‐stearic acid and cis‐parinaric acid with KD values near 0.01 and 0.7 µM, respectively. The brain B‐FABP doublet was immunoreactive with affinity‐purified antibodies against recombinant mouse brain B‐FABP, but not with affinity‐purified antibodies against heart H‐FABP. [3H]Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B‐FABP and brain H‐FABP represented 0.1 and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H‐FABP and brain B‐FABP.
Time‐Dependent Increase in Protein Phosphorylation Following One‐Trial Enhancement in Hermissenda
Authors: Crow Terry , Siddiqi Vilma , Zhu Qing , Neary Joseph T.
Published: July 2010

Abstract: One‐trial conditioning of the nudibranch mollusk Hermissenda produces short‐ and long‐term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of 32PO4 into proteins were determined using two‐dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46‐ and 55‐kDa phosphoproteins detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long‐term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55‐, 46‐, and 22‐kDa phosphoproteins, at 1 h for the 55‐ and 46‐kDa phosphoproteins, and at 2 h for the 55‐, 46‐, and 22‐kDa phosphoproteins. The 46‐ and 55‐kDa phosphoproteins are putative structural proteins, and the 22‐kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time‐dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one‐trial conditioning.
Transcriptional Regulation of CREB (Cyclic AMP Response Element‐Binding Protein) Expression in CATH.a Cells
Authors: Widnell Katherine L. , Chen Jing‐Shan , Iredale Philip A. , Walker William H. , Duman Ronald S. , Habener Joel F. , Nestler Eric J.
Published: July 2010

Abstract: We have recently demonstrated that mRNA expression of cyclic AMP (cAMP) response element‐binding protein (CREB) is down‐regulated in CATH.a cells (a neural‐derived cell line) by activation of the cAMP pathway. We now demonstrate that this down‐regulation can be accounted for by a decrease in the rate of CREB gene transcription. It was found that cycloheximide, a protein synthesis inhibitor, prevented the forskolin‐induced decrease in CREB mRNA levels in CATH.a cells. Nuclear run‐on assays demonstrated that forskolin decreased the rate of CREB transcription by close to 50%. Moreover, forskolin decreased chloramphenicol acetyltransferase (CAT) activity in CATH.a cells transiently transfected with a construct containing 1,240 bp of CREB promoter fused to a CAT reporter plasmid. Possible mechanisms by which activation of the cAMP pathway leads to a decrease in CREB gene transcription are discussed.
Increased Expression of Ubiquitin During Adenosine 3′,5′‐Cyclic Monophosphate‐Induced Differentiation of Neuroblastoma Cells in Culture
Authors: La Rosa Francisco G. , Kumar Sanjay , Prasad Kedar N.
Published: July 2010

Abstract: The role of ubiquitin in proliferation and differentiation of nerve cells has not been studied. An elevation of the intracellular level of adenosine 3′,5′‐cyclic monophosphate (cAMP) in neuroblastoma cells induces terminal differentiation in these cells. Therefore, in this study we investigated the changes in the level and subcellular distribution of ubiquitin during proliferation and differentiation of neuroblastoma cells. Prostaglandin E1, a stimulator of adenylate cyclase, plus β‐carotene, and 4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro 20‐1724), an inhibitor of cyclic nucleotide phosphodiesterase, plus β‐carotene were used to induce terminal differentiation in >90% of neuroblastoma cells. Changes in ubiquitin level were studied by immunofluorescent staining using either a mouse monoclonal antibody or a rabbit polyvalent antibody to ubiquitin. Results showed that the dividing neuroblastoma cells contained very low levels of ubiquitin localized primarily in the cytoplasm. The intensity of cytoplasmic staining for ubiquitin markedly increased during cAMP‐induced differentiation of neuroblastoma cells, being the highest at 4 days after treatment. The neurites of these differentiated cells were also stained, but the nuclei were not. We propose a hypothesis that higher levels of cytoplasmic ubiquitin are needed during cAMP‐induced differentiation of neuroblastoma cells for the removal of proteins responsible for cell proliferation through rapid degradation and/or inhibition of transcription, later leading to terminal differentiation.
Role of Protein Kinase C α in Nerve Growth Factor‐Induced Arachidonic Acid Release from PC12 Cells
Authors: Zheng Wen‐Hua , Fink Donald W. , Guroff Gordon
Published: July 2010

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down‐regulated by long‐term treatment of the cells with phorbol 12‐myristate 13‐acetate (PMA). Such prolonged exposure to PMA (1 µM) resulted in the inhibition of NGF‐induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF‐induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down‐regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF‐induced AA release, an isoform‐specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF‐induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF‐induced AA release.
Effects of Glutamate Antagonists on Methamphetamine and 3,4‐Methylenedioxymethamphetamine‐Induced Striatal Dopamine Release In Vivo
Authors: Finnegan Kevin T. , Taraska Terri
Published: July 2010

Abstract: Several amphetamine analogues are reported to increase striatal glutamate efflux in vivo, whereas other data indicate that glutamate is capable of stimulating the efflux of dopamine (DA) in the striatum via a glutamate receptor‐dependent mechanism. Based on these findings, it has been proposed that the ability of glutamate receptor‐blocking drugs to antagonize the effects of amphetamine may be explained by their capacity to inhibit DA release induced by glutamate. To examine this possibility further, we investigated in vivo the ability of glutamate antagonists to inhibit DA release induced by either methamphetamine (METH) or 3,4‐methylenedioxymethamphetamine (MDMA). Both METH and MDMA increased DA efflux in the rat striatum and, in animals killed 1 week later, induced persistent depletions of DA and serotonin in tissue. Pretreatment with MK‐801 or CGS 19755 blocked the neurotoxic effects of METH and MDMA but, did not significantly alter striatal DA efflux induced by either stimulant. Infusion of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione into the striatum likewise did not alter METH‐induced DA overflow, and none of the glutamatergic antagonists affected the basal release of DA when given alone. The findings suggest that the neuroprotective effects of NMDA antagonists do not involve an inhibition of DA release, nor do the data support the proposal that glutamate tonically stimulates striatal DA efflux in vivo. Whether phasic increases in glutamate content might stimulate DA release, however, remains to be determined.
Stress‐ and Yohimbine‐Induced Release of Cholecystokinin in the Frontal Cortex of the Freely Moving Rat: Prevention by Diazepam but Not Ondansetron
Authors: Nevo Igal , Becker Christel , Hamon Michel , Benoliel Jean‐Jacques
Published: July 2010

Abstract: The in vivo release of cholecystokinin (CCK)‐like material (CCKLM) was measured in the frontal cortex of freely moving rats using the microdialysis technique combined with a sensitive radioimmunoassay. Local perfusion of K+ (100 mM)‐enriched artificial CSF resulted in a 10‐fold increase in CCKLM outflow, as compared with that occurring under basal resting (K+ = 3.0 mM) conditions, and this effect could be completely prevented by removal of Ca2+ in the perfusing fluid. Chromatographic analyses demonstrated that CCK‐8S contributed to 70% of CCKLM. Stressful stimuli such as a 2‐min exposure to diethyl ether and a 30‐min restraint produced a marked but transient increase in cortical CCKLM release. In addition, anxiety‐like behavior induced by the systemic administration of yohimbine (5 mg/kg i.p.) was associated with a long‐lasting enhancement in the peptide outflow. Pretreatment with the potent anxiolytic drug diazepam (5 mg/kg i.p., 5 min before each condition), which exerted no effect on its own, completely prevented CCKLM overflow due to diethyl ether, restraint, or yohimbine administration. In contrast, neither the systemic injection (0.1 mg/kg i.p.) nor the local application (100 µM through the microdialysis probe) of the serotonin 5‐HT3 antagonist ondansetron affected the increased release of CCKLM in rats restrained for 30 min or treated with yohimbine. These results indicate that cortical CCKergic neurotransmission is increased during stress or anxiety‐like behavior in rats. Prevention of this effect by diazepam suggests that an inhibitory influence of benzodiazepines on cortical CCKergic neurons might participate in the anxiolytic action of these drugs.
Heteromers of Glutamate Decarboxylase Isoforms Occur in Rat Cerebellum
Authors: Sheikh Sabina N. , Martin David L.
Published: July 2010

Abstract: The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD65 and GAD67). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising <3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD65 and GAD67, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD65 and GAD67, whereas some GAD67 was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD65 and GAD67, whereas some GAD65 remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD65 and GAD67 even after extensive washing. These results are consistent with the presence of heteromultimers of GAD65 and GAD67 in cerebellum in addition to homomers of each form.
Species‐ and Brain Region‐Specific Dopamine Transporters: Immunological and Glycosylation Characteristics
Authors: Vaughan Roxanne A. , Brown Vickie L. , McCoy Michael T. , Kuhar Michael J.
Published: July 2010

Abstract: Dopamine transporters (DATs) from the caudate nucleus of four species (rat, mouse, dog, and human) and four regions of rat brain (striatum, nucleus accumbens, prefrontal cortex, and midbrain) were photoaffinity labeled and analyzed by immunoprecipitation and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for cross‐reactivity to four epitope‐specific rat antipeptide antibodies. Each of these antibodies varied in its efficiency at recognizing DAT. The DATs from the rat brain regions exhibited the same degree of recognition by each of the four sera, a result compatible with these proteins being the product of a single gene. The DATs from the different species were recognized by all four sera but with different efficiencies, possibly relating to amino acid sequence differences within the immunizing epitope. All of the photolabeled, immunoprecipitated DATs migrated with a molecular mass of ∼80 kDa, and no lower molecular mass forms were found. The DATs from all species and brain regions tested were shown by enzymatic deglycosylation to contain N‐linked carbohydrates and sialic acids in amounts comparable with rat striatal DATs. The finding that no photolabeled DAT forms <80 kDa were isolated from membranes indicates that partially or incompletely glycosylated forms are not present, even in the midbrain cell bodies where immature forms might be expected to be found. These findings verify the utility of these anti‐rat antibodies as biochemical tools for studying DATs from other species and extend our knowledge of biochemical characteristics of DATs from these species and brain regions.
Disposition of Histamine, Its Metabolites, and pros‐Methylimidazoleacetic Acid in Brain Regions of Rats Chronically Infused with α‐Fluoromethylhistidine
Authors: Prell George D. , Douyon Erwin , Sawyer William F. , Morrishow Albert Marcus
Published: July 2010

Abstract: In mammalian brain, histamine is known to be metabolized solely by histamine methyltransferase (HMT), forming tele‐methylhistamine (t‐MH), then tele‐methylimidazoleacetic acid (t‐MIAA). We previously showed that imidazoleacetic acid (IAA), a GABA agonist, and histamine's metabolite in the periphery, is present in brain where its concentration increased after inhibition of HMT. Also, when [3H]histamine was given intracerebroventricularly to rats, a portion was converted to IAA, a process increased by inhibition of HMT. These results indicated that brain has the capacity to oxidize histamine but did not show whether this pathway is operative under physiological conditions. To address this question, rats were infused for >4 weeks with α‐fluoromethylhistidine (α‐FMHis), an irreversible inhibitor of histamine's synthetic enzyme, l‐histidine decarboxylase. Compared with controls (untreated and saline‐treated rats), brain levels of histamine, t‐MH, and t‐MIAA in all regions were markedly reduced in treated rats. As a percentage of controls, depletion of t‐MIAA > t‐MH > histamine in all regions, and regional depletions of histamine corresponded to its turnover rates in regions of rat brain. In contrast, levels of IAA were unchanged as were levels of pros‐methylimidazoleacetic acid, an isomer of t‐MIAA unrelated to histamine metabolism. Results suggest that in brains of rats, unlike in the periphery, most IAA may not normally derive from histamine. Because histamine in brain can be converted to IAA under certain conditions, direct oxidation of histamine may be a conditional phenomenon. Our results also support the existence of a very slow turnover pool of brain histamine and use of chronic α‐FMHis infusion as a model to probe the histaminergic system in brain.
Preparation and Characterization of Monoclonal Antibodies Specific for Lauroylated Isoform of Bovine Transducin α‐Subunit: Immunohistochemical Analysis of Bovine Retinas
Authors: Shouno Osamu , Kokame Koichi , Araki Masasuke , Takao Toshifumi , Shimonishi Yasutsugu , Murata Masayuki , Yoshizawa Tôru , Fukada Yoshitaka
Published: July 2010

Abstract: The photoreceptor G protein transducin [α‐ and βγ‐subunits (Tα/Tβγ)] plays a central role in the visual transduction process. The amino‐terminus of bovine Tα is modified by one of four distinct fatty acids—laurate (C12:0), myristate (C14:0), C14:1 (5‐cis), and C14:2 (5‐cis, 8‐cis)—but the biological significance and the localization of the four isoforms of Tα are poorly understood. To investigate the cellular distribution of each isoform, we prepared monoclonal antibodies against a synthetic C12:0‐, C14:0‐, C14:1‐, or C14:2‐nonapeptide corresponding to the N‐terminal region of Tα. Among several types of antibodies isolated, only one type, represented by LA4, reacted specifically with the C12:0‐peptide as well as purified Tα but not with the other proteins in bovine retinal homogenate, including recoverin, indicating that the epitope comprises both C12:0 and the N‐terminal amino acids of Tα. Immunohistochemical analyses of bovine retinal sections by LA4 showed the uniform distribution of C12:0‐Tα in almost all the rod outer segments. Hence, it seemed unlikely that each isoform of Tα was localized in specific cells. This observation, together with evidence for a possible functional diversity among the isoforms, suggests that the four isoforms of Tα in a single rod cell may contribute simultaneously to a fine tuning of the photon‐signal transduction process.
Overexpressions of cDNAs for β‐Amyloid Precursor Proteins 695, 751, and 770 Enhance the Secretion of β‐Amyloid Precursor Protein Derivatives and the Survival of P19‐Derived Neurons
Authors: Fukuchi Ken‐ichiro , Ohman Tauni , Dang Nocthao , Smith Anetta C. , Furlong Clement E. , Martin George M.
Published: July 2010

Abstract: P19 is a C3H mouse‐derived line of multipotent embryonic carcinoma cells that differentiate into neural cells. P19 cell clones overexpressing the three major forms of β‐amyloid precursor protein from their cDNA constructs were established. Unlike a previous study in which P19‐derived neurons had a limited α‐secretase activity, all of these clones produced significant amounts of secreted β‐amyloid precursor protein. When treated with retinoic acid, these transformed lines differentiated into neurons and survived better than did nontransformed parental P19 cells. Furthermore, P19‐derived neurons survived better in medium conditioned by the transformed P19 line, and survival was reduced by immunoabsorption with an antibody to β‐amyloid precursor protein. These results suggest neurotrophic effects of secreted β‐amyloid precursor protein and contrast with a previous report in which overexpression of a full‐length cDNA for β‐amyloid precursor protein led to degeneration of P19‐derived neurons. Western blot analysis suggested that this difference might result from different levels of expression of putative neurotoxic C‐terminal fragments of β‐amyloid precursor protein; moreover, P19‐derived neurons differ from P19 stem cells in the processing of these C‐terminal fragments.
Sialidase Activity in Nuclear Membranes of Rat Brain
Authors: Saito Megumi , Fronda Christian L. , Yu Robert K.
Published: July 2010

Abstract: A highly purified nuclear membrane preparation was obtained from adult rat brain and examined for sialidase activity using GM3, GD1a, GD1b, or N‐acetylneuramin lactitol as the substrate. The nuclear membranes contained an appreciable level of sialidase activity; the specific activities toward GM3 and N‐acetylneuramin lactitol were 20.5 and 23.8% of the activities in the total brain homogenate, respectively. The sialidase activity in nuclear membranes showed substrate specificity distinct from other membrane‐bound sialidases localized in lysosomal membranes, synaptosomal plasma membranes, or myelin membranes. These results strongly suggest the existence of a sialidase activity associated with the nuclear membranes from rat brain.