bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Characterization of a cDNA Encoding a Novel Fatty Acid Binding Protein from Rat Brain
Authors: Bennett Ellen , Stenvers Kaye L. , Lund P. Kay , Popko Brian
Published: July 2010

Abstract: We have adopted a polymerase chain reaction approach to identify and clone a cDNA that contains the complete coding sequence of a novel fatty acid binding protein (FABP) from a rat brain λgt10 library. Sequencing of the brain FABP (B‐FABP) cDNA revealed an open reading frame coding for a protein with 132 amino acids and a predicted size of ∼15,000 Da. This putative protein shares extensive sequence homology with other members of the FABP family. Northern blot analysis using the B‐FABP cDNA as a probe established the presence of an abundant mRNA ∼0.8 kb long in rat brain and in the MOCH‐1 oligodendrocyte cell line. This transcript was also present in rat liver but not in other tissues examined. A developmental profile of this mRNA in rat brain demonstrated detectable expression in 15‐day‐old embryos with levels peaking in 1‐day postnatal neonates and declining thereafter, reaching a low steady‐state level at 3 weeks of age. In situ hybridization histochemistry revealed B‐FABP mRNA in various brain regions, with the highest levels in fiber tracts. The B‐FABP message was also detected at a lower level in several gray matter regions. The cloning approach used in this study would likely be useful in the identification and isolation of FABP‐encoding genes from other tissues and species.
Cytoskeletal Alterations in Human Fetal Astrocytes Induced by Interleukin‐1β
Authors: Liu Wei , Shafit‐Zagardo Bridget , Aquino Dennis A. , Zhao Meng‐Liang , Dickson Dennis W. , Brosnan Celia F. , Lee Sunhee C.
Published: July 2010

Abstract: Previous studies in this and other laboratories have shown that interleukin‐1β (IL‐1β) is a selective and potent activator of human astrocytes with respect to induction of cytokines and hematopoietic growth factors. To study the effect of recombinant human IL‐1β (rhIL‐1β) on astrocyte morphology, glial fibrillary acidic protein (GFAP) and vimentin expression, and actin organization, we conducted a systematic survey using dissociated human fetal astrocyte cultures. Within hours of stimulation with IL‐1β, the majority of astrocytes converted from flat, polygonal cells to small, contracted, highly branched cells. This change in morphology was more striking when serum was eliminated from the medium. Complete dissolution of filamentous actin occurred simultaneously with the change in cell shape, as demonstrated by fluorescein‐phalloidin binding. These “activated” astrocytes displayed intense GFAP and vimentin immunoreactivity in the small perikarya and processes. In contrast, the large, flat astrocytes in control cultures showed diffuse pale immunoreactivity for GFAP and vimentin. To quantify the changes in GFAP and vimentin content with IL‐1β stimulation, densitometric analyses of northern and western blots were performed. Northern blot analysis of IL‐1β‐stimulated astrocytes revealed a transient, marked decrease in steady‐state levels of mRNA for GFAP, vimentin, and microtubule‐associated protein 4. The decrease in mRNA levels was evident by 4–8 h and fell to the lowest level at 16–24 h (80–98% decrease by densitometry) with partial recovery by 72 h. By immunoblotting, a significant decrease in both GFAP and vimentin protein content was observed after IL‐1β stimulation. Furthermore, metabolic labeling studies revealed an almost total loss of GFAP synthesis following stimulation with IL‐1β for 16 h. These observations are consistent with the idea that increases in immunoreactivity were related to factors such as redistribution of epitope, rather than increases in total protein content. We hypothesize that in IL‐1β‐stimulated astrocytes, synthesis of other proteins, e.g., inflammatory cytokines, occurs at the expense of structural proteins and that the decrease in content of cytoskeletal proteins may reflect an “activated” state of astrocytes.
Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: Schläfer Matthias , Volknandt Walter , Zimmermann Herbert
Published: July 2010

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34‐kDa protein that binds azido‐ATP, azido‐AMP, and N‐ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S‐1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP‐binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP‐Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35‐kDa protein of Torpedo vesicles. An N‐terminal sequence obtained against the 34‐kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde‐3‐phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde‐3‐phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde‐3‐phosphate dehydrogenase.
Electroconvulsive Shock Reduces Inositol 1,4,5‐Trisphosphate 3‐Kinase mRNA Expression in Rat Dentate Gyrus
Authors: Kim Hyun , Ko Jae Pil , Kang Ung Gu , Park Joo‐Bae , Kim Hyung‐Lae , Lee Young Han , Kim Yong Sik
Published: July 2010

Abstract: We investigated the expression of inositol 1,4,5‐trisphosphate (InsP3) 3‐kinase mRNA after a single electroconvulsive shock (ECS) with in situ hybridization histochemistry in rat brain. At 6 h after ECS, the expression was markedly decreased in the dentate gyrus, and the decrease was maintained until 9 h with a slight recovery. The InsP3 3‐kinase mRNA content returned to basal levels after 12 h. We could not detect any apparent changes in the expression of InsP3 3‐kinase mRNA in the CA1–CA3 areas of hippocampus, the striatum, and the cerebral cortex at any time point examined. In the temporal pattern, the reduction of the expression in the dentate gyrus was preceded by the induction of c‐ fos after ECS. These observations suggest that the InsP3 3‐kinase might be one of the genes whose expression can be altered by ECS.
Identification of an Ectokinase Activity in Cerebellar Granule Primary Neuronal Cultures
Authors: Volonté Cinzia , Merlo Daniela , Ciotti Maria Teresa , Calissano Pietro
Published: July 2010

Abstract: Primary neuronal cultures from 8‐day‐old rat cerebellum were incubated in the presence of exogenously added 16 nM [γ‐32P]ATP. Phosphorylation of a 45‐kDa endogenous protein was detected within 1 min and increased linearly for ∼20 min. Unlike what was seen with [γ‐32P]ATP, in the presence of [32P] orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45‐kDa protein was reduced by 80–90% in the presence of 1 µM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO43−. Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 µM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45‐kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45‐kDa protein by 80–90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45‐kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto‐protein enzymes and intracellular kinases.
Depolarization Differentially Regulates the Effects of Bone Morphogenetic Protein (BMP)‐2, BMP‐6, and Activin A on Sympathetic Neuronal Phenotype
Authors: Fann Ming‐Ji , Patterson Paul H.
Published: July 2010

Abstract: As a first step in defining the role of the transforming growth factor‐β (TGF‐β) superfamily in the development of the sympathetic nervous system, we analyzed effects of several members of this family on neuronal gene expression in dissociated cell culture using a reverse transcription‐polymerase chain reaction method. We found that, in addition to activin A, bone morphogenetic protein (BMP)‐2 and BMP‐6 also induce mRNAs for distinct sets of neuropeptides and neurotransmitter synthetic enzymes in sympathetic neurons. TGF‐β1 and TGF‐β3 are, however, without detectable effect in this assay. Surprisingly, we find that the patterns of neuropeptide genes induced by activin A, BMP‐2, and BMP‐6 are each affected differently by neuronal depolarization. Depolarization can either promote or block the effects of different cytokines on the same neuropeptide gene, and depolarization can either promote or block the effects of a given cytokine on different neuropeptide genes. This evidence suggests that neuronal activity may be a key mediator of cytokine modulation of neuronal gene expression.
Ascorbic Acid Inhibits 125I‐SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors
Authors: Kimura Kazuhiro , Sidhu Anita
Published: July 2010

Abstract: Effects of ascorbic acid (AA) on 125I‐SCH 23982 binding to D1 dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µM–0.33 mM inhibited 75% of specific binding of 125I‐SCH 23982 in a dose‐dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 mM AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 mM AA, but reduced the inhibition by 3.3 mM AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 mM EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high‐affinity (Kh = 224.9 ± 48.9 nM) and low‐affinity (Kl = 21,100 ± 2,400 nM) binding sites. Although the maximum binding of 125I‐SCH 23982 decreased to 40% without affecting the KD value in the presence of 1.67 mM AA, the value of the high‐affinity site for dopamine was increased (Kh = 23.3 ± 9.4 nM) and that of the low‐affinity site was decreased (Kl = 136,800 ± 40,900 nM). These results suggest that AA may affect D1 dopamine receptor function by lipid peroxidation, competition with dopamine for low‐affinity sites, and reduced oxidation of dopamine.
Carbohydrate Structures of β‐Trace Protein from Human Cerebrospinal Fluid: Evidence for “Brain‐Type”N‐Glycosylation
Authors: Hoffmann Andrea , Nimtz Manfred , Wurster Ulrich , Conradt Harald S.
Published: July 2010

Abstract: The carbohydrate structures of β‐trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N‐linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N‐glycans were studied by compositional and methylation analyses, high‐pH anion‐exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N‐acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N‐acetylglucosamine residues of nearly all structures were found to be α1,6‐fucosylated. Peripheral fucose (about 20%α1,3‐linked to N‐acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N‐acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N‐acetylglucosamine only were present. At least 20 different N‐glycans were identified. Analysis of the site‐specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N‐acetylglucosamine, as well as terminal N‐acetylglucosamine and galactose residues, and significant amounts of N‐acetylneuraminic acid in α2,3 linkage), this protein can be classified as “brain‐type” glycosylated.
Autoradiographic Distribution and Characteristics of High‐ and Low‐Affinity Polyamine‐Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin
Authors: Nicolas C. , Carter C.
Published: July 2010

Abstract: We have studied the regional distribution and characteristics of polyamine‐sensitive [3H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [3H]ifenprodil (40 nM) in a biphasic manner with IC50 values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC50 values in the high micromolar range. Wiping studies using forebrain slices (containing both high‐ and low‐affinity sites) or cerebellar slices (containing only the low‐affinity site) showed that high‐ and low‐affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [3H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [3H]ifenprodil from its low‐affinity site in hindbrain and forebrain regions. High‐affinity [3H]ifenprodil sites, defined either by ifenprodil displacement curves or by [3H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low‐affinity [3H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [3H]Ifenprodil was found to bind to calmodulin‐agarose with very low affinity (IC50 of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [3H]ifenprodil from its low‐affinity site in brain sections. However, the localization of the low‐affinity [3H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high‐affinity [3H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.
Subcellular Localisation of 14‐3‐3 Isoforms in Rat Brain Using Specific Antibodies
Authors: Martin H. , Rostas J. , Patel Y. , Aitken A.
Published: July 2010

Abstract: The 14‐3‐3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14‐3‐3 proteins are cytosolic, a small but significant proportion of 14‐3‐3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform‐specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.
2,5‐Hexanedione Alters Elemental Composition and Water Content of Rat Peripheral Nerve Myelinated Axons
Authors: LoPachin Richard M. , Lehning Ellen J. , Stack Edward C. , Hussein Steven J. , Saubermann Albert J.
Published: July 2010

Abstract: Effects of 2,5‐hexanedione on elemental concentrations and water content of peripheral nerve myelinated axons were determined using electron probe x‐ray microanalysis. Axons (small, medium, and large) were analyzed in unfixed cryosections from rat tibial and proximal sciatic nerve samples. Animals were intoxicated with 2,5‐hexanedione by two dosing paradigms: intraperitoneal or oral. Regardless of the route of exposure, internodal axoplasm of small and medium axons from both nerve regions exhibited selective, progressive reductions in dry weight K concentrations and water content. When calculated on a wet weight basis, K levels were comparable to or slightly above control values in tibial nerve, whereas in sciatic nerve, small transient decreases in wet weight K were evident. These changes in K and water correlated with the development of axonal atrophy. The wet and dry weight internodal elemental changes reported here do not suggest a metabolic or axolemmal defect, but rather imply a homeostatic response possibly related to the process of axonal atrophy. Giant axonal swellings were primarily associated with oral 2,5‐hexane‐dione intoxication, and corresponding analyses revealed few changes in element or water content compared with control. The absence of significant alterations in these swellings is consistent with mechanical expansion of the axon probably as a function of accumulating neurofilaments.
Identification of Endogenously Phosphorylated KSP Sites in the High‐Molecular‐Weight Rat Neurofilament Protein
Authors: Elhanany Eytan , Jaffe Howard , Link William T. , Sheeley Douglas M. , Gainer Harold , Pant Harish C.
Published: July 2010

Abstract: The high‐molecular‐weight neurofilament protein (NF‐H) is highly phosphorylated in vivo, with estimates as high as 16–51 mol of Pi/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy‐terminal tail region of the molecule. Because the extent and site‐specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, Pi determinations, microsequencing, and mass‐spectral analysis to determine the endogenously phosphorylated sites in the NF‐H tail isolated from rat spinal cord. Twenty Ser residues in NF‐H carboxy‐terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 “non‐KSP” Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27‐kDa KSP‐rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of Pi/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu‐C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal‐derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF‐H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.
Changes in the Tyrosine Phosphorylation of Mitogen‐Activated Protein Kinase in the Rat Hippocampus During and Following Severe Hypoglycemia
Authors: Kurihara Junichi , Hu Bing‐Ren , Wieloch Tadeusz
Published: July 2010

Abstract: The changes in the levels of tyrosine‐phosphorylated proteins in the cytosolic fraction of the rat hippocampus subjected to severe hypoglycemia were analyzed. A marked increase in tyrosine phosphorylation of a 43‐kDa protein was observed at 30 min of isoelectric EEG and 30 min and 1 h of recovery. Immunostaining of the same blot with antibody against mitogen‐activated protein (MAP) kinase demonstrated a double band of ∼42 and 43 kDa. The increased tyrosine phosphorylation of MAP kinase during hypoglycemic coma and the early recovery period suggests that MAP kinase may be involved in neuronal degeneration and repair.
Characterization and Distribution of a Cloned Rat μ‐Opioid Receptor
Authors: Bunzow James R. , Zhang Ge , Bouvier Claudia , Saez Carmen , Ronnekleiv Oline K. , Kelly Martin J. , Grandy David K.
Published: July 2010

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ‐opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS‐7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 ± 0.2 nM) ≈β‐endorphin (Ki = 0.7 ± 0.5 nM) ≈ morphine (Ki = 0.8 ± 0.5 nM) ≈ [d‐Ala2, N‐Me‐Phe4,Gly‐ol5]‐enkephalin (DAMGO; Ki = 1.6 ± 0.5 nM) ⋙ U50,488 (Ki = 910 ± 0.78 nM) > [d‐Pen2,5]‐enkephalin (Ki = 3,170 ± 98 nM) > dextrorphan (Ki = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a μ‐opioid binding site. Two additional experiments provided evidence that this opioid‐binding site is functionally coupled to G proteins: (a) In COS‐7 cells 50 µM 5′‐guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 ± 0.5 nM) to a lower‐affinity state (IC50 = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ‐opioid receptor to DAMGO resulted in a dose‐dependent, naloxone‐sensitive inhibition of forskolin‐stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ‐opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ‐receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.
Molecular Cloning of a Novel G Protein‐Coupled Receptor Related to the Opiate Receptor Family
Authors: Lachowicz Jean E. , Shen Yong , Monsma Frederick J. , Sibley David R.
Published: July 2010

Abstract: A cDNA clone encoding a novel G protein‐linked receptor was isolated from a rat cerebral cortex cDNA library using a polymerase chain reaction‐amplified cDNA fragment as a probe. This 2.4‐kb clone encodes a 367 amino acid protein with seven putative transmembrane spanning domains. The protein is highly homologous to the cloned μ, δ, and κ opioid receptors and shares with them structural features such as three glycosylation sites in the amino terminus, a cyclic AMP‐dependent kinase phosphorylation site in the third cytoplasmic loop, an aspartic acid residue in the second transmembrane domain, and a palmitoylation site on the intracellular carboxy terminus. The receptor is also homologous with members of the somatostatin receptor family, yet it binds neither opiate nor somatostatin ligands. Northern blot analysis reveals two transcripts of 3.2 and 7.6 kb that are predominantly expressed in the cerebral cortex and hypothalamus. In situ hybridization analysis also shows a high abundance of mRNA in the cerebral cortex, hippocampus, amygdala, hypothalamus, thalamus, and dorsal raphe nuclei. It is suggested that the endogenous ligand for this receptor may represent a novel neuropeptide that may be closely related to the opiate peptide family.
Developmental Characterization of Thymosin β4 and β10 Expression in Enriched Neuronal Cultures from Rat Cerebella
Authors: Voisin Pierre J. , Pardue Sibile , Morrison‐Bogorad Marcelle
Published: July 2010

Abstract: The β4 and β10 thymosins are G‐actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β4 is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β10 is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β‐thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β‐thymosins were initially expressed in granule cells, although expression, especially of thymosin β4, was truncated compared with the in vivo time course. As in vivo, thymosin β4 was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β‐thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β‐thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.
Persistent Enhancement of Sustained Calcium‐Dependent Glutamate Release by Phorbol Esters: Requirement for Localized Calcium Entry
Authors: Terrian David M.
Published: July 2010

Abstract: Sustained activation of protein kinase C significantly enhanced a secondary (slow) phase in the depolarization‐induced release of glutamate from isolated hippocampal nerve endings. The phorbol ester, 4β‐phorbol 12,13‐dibutyrate, was used to sustain the activation of presynaptic protein kinase C for a prolonged (10‐min) period, and then this relatively water‐soluble phorbol ester was removed by superfusion before a 2‐min stimulus of continuous membrane depolarization. These conditions were used to investigate the persistent effects of sustained protein kinase C activation on the magnitude of the slow phase of evoked glutamate release, in which the efficiency of synaptic vesicle mobilization and recycling may be primary determinants of response magnitude. It is reported here that sustained protein kinase C activation selectively increased the Ca2+‐dependent component of glutamate release during a prolonged phase of K+‐induced depolarization. The magnitude of this persistent effect on Ca2+‐dependent glutamate release was directly related to the dose of 4β‐phorbol 12,13‐dibutyrate and the duration of exposure that was used to prime the release apparatus, was observed using two alternative synaptosomal preparations, and was evident regardless of the depolarizing stimulus used (elevated [KCl] or 4‐aminopyridine). However, 4β‐phorbol 12,13‐dibutyrate did not alter the release induced by the Ca2+ ionophore ionomycin. Thus, the persistent effects of protein kinase C activation on a prolonged phase of glutamate release were dependent on the route of Ca2+ influx. The finding that voltage‐regulated Ca2+ channel blockers were able to neutralize completely the 4β‐phorbol 12,13‐dibutyrate‐dependent facilitation of K+‐evoked glutamate release provided further support for this conclusion. Thus, 4β‐phorbol 12,13‐dibutyrate significantly potentiated the sustained release of glutamate without altering the strict requirement that is normally displayed by synaptosomes for localized and voltage‐regulated Ca2+ entry.
Persistent Enhancement of Sustained Calcium‐Dependent Glutamate Release by Phorbol Esters: Role of Calmodulin‐Independent Serine/Threonine Phosphorylation and Actin Disassembly
Authors: Terrian David M. , Ways D. Kirk
Published: July 2010

Abstract: The phorbol ester 4β‐phorbol 12,13‐dibutyrate increases the final extent of Ca2+‐dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca2+. Previous work from our laboratory demonstrated that this 4β‐phorbol 12,13‐dibutyrate‐dependent enhancement of synaptic vesicle recycling persists following the removal of 4β‐phorbol 12,13‐dibutyrate, requires localized Ca2+ entry through voltage‐regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C‐ and multifunctional Ca2+/calmodulin‐dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β‐phorbol 12,13‐dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca2+/calmodulin‐dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester‐dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca2+‐dependent glutamate release through a kinase‐independent mechanism. Given our finding that the actin‐depolymerizing agent cytochalasin D totally occluded the effect of 4β‐phorbol 12,13‐dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.
Inflammatory Mediator Stimulation of Astrocytes and Meningeal Fibroblasts Induces Neuronal Degeneration via the Nitridergic Pathway
Authors: Skaper Stephen D. , Facci Laura , Leon Alberta
Published: July 2010

Abstract: The role of inflammatory cytokines in the pathogenesis of neurological disorders is not entirely clear. The neurotoxic effects of cytokines, and perhaps indirectly bacterial endotoxins, could be mediated by the stimulation of immunocompetent cells in the brain to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. NO is a short‐lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. Both constitutive and inducible NO synthase has been described in astrocytes in vitro. Here we demonstrate that newborn mouse cortical astrocytes, when coincubated with neonatal mouse cerebellar granule cells or hippocampal neurons, induced neurotoxicity upon stimulation with endotoxin (lipopolysaccharide) (ED50 30 ng/ml). Astrocytes were unresponsive to the cytokines tumor necrosis factor‐α or interleukin‐1β individually, but exhibited a marked synergistic stimulation in their combined presence. Moreover, meningeal fibroblasts treated with tumor necrosis factor‐α, but not interleukin‐1β or lipopolysaccharide, elaborated neurotoxicity for cocultured granule cells (ED50 30 U/ml). In cocultures of immunostimulated astrocytes or meningeal fibroblasts, neurotoxicity was blocked by the NO synthase inhibitors Nω‐nitro‐l‐arginine and Nω‐nitro‐d‐arginine methyl ester, and by oxyhemoglobin, which inactivates NO. Astroglial‐induced neurotoxicity was not affected by N‐methyl‐d‐aspartate receptor antagonists. Superoxide dismutase, which degrades superoxide anion, attenuated astrocyte‐ and fibroblast‐mediated neurotoxicity, indicating that endogenous superoxide anion may react with NO to form toxic peroxynitrite and its breakdown products. These findings suggest a potentially important role for glial‐ and meningeal fibroblast‐induced NO synthase in the pathophysiology of CNS disease states of immune or inflammatory origin.
Persistent Translocation of Ca2+/Calmodulin‐Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia
Authors: Hu Bing‐Ren , Wieloch Tadeusz
Published: July 2010

Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca2+/calmodulin‐dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.
Time‐Related Cortical Amino Acid Changes After Basal Forebrain Lesion: A Microdialysis Study
Authors: Boatell M. Ll. , Bendahan G. , Mahy N.
Published: July 2010

Abstract: Cholinergic basal forebrain (BF) lesions in experimental animals have been used as a potential model for cholinergic deficits in cortex and hippocampus that occur in normal aging and Alzheimer's disease (AD). Glutamatergic cortical neurons are also affected in AD and could be part of the neurodegenerative process. In the present study, the effect of bilateral BF lesion with ibotenic acid microinjection on cortical extracellular amino acid levels was determined. Samples were collected every 20 min with microdialysis probes in awake, freely moving rats under basal and potassium stimulation conditions and measured by HPLC with fluorescence detection. Microdialysis experiments were performed 13 days, 21 days, and 30 days after BF lesion. The effectiveness of the lesion was shown by a significant 30% depletion in acetyl‐CoA:choline O‐acetyltransferase (EC 2.3.1.6) activity in the frontal cortex. Under basal conditions at 13 days only extracellular levels of taurine (Tau) and Glu were significantly reduced. Tau and Glu levels were recovered after 21 days and 30 days, respectively. In contrast, increase in Gly levels reaches its significance only at 30 days after lesion. Significant increases of Gln levels were observed at 21 days and 30 days. Asp and Ser levels remained constant throughout the period studied. Potassium stimulation led to increased Asp, Glu, Gly, and Tau levels, whereas Gln content decreased and Ser remained unaltered. As Ser is not believed to be a neurotransmitter, its lack of variation in any of the experimental conditions studied supports specific neuronal changes of the other amino acids. Results are discussed with reference to data observed in AD patients and possible mechanisms underlying the changes are suggested.
Structural Characterization by Affinity Cross‐Linking of Glucagon‐Like Peptide‐1(7–36)Amide Receptor in Rat Brain
Authors: Calvo José C. , Yusta Bernardo , Mora Francisco , Blázquez Enrique
Published: July 2010

Abstract: Specific binding of glucagon‐like peptide (GLP)‐1(7–36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of 125I‐GLP‐1(7–36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon‐derived peptides. The rank order of potency was GLP‐1(7–36)amide ≫ GLP‐1(1–36)amide > GLP‐1(1–37) ≅ GLP‐2 > glucagon. Scatchard analysis of the steady‐state binding data was consistent with the presence of both high‐ and low‐affinity binding sites in hypothalamus and brainstem. Brain 125I‐GLP‐1(7–36)amide‐binding protein complexes were covalently cross‐linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A single radiolabeled band of Mr 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP‐1(7–36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited Mr 56,000 complex labeling with the following order of potency: GLP‐1(1–36)amide > GLP‐1(1–37) > GLP‐2. The binding of 125I‐GLP‐1(7–36)amide and the intensity of the cross‐linked band were similarly inhibited in a dose‐response manner by increasing concentrations of unlabeled GLP‐1(7–36)amide. Covalent Mr 56,000 125I‐GLP‐1(7–36)amide‐binding protein complexes solubilized by Triton X‐100 were adsorbed onto wheat germ agglutinin. Our results suggest that the GLP‐1(7–36)amide receptor in rat brain is a glycoprotein with a single binding subunit that has a greater molecular weight but binding features and ligand specificity similar to those of its peripheral tissue counterparts.
Iron Levels Modulate α‐Secretase Cleavage of Amyloid Precursor Protein
Authors: Bodovitz Steven , Falduto Michael T. , Frail Donald E. , Klein William L.
Published: July 2010

Abstract: The amyloid precursor protein (APP) is a membrane‐spanning glycoprotein that is the source of βA4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the βA4 sequence to release a soluble N‐terminal derivative (APPsol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C‐terminal, βA4, and N‐terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The data show a specific effect on the production of APPsol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APPsol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C‐terminal α‐secretase derivative C83, βA4, and p3 peptides. The specificity for modulatory changes in APPsol suggests that iron acts at the level of α‐secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP. If APPsol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in iron and iron‐regulating proteins in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APPsol.
Lipid Composition of Neuronal Cell Bodies and Neurites from Cultured Dorsal Root Ganglia
Authors: Calderon R. O. , Attema B. , DeVries G. H.
Published: July 2010

Abstract: The lipid composition of neuronal somata and neuritic processes of cultured root ganglia has been determined. Neuronal soma contained 37% of dry weight as lipid (15.4% cholesterol, 4.8% galactolipid, and 57.1% phospholipid). The major phospholipids were phosphatidylcholine and phosphatidyl ethanolamine. Galactolipids consisted of cerebroside and sulfatide in molar ratio 2:1. The neuronal soma contained tetrasialo‐, disialo‐, and monosialoganglioside. In contrast, neurites contained 15% of the dry weight as lipid (22.1% cholesterol, 7.7% galactolipid with cerebroside and sulfatide in molar ratio 2:1, and 56.4% total phospholipid). The neuritic galactolipid content was higher, as was the percentage of sphingomyelin, and phosphatidyl serine. The higher cholesterol content in neuritic lipid reflected the higher percentage of plasma membrane in this compartment. The ganglioside pattern of neurites was distinct from that of the neuronal soma and consisted entirely of gangliosides GQ1b, GT1b, GD1b, GD1a, and GD3, with no monosialogangliosides. The results indicate a preferential phospholipid and glycolipid sorting to the neuritic plasma membrane that may be related to the distinctive functions of this neuronal compartment.
Down‐Regulation of AMPA Receptor Subunit GluR2 in Amygdaloid Kindling
Authors: Prince Heather K. , Conn P. Jeffrey , Blackstone Craig D. , Huganir Rick L. , Levey Allan I.
Published: July 2010

Abstract: Alterations in glutamatergic transmission are postulated to be important in kindling and epilepsy. The levels of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) receptor subunits (GluR1, 2, and 4) were compared in amygdalakindled and sham‐operated animals using subunit‐specific antibodies and quantitative western blotting. Four limbic regions were examined: limbic forebrain, piriform cortex/amygdala, hippocampus, and entorhinal cortex. When subunit levels were examined 24 h after the last stage 5 seizure, levels of GluR2 were found to be selectively reduced in limbic forebrain (30%) and piriform cortex/amygdala (25%), with no changes in other regions examined. In addition, no changes in the other subunits were observed in any region. The decrease in GluR2 that was observed in kindled animals at 24 h was no longer present at 1 week and 1 month after the last stage 5 seizure. Because the GluR2 subunit uniquely determines the calcium permeability of these receptors and because the piriform cortex has been implicated as a source of excitatory drive for limbic seizures, reduced GluR2 expression may be important in increasing neuronal excitability in kindling‐induced epilepsy, or may reflect a compensatory mechanism resulting from kindling.
Retrovirus‐Mediated Expression of an Artificial β‐Endorphin Precursor in Primary Fibroblasts
Authors: Beutler Andreas S. , Banck Michaela S. , Bach Flemming W. , Gage Fred H. , Porreca Frank , Bilsky Edward J. , Yaksh Tony L.
Published: July 2010

Abstract: Peptides are of potential interest in the field of gene therapy but require modification by genetic engineering to facilitate their secretion. Amino terminal addition of a signal peptide is not always sufficient to achieve this goal, as found in this study for β‐endorphin. To overcome this problem, addition of the pre‐pro‐sequence of mouse nerve growth factor to β‐endorphin was tested. Retrovirus‐mediated expression of a hybrid construct of the pre‐pro‐sequence of nerve growth factor and human β‐endorphin in primary fibroblasts resulted in the secretion of β‐endorphin immunoreactivity at a rate of 620 pg/h/106 cells. Analysis of the secreted β‐endorphin immunoreactivity with reverse‐phase HPLC, immunoassays using three different antibodies, and an assay for the specific displacement of [3H][d‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin from μ‐opioid receptors suggests that the pre‐pro‐sequence is cleaved off from the pre‐pro‐sequence/β‐endorphin construct prior to secretion, resulting in bona fide β‐endorphin. Transplantation of β‐endorphin‐secreting cells into brain or spinal cord may provide a gene therapy approach for the treatment of chronic, opioid‐sensitive pain states.
Regulation of Cytochrome c Oxidase Subunit mRNA and Enzyme Activity in Rat Brain Reward Regions During Withdrawal from Chronic Cocaine
Authors: Walker John R. , Sevarino Kevin A.
Published: July 2010

Abstract: A subtractive hybridization and differential screening procedure was used to detect up‐regulation of cytochrome c oxidase (CO) subunits I, III, and IV mRNA in the nucleus accumbens (NAc) of rats chronically treated with cocaine. Northern blot analyses of mRNA isolated from individual rats confirmed that CO subunit I was up‐regulated by chronic, but not acute, cocaine in two brain regions, the NAc (33%) and caudate‐putamen (CP)(35%). CO activity, used as a measure of metabolic activity, was increased by 88% in the NAc, and decreased by 20% in the medial prefrontal cortex (mPFC), the day after chronic treatment was terminated. CO enzyme activity was not regulated in the CP, or in other brain regions not involved in drug reward. CO activity in both the NAc and mPFC showed unique time‐dependent patterns of regulation during the week after chronic cocaine treatment.
K‐252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells
Authors: Maroney Anna Coco , Lipfert Lorraine , Forbes M. Elizabeth , Glicksman Marcie A. , Neff Nicola T. , Siman Robert , Dionne Craig A.
Published: July 2010

Abstract: The protein kinase inhibitor K‐252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K‐252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH‐SY5Y. K‐252a induced neurite outgrowth in a dose‐dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125‐ and 140‐kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down‐regulation of protein kinase C by long‐term treatment with phorbol ester did not prevent K‐252a‐induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF‐109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K‐252a was also observed in LA‐N‐5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C‐independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K‐252a‐induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.