bluuebunny/crossref_metadata_2025 huggingface dataset:
The Quail Tyrosine Hydroxylase Gene Promoter Contains an Active Cyclic AMP‐Responsive Element
Authors: Fauquet Mireille , Boni Claudette
Published: October 2006

Abstract: A 1.5‐kb genomic DNA fragment situated upstream from the quail tyrosine hydroxylase (TH) gene transcription site was isolated. This upstream region starts 15 bp from the translation initiation site. It contains two canonical TATA boxes, at positions −37 and −297, three putative glucocorticoid‐responsive elements, at positions −1487, −1329, and −1268, and one putative cyclic AMP (cAMP)‐responsive element (CRE) at position −53, as well as a putative negative regulatory element consensus sequence at position −735. The consensus POU‐Oct site is partly conserved. Comparison of the 5′flanking sequences of quail and mammalian (bovine, human, and rat) TH genes revealed a strong sequence conservation within the 230 nucleotides upstream of the TATA box, with a distinct conservation of the CRE region. Constructs in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was linked to promoter stretches of increasing lengths were transfected into three cell lines, two of them originating from quail and rat neural crest and the third derived from mouse fibroblasts. Reporter gene expression was specifically high in the quail and rat neural crest‐derived cells compared to the fibroblast cell line. The physiological activity of this putative quail CRE was analyzed further in transfected neural crest cells of quail origin. Both cAMP analogues and agents that enhance intracellular cAMP increased CAT activity. The physiological relevance of this finding is sustained by the presence, in quail nuclear extracts, of a protein(s) that binds CRE consensus sequences.
Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH‐SY5Y Neuroblastoma Cells: Modifications During Differentiation
Authors: Leli Ubaldo , Shea Thomas B. , Cataldo Anne , Hauser George , Grynspan Frida , Beermann Mary Lou , Liepkalns Vis A. , Nixon Ralph A. , Parker Peter J.
Published: October 2006

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H‐7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH‐SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH‐SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH‐SY5Y cells, as well as in rat brain. Protein kinase C‐α and ‐β1 were the most abundant isoforms in SH‐SY5Y cells, and immunoreactive protein kinase C‐δ and ‐ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol or staurosporine, and that protein kinase C‐ɛ is predominantly membrane‐bound. Its localization did not change during differentiation. Western blots of total SH‐SY5Y cell extracts and of subcellular fractions probed with isoform‐specific polyclonal antibodies showed that when SH‐SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C‐α and ‐ɛ decreased, and protein kinase C‐β1, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.
Cloning and Expression of a Rat Acetylcholinesterase Subunit: Generation of Multiple Molecular Forms and Complementarity with a Torpedo Collagenic Subunit
Authors: Legay Claire , Bon Suzanne , Vernier Philippe , Coussen Françoise , Massoulié Jean
Published: October 2006

Abstract: We obtained a cDNA clone encoding one type of catalytic subunit of acetylcholinesterase (AChE) from rat brain (T subunit). The coding sequence shows a high frequency of (G + C) at the third position of the codons (66%), as already noted for several AChEs, in contrast with mammalian butyrylcholinesterase. The predicted primary sequence of rat AChE presents only 11 amino acid differences, including one in the signal peptide, from that of the mouse T subunit. In particular, four alanines in the mouse sequence are replaced by serine or threonine. In northern blots, a rat AChE probe indicates the presence of major 3.2‐and 2.4‐kb mRNAs, expressed in the CNS as well as in some peripheral tissues, including muscle and spleen. In vivo, we found that the proportions of G1, G2, and G4 forms are highly variable in different brain areas. We did not observe any glycolipid‐anchored G2 form, which would be derived from an H subunit. We expressed the cloned rat AChE in COS cells: The transfected cells produce principally an amphiphilic G1a form, together with amphiphilic G2a and G4a forms, and a nonamphiphilic G4na form. The amphiphilic G1a and G2a forms correspond to type II forms, which are predominant in muscle and brain of higher vertebrates. The cells also release G4na, G2a, and G1a in the culture medium. These experiments show that all the forms observed in the CNS in vivo may be obtained from the T subunit. By cotransfecting COS cells with the rat T subunit and the Torpedo collagenic subunit, we obtained chimeric collagentailed forms. This cross‐species complementarity demonstrates that the interaction domains of the catalytic and structural subunits are highly conserved during evolution.
Inhibition of Protein Synthesis During Repetitive Cortical Spreading Depression
Authors: Mies Günter
Published: October 2006

Abstract: The effect of cortical spreading depression (CSD) on cerebral protein synthesis (CPS) was examined. CSD was evoked in normal rats with KCI, and CPS was measured autoradiographically with [1‐14C]leucine. Average rates (mean ± SD) of CPS in layers I‐IV of cortex decreased significantly from 10.7 ± 0.6 (sham‐operated controls; n = 4) to 6.7 ± 0.7 nmol/g/min (n = 4; p < 0.01) and in layers V‐VI from 10.9 ± 0.5 to 9.4 ± 0.4 nmol/g/min (p < 0.05) during 60 min of repetitive CSD. Spreading depression did not affect CPS rates in other subcortical brain regions. These results indicate that KCl‐evoked CSD induces inhibition but not suppression of cortical protein synthesis.
ADP‐Ribosylation of the Neuronal Phosphoprotein B‐50/GAP‐43
Authors: Coggins Philip J. , McLean Kim , Nagy Andy , Zwiers Henk
Published: October 2006

Abstract: The neuronal phosphoprotein B‐50/GAP‐43 is associated with growth and regeneration within the nervous system and its posttranslational status can be correlated with its cellular localization during growth and regeneration. Recently, B‐50 has been shown to interact with certain G protein subunits. Regulation of G protein‐mediated signal transduction may involve ADP‐ribosylation in vivo. In the present study we have demonstrated that B‐50 is a substrate for endogenous ADP‐ribosyltransferases. The results are discussed with respect to the possible interaction of B‐50 with G proteins, but also with regard to the posttranslational modification of B‐50 by all major regulatory mechanisms that act at, or through, the neuronal membrane.
Regulated Expression of the Neurofibromin Type I Transcript in the Developing Chicken Brain
Authors: Baizer Lawrence , Ciment Gary , Hendrickson Susan K. , Schafer Gwen L.
Published: October 2006

Neurofibromatosis type 1 (NF‐1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF‐1 gene is similar in sequence to the ras‐GTPase activator protein (ras‐GAP), and investigations have confirmed that the NF‐1 gene product (now known as neurofibromin) stimulates ras‐GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63‐bp exon inserted in the GAP‐related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon‐intron boundaries. RNase protection and RNA‐polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is enriched in neurons.
Alterations in Actin‐Binding β‐Thymosin Expression Accompany Neuronal Differentiation and Migration in Rat Cerebellum
Authors: Border Barbara G. , Lin Sheng‐Cai , Griffin W. Sue T. , Pardue Sibile , Morrison‐Bogorad Marcelle
Published: October 2006

The β4‐and β10‐thymosins, recently identified as actin monomer‐sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohis‐tochemical techniques to investigate the distribution of the β‐thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both β‐thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both β‐thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin β4, but not thymosin β10, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin β4 was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin β10 was detected in an even smaller population of glia. Expression of thymosin β4 and thymosin β10 in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both β‐thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.
A Neurofilament‐Associated Kinase Phosphorylates Only a Subset of Sites in the Tail of Chicken Midsize Neurofilament Protein
Authors: Hollander Brian A. , Ayyub Champakali , Shaw Gerry , Bennett Gudrun S.
Published: October 2006

Although neurofilaments are among the most highly phosphorylated proteins extant, relatively little is known about the kinases involved in their phosphorylation. The majority of the phosphates present on the two higher‐molecular‐mass neurofilament subunits are added to multiply repeated sequence motifs in the tail. We have examined the specificity of a neurofilament‐associated kinase (NFAK) partially purified from chicken spinal cord that selectively phosphorylates the middle‐molecular‐mass neurofilament subunit, NF‐M. Two‐dimensional phosphopeptide mapping of 32P‐labeled NF‐M shows that, in vitro, NFAK phosphorylates a subset of peptides phosphorylated in vivo in cultured neurons. The absence of a complete complement of labeled phosphopeptides following in vitro phosphorylation, compared with phosphorylation in vivo, is not due to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF‐M is used as an in vitro substrate. Phosphopeptide maps from in vitro‐phosphorylated NF‐M and those from a recombinant fusion protein containing only a segment of the tail piece of chicken NF‐M reveal identical labeled peptides. The fusion protein lacks a segment containing 17 KXX(S/T)P putative phosphorylation sites contained in the tail of chicken NF‐M but contains a segment that includes four KSPs and a KSD site also present in the intact tail. These results suggest (a) that NFAK mediates the phosphorylation of some, but not all, potential phosphorylation sites within the tail of NF‐M and (b) that multiple kinases are necessary for complete phosphorylation of the NF‐M tail.
Growth Conditions Differentially Regulate the Expression of α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate (AMPA) Receptor Subunits in Cultured Neurons
Authors: Condorelli D. F. , Dell'Albani P. , Aronica E. , Genazzani A. A. , Casabona G. , Corsaro M. , Balazs R. , Nicoletti F.
Published: October 2006

We have studied the expression of a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K± (10, 25, or 40 mM) with or without 100 μM N‐methyl‐D‐aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K± (K10) and increased dramatically in cultures grown in 25 mM K± (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mM K± (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and ‐3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA‐stimulated 4SCa2± influx and 40‐[3H]phorbol 12, 13‐dibutyrate binding in intact cells. Both functional parameters increased along with the K± concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.
Neither Moderate Hypoxia nor Mild Hypoglycaemia Alone Causes Any Significant Increase in Cerebral [Ca2±i: Only a Combination of the Two Insults Has This Effect.: A 31P and 19F NMR Study
Authors: Badar‐Goffer Ronnitte S. , Thatcher Nicola M. , Morris Peter G. , Bachelard Herman S.
Published: October 2006

(1) The energy state and free intracellular calcium concentration ([Ca2±i) of super‐fused cortical slices were measured in moderate hypoxia (∼65 μM O2), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using 19F and 31P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca2±i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca2±, and a decrease in PCr level to ∼25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca2±i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca2±i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca2±i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.
Chicken Tyrosine Hydroxylase Gene: Isolation and Functional Characterization of the 5′ Flanking Region
Authors: Carrier Alice , Devignes Marie‐Dominique , Renoir Dominique , Auffray Charles
Published: October 2006

Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the biosynthesis of catecholamines. We describe here the isolation of the chicken TH gene and the analysis of 3 kb of its 5′ flanking region. The chicken TH transcription unit spans 19 kb. The 60‐bp proximal promoter contains a TATA box and a cyclic AMP response element (CRE) sequence. The 5′ flanking region contains several AP1‐, AP2‐, and octamer‐like sequences as well as a glucocorticoid response element at position ‐1.4 kb. A construct containing the 3‐kb 5′ flanking DNA fused to the chloramphenicol acetyltransferase (CAT) gene was transiently transfected into PC12 cells, and the effect of various effectors was tested. Only forskolin increased the CAT activity, likely owing to the presence of the CRE sequence. Constructs prepared by progressively deleting the 5′ flanking DNA were transfected into PC12 and QT6 (quail transformed fibroblasts) cells. In both cell types, the transcriptional activity increased with deletion of the 5′ flanking region. These results show that the 60‐bp region containing the TATA box and the CRE is sufficient to act as a constitutive promoter for the chicken TH gene and that this region appears to be negatively controlled by upstream sequences.
A Previous Potassium Stimulation Enhances the Increases of Striatal Extracellular Dopamine and 5‐Hydroxytryptamine During Global Ischaemia Under Simulated Penumbral Conditions
Authors: Richards D. A. , Obrenoyitch T. P. , Johonson‐Mora A. , Mase M. , Symon L. , Curzon G.
Published: October 2006

The effect of a previous K± stimulation on striatal extracellular monoamine levels during global ischaemia, under simulated penumbral conditions, was investigated. Rats were implanted with microdialysis probes in both striata, monoamine release was stimulated unilaterally by adding K± (100 mM, 20 min) to the artificial CSF perfused through one probe, and bilateral partial ischaemia was imposed after monoamine levels had returned to basal values or below. Resultant increases in dialysate levels of dopamine and 5‐hydroxytryptamine were markedly and significantly greater on the side previously exposed to K±, even though electrophysiological measurements indicated similarly severe ischaemia on both sides. Associated monoamine metabolite changes did not differ significantly between the two sides. There was no evidence of greater neuronal loss in the K±‐stimulated striata 7 days after ischaemia. However, striatal tissue probably exposed to the highest concentrations of K± could not be examined because of extensive gliosis around the probe.
ucb 11056, a New Potential Nootropic Drug, Amplifies Induced Cyclic AMP Formation in Rat Brain Tissue
Authors: Tamer A. El , Raikoff K. , Corey J. , Wulfert E. , Hanin I.
Published: October 2006

ucb 11056 [2‐(4‐morpholino‐6‐propyl‐1, 3, 5‐triazin‐2‐yl)aminoethanol] induced a significant (∼25%) increase in cyclic AMP levels in different brain areas following its intraperitoneal injection. This effect started as early as 2 min postinjection and lasted for 30 min, after which cyclic AMP levels returned to normal. In hippocampal slice preparations in vitro, ucb 11056 exerted a strong potentiation of cyclic AMP levels when it was combined with agents such as norepinephrine, forskolin, and isoproterenol. Only a slight effect on cyclic AMP levels was measured when ucb 11056 was incubated alone with hippocampal slices. The potentiating effect of ucb 11056 on norepinephrine‐stimulated cyclic AMP formation was partially reduced when slices were pretreated with yohimbine and totally abolished when slices were treated with propranolol. These combined data indicate that (a) ucb 11056 rapidly increases cyclic AMP levels in the rat brain in vivo and (b) ucb 11056 potentiates stimulated cyclic AMP formation in vitro. The data also suggest that the central effect of ucb 11056 might be via the modulation of cyclic AMP generation, most probably mediated through adenylate cyclase activation mechanisms combined with a weak inhibitory activity on the cyclic nucleotide phosphodiesterase activity.
Clearance of Exogenous Dopamine in Rat Dorsal Striatum and Nucleus Accumbens: Role of Metabolism and Effects of Locally Applied Uptake Inhibitors
Authors: Cass Wayne A. , Zahniser Nancy R. , Flach Karen A. , Gerhardt Greg A.
Published: October 2006

In vivo electrochemistry was used to investigate the mechanisms contributing to the clearance of locally applied dopamine in the dorsal striatum and nucleus accumbens of urethane‐anesthetized rats. Chronoamperometric recordings were continuously made at 5 Hz using Nafion‐coated carbon fiber electrodes. When a finite amount of dopamine was pressure‐ejected at 5‐min intervals from a micropipette adjacent to the electrode, transient and reproducible dopamine signals were detected. Substitution of L‐a‐methyldopamine, a substrate for the dopamine transporter but not for monoamine oxidase, for dopamine in the micropipette did not substantially alter the time course of the resulting signals. This indicates that metabolism of locally applied dopamine to 3,4‐dihydroxyphenylacetic acid is not responsible for the decline in the dopamine signal. Similarly, changing the applied oxidation potential from ±0.45 to ±0.80 V, which allows for detection of 3‐methoxytyramine formed from dopamine via catechol‐O‐methyltransferase, had little effect on signal amplitude or time course. In contrast, lesioning the dopamine terminals with 6‐hydroxydopamine, or locally applying the dopamine uptake inhibitors cocaine or nomifensine before pressure ejection of dopamine, significantly increased the amplitude and time course of the dopamine signals in both regions. The effects of cocaine and nomifensine were greater in the nucleus accumbens than in the dorsal striatum. Local application of lidocaine and procaine had no effect on the dopamine signals. Initial attempts at modeling resulted in curves that were in qualitative agreement with our experimental findings. Taken together, these data indicate that (1) uptake of dopamine by the neuronal dopamine transporter, rather than metabolism or diffusion, is the major mechanism for clearing locally applied dopamine from the extracellular milieu of the dorsal striatum and nucleus accumbens, and (2) the nucleus accumbens is more sensitive to the effects of inhibitors of dopamine uptake than is the dorsal striatum.
Ceramide 1‐Phosphate Phosphatase Activity in Brain
Authors: Shinghal Rajesh , Scheller Richard H. , Bajjalieh Sandra M.
Published: October 2006

Recent studies have implicated sphingolipids in a variety of intracellular signaling systems. The finding that a calcium‐stimulated ceramide kinase copurifies with neurotransmitter‐containing vesicles suggests that ceramide, or one of its metabolites, has a role in neurotransmitter release. As a step toward understanding the role of ceramide kinase in vesicle functioning, this study sought to determine the metabolic fate of the product, ceramide 1‐phosphate. We report that ceramide 1‐phosphate is not deacylated by brain ceramidases to produce sphingosine 1‐phosphate. It is, however, the substrate for a phosphatase activity that we name ceramide 1‐phosphate phosphatase (CPPase). Subcellular fractionation studies suggest that CPPase is found in the synaptic terminal and is associated with both synaptic vesicle and plasma membranes. Divalent cations, most notably calcium, inhibit CPPase activity although not at concentrations that activate ceramide kinase. The existence of both ceramide kinase and CPPase activities at the synapse suggests that ceramide 1‐phosphate production regulates some aspect of synaptic vesicle functioning.
NMDA and AMPA Receptors in Transgenic Mice Expressing Human β‐Amyloid Protein
Authors: Sandhu Faheem A. , Porter Richard H. P. , Eller Robert V. , Zain Sayeeda B. , Salim Mohammad , Greenamyre J. Timothy
Published: October 2006

The human β‐amyloid protein may play an important, possibly primary, role in the pathogenesis of Alzheimer's disease (AD), and it appears to potentiate the susceptibility of neurons to excitotoxicity. AD is associated with alterations in the W‐methyl‐D‐aspartate (NMDA) and a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionic acid (AMPA) subtypes of glutamate receptors, and it has been suggested that excitotoxicity may play a role in neuronal damage in AD. In this study, we have used quantitative receptor autoradiography to examine NMDA and AMPA receptors in transgenic mice that contain the gene for the carboxyl‐terminal 100 amino acids of the human amyloid precursor protein, beginning with the β‐amyloid region, which is under the control of the JC viral early region promoter. Reverse transcriptase‐polymerase chain reaction confirmed that the brains of transgenic mice expressed β‐amyloid mRNA and that control mice did not. NMDA receptors, assessed with [3H]MK‐801, were unchanged in the transgenic compared with the control mice. In the transgenic mice, there were no significant changes in [3H]AMPA receptor binding compared with controls. This study represents the first attempt to evaluate in transgenic mice the in vivo interaction between β‐amyloid expression and excitatory amino acid receptors.
Expression of the Baculovirus p35 Gene Inhibits Mammalian Neural Cell Death
Authors: Rabizadeh S. , LaCount D. J. , Friesen P. D. , Bredesen D. E.
Published: October 2006

Expression of the apoptosis suppressor genep35, derived from the baculovirus Autographa californica nuclear polyhedrosis virus, markedly inhibited the cell death of stably transfected mammalian neural cells whether the cell death was induced by glucose withdrawal, calcium ionophore, or serum withdrawal. The p35 protein, which is required to block virus‐induced apoptosis of cultured insect cells, is only the second gene product shown to block mammalian neural cell death, with Bcl‐2 being the first. Because there is no apparent homology between p35 and Bcl‐2, the existence of a cellular death program that may be modulated at multiple points is suggested/Furthermore, these findings demonstrate that the putative cellular death program is conserved across species and cell types.
Superoxide Dismutase Activity, Oxidative Damage, and Mitochondrial Energy Metabolism in Familial and Sporadic Amyotrophic Lateral Sclerosis
Authors: Bowling Allen C. , Schulz Jorg B. , Brown Robert H. , Beal M. Flint
Published: October 2006

The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is unknown. Recently, it was found that some patients with autosomal‐dominant familial ALS (FALS) have point mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1). In this study of postmortem brain tissue, we examined SOD activity and quantified protein carbonyl groups, a marker of oxidative damage, in samples of frontal cortex (Brodmann area 6) from 10 control patients, three FALS patients with known SOD1 mutations (FALS‐1), one autosomal‐dominant FALS patient with no identifiable SOD1 mutations (FALS‐0), and 11 sporadic ALS (SALS) patients. Also, we determined the activities of components of the electron transport chain (complexes I, II‐III, and IV) in these samples. The cytosolic SOD activity, which is primarily SOD1 activity, was reduced by 38.8% (p < 0.05) in the FALS‐1 patients and not significantly altered in the SALS patients or the FALS‐0 patient relative to the control patients. The mitochondrial SOD activity, which is primarily SOD2 activity, was not significantly altered in the FALS‐1, FALS‐0, or SALS patients. The protein carbonyl content was elevated by 84.8% (p < 0.01) in the SALS patients relative to the control patients. Finally, the complex I activity was increased by 55.3% (p < 0.001) in the FALS‐1 patients relative to the control patients. These results from cortical tissue demonstrate that SOD1 activity is reduced and complex I activity is increased in FALS‐1 patients and that oxidative damage to proteins is increased in SALS patients.
Botulinum A Like Type B and Tetanus Toxins Fulfils Criteria for Being a Zinc‐Dependent Protease
Authors: Paiva Anton de , Ashton Anthony C. , Foran Patrick , Schiavo Giampetro , Montecucco Cesare , Dolly J. Oliver
Published: October 2006

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc‐binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc‐dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxins’ neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins’ degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc‐dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins’ effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.
N‐Methyl‐d‐Aspartic Acid/Glycine Interactions on the Control of 5‐Hydroxytryptamine Release in Raphe Primary Cultures
Authors: Becquet D. , Héry M. , Deprez P. , Faudon M. , Fache M. P. , Giraud P. , Héry F.
Published: October 2006

Abstract: Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg2+, NMDA (50 μM) significantly stimulated (by 69%) release of newly synthesized 5‐[3H]hydroxytryptamine ([3H]5‐HT). d‐2‐Amino‐5‐phosphonopentanoate (AP‐5; 50 μM) blocked the stimulatory effect of NMDA. AP‐5 by itself inhibited [3H]5‐HT release (by 25%), suggesting a tonic control of 5‐HT by glutamate. In the absence of Mg2+, basal [3H]5‐HT release was 60% higher as compared with release with Mg2+. AP‐5 blocked the increased [3H]5‐HT release observed without Mg2+, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 μM) inhibited [3H]5‐HT release in the absence of Mg2+. Strychnine (50 μM) blocked the inhibitory effect of glycine, indicating an action through strychnine‐sensitive inhibitory glycine receptors. The [3H]5‐HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA‐evoked [3H]5‐HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA‐associated glycine receptor, 7‐chlorokynurenate (100 μM). 7‐Chlorokynuren‐ate by itself induced a drastic decrease in [3H]5‐HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [3H]5‐HT release in both the presence or absence of Mg2+. Use of selective antagonists allowed differentiation of a strychnine‐sensitive glycine response (inhibition of [3H]5‐HT release) from a 7‐chlorokynurenate‐sensitive response (potentiation of NMDA‐evoked [3H]5‐HT release).
Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin‐Related Peptide
Authors: Kihara Tsuyoshi , Ikeda Masato , Miyazaki Hiroshi , Matsushita Akira
Published: October 2006

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin‐related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K+ concentrations. Increasing the K+ concentration in the perfusate from 4 to 15 or 17.5 mM did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 mM K+, dose‐dependent reductions of both basal and MFB‐stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB‐stimulated DA release with 4 or 15 mM K+ in the perfusate. However, after perfusion with 17.5 mM K+, ceruletide significantly attenuated the basal and MFB‐stimulated DA release. Carbachol (10 μM) locally applied via the dialysis probe also attenuated MFB‐stimulated DA release after perfusion with 17.5 mM K+. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.
Pertussis Toxin Enhances Proenkephalin Synthesis in Bovine Chromaffin Cells
Authors: Wilson Steven P.
Published: October 2006

Abstract: The synthesis of the neuropeptide precursor proenkephalin was measured in bovine adrenal Chromaffin cells following radiolabeling with [35S]methionine. Treatment of Chromaffin cells with pertussis toxin (100 ng/ml) approximately doubled proenkephalin synthesis without altering total protein synthesis. Pertussis toxin pretreat‐ment also increased proenkephalin synthesis in chromaf‐fin cells exposed to vasoactive intestinal peptide (VIP) and 3‐isobutyl‐1 ‐methylxanthine (IBMX). Combinations of IBMX plus nicotine, VIP, or histamine also synergistically enhanced proenkephalin synthesis, with no further elevation when the cells were also pretreated with pertussis toxin. The action of forskolin, a direct activator of adenyl‐ate cyclase, on proenkephalin synthesis was similarly potentiated by pertussis toxin or IBMX, presumably reflecting the abilities of both the toxin and this phosphodiester‐ase inhibitor to enhance the cyclic AMP response to forskolin. In contrast, increased synthesis of proenkephalin in response to phorbol esters was not affected by pertussis toxin treatment. These results suggest that pertussis toxin potentiates proenkephalin synthesis primarily through inactivation of guanine nucleotide‐binding proteins that inhibit adenylate cyclase, although other signaling pathways may also be involved.
Prenatal Ethanol Exposure: Changes in Regional Brain Catecholamine Content Following Stress
Authors: Rudeen P. Kevin , Weinberg Joanne
Published: October 2006

Abstract: Previous studies have shown that fetal ethanol exposure (FEE) may have long‐term effects on the function of catecholaminergic neurons in different regions of the CNS. The present study is the first to examine the effects of FEE on regional brain catecholamine responses following acute stress (a single 60‐min restraint stress), repeated stress (single periods of restraint stress on 1, 5, or 10 consecutive days), and recovery from stress (recovery for up to 60 min in the home cage following a single 60‐min period of restraint stress). Both male and female offspring from FEE, pair‐fed (PF), and ad libitum‐fed control (C) groups were tested in adulthood to determine catecholamine content in the cortex, hypothalamus, and hippocampus. A single period of restraint reduced cortical norepinephrine (NE) content in FEE and PF animals compared with that in the cortex of C animals, and reduced hypothalamic NE content in FEE female offspring below that found in animals in all other groups. In contrast, hippo‐campal NE content was higher in FEE than in C animals following a single period of restraint; PF animals had intermediate levels of hippocampus NE and did not differ significantly from either FEE or C animals. Following repeated periods of restraint, cortical NE content was lower in FEE than in C animals; PF animals once again had intermediate levels of NE. Importantly, basal (non stressed) NE content did not differ among groups in any brain area examined. In addition, several significant changes in regional brain catecholaminergic responses to acute stress were observed in animals across all treatment groups. Females generally had significantly lower cortical NE levels than males following both single and multiple exposures to restraint. In addition, the cortical NE content decreased below non‐stressed levels in all groups following a single restraint period, and remained significantly below basal levels during the 60‐min recovery period, whereas the hypothalamic NE content was significantly decreased immediately following the restraint period but showed some recovery toward basal levels by 60 min. There were no significant changes over time in hippocampal NE level or in cortical or hypothalamic dopamine (DA) content following a single restraint stress. Following multiple periods of restraint, hippocampal NE levels were significantly increased and hypothalamic DA levels were significantly decreased in all animals compared with basal levels. These data suggest that the brain noradrenergic response to acute stress is particularly sensitive to the effects of FEE, and that with regard to the hypothalamus, male and female offspring were differentially affected. Furthermore, nutritional effects appear to play some role in mediating the changes in regional brain catecholamine content that are observed. In addition, stress effects on brain catecholamine content across all treatment groups were found to be both region and sex specific.
Stress‐Induced Dopamine Release in the Neostriatum: Evaluation of the Role of Action Potentials in Nigrostriatal Dopamine Neurons or Local Initiation by Endogenous Excitatory Amino Acids
Authors: Keefe Kristen A. , Sved Alan F. , Zigmond Michael J. , Abercrombie Elizabeth D.
Published: October 2006

Abstract: It has been hypothesized that excitatory amino acids can initiate dopamine release in neostriatum. We examined whether the increase in extracellular dopamine in neostriatum produced by acute stress reflects presynaptic initiation of dopamine release by endogenous excitatory amino acids. Thirty minutes of intermittent tail‐shock stress significantly elevated extracellular concentrations of dopamine, glutamate, aspartate, and γ‐aminobutyric acid in neostriatum of freely moving rats as measured with in vivo microdialysis. Local infusion of the N‐methyl‐d‐aspartate receptor antagonist 2‐amino‐5‐phosphonovaler‐ate or the non‐N‐methyl‐d‐aspartate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione via the dialysis probe did not attenuate the stress‐induced increase in extra cellular dopamine. In fact, the increase was prolonged in rats treated with specific excitatory amino acid receptor antagonists. Infusion of tetrodotoxin into medial forebrain bundle increased extra cellular glutamate and aspartate in neostriatum yet reduced basal dopamine in extra cellular fluid to below the limit of detection of the assay and eliminated the stress‐induced increase in extra cellular dopamine. These findings fail to support the hypothesis that the stress‐induced increase in extra cellular dopamine in neostriatum is initiated locally by excitatory amino acids. Rather, the effects of stress on extra cellular dopamine seem to be determined by impulse propagation in dopamine neurons.
Rapid Communication: ras‐Independent Induction of Rat Brain Type II Sodium Channel Expression in Nerve Growth Factor‐Treated PC 12 Cells
Authors: Fanger Gary R. , Erhardt Peter , Cooper Geoffrey M. , Maue Robert A.
Published: October 2006

Abstract— Nerve growth factor (NGF) plays an important role in the development of the nervous system, and there is considerable interest in understanding the molecular mechanisms underlying its effects on neuronal differentiation. To determine if the activity of proteins of the ras gene family is necessary for the NGF‐mediated induction of sodium channel expression in pheochromocytoma (PC12) cells, sodium channel expression was analyzed in PC12 sublines stably overexpressing the dominant inhibitory mutant c‐Ha‐ras(Asn‐17). Northern blot analysis, RNase protection assays, and whole‐cell patch clamp recordings indicate that the NGF‐mediated increase in type II sodium channel mRNA and sodium current density can occur independent of ras activity and by doing so provide strong evidence for the importance of ras‐independent mechanisms in NGF‐mediated neuronal differentiation.
Domain Poisoning: The Redundancy of Current Models of Assessment through Art
Authors: Hardy Tom
Published: October 2006

With the National Foundation for Educational Research concluding that schools which include Contemporary Art Practice (CAP) in their curriculum add significant value to their students' art experience, [1] and at a time when much of the discussion around contemporary art questions the value of the art object itself, this article addresses the question: how are we to engage students with the contemporary and, at the same time, make value judgments of their own work?

And, while the professional fine art world subscribes increasingly to the ‘rhizomatic’ [2] template of art processes, how do we square this with current assessment criteria which require that students produce work where the preparation and finished product occupy separate domains and rely on ‘procedures and practices that reach back to the nineteenth century’? [3] By way of a postscript to the inconclusive findings of the Eppi‐centre art and design review group [4], this article will also address what we have lost in the drive for domain‐based assessment and how to regain some of the ground lost since the introduction of Curriculum 2000.
Art Education and the Visual Arts in Botswana
Authors: Brennan Geraldine
Published: October 2006

The history of art and design education in Botswana has evolved in a unique way and reflects its British colonial history and post‐independence development. It has involved constant exchange and dialogue with other countries through the employment of teachers, teacher trainers and university lecturers from a variety of European, Asian and other African countries. This dialogue has continued as locally trained art teachers pursue their degrees overseas and return with new ideas and influences. At the same time, the development of local crafts for a global market and the inter‐cultural exchange in the visual arts outside the formal education system are thriving with the help of various organisations and the museum and gallery sector.

This article will look more closely at the country context, the history and development of art education and the interrelationships that have evolved over the last two decades. It will show how students engage with the challenge of integrating their African heritage and changing traditions with their development as teachers and artists. It will also consider how closer links between the formal and informal art sectors might be mutually beneficial and demonstrate the potential for art and design to play a role in the social, economic and cultural development of the country.
Raising Pupils' Self‐Esteem through Leadership Activities in Art
Authors: Hickman Richard
Published: October 2006

This article is about the ways in which young people who have disengaged from learning in school can find a way back through leadership activities in art. It is based on a project which was funded by a small grant from the Wallenberg Foundation. The project explored the potential of an approach to developing positive leadership qualities in pupils who were not consistently committed to the school's learning purposes. This account describes and comments upon two pupils' guided attempts at peer teaching in art and its subsequent effect upon their selfesteem and attitude towards school. It was found that pupils who taught art to other pupils had an increased sense of self‐worth and were more positively affected towards learning. However, broader issues, such as the negative nature of some school systems and their role in de‐motivating pupils were highlighted.
Complexity of Anti‐immunosenescence Strategies in Humans
Authors: Capri Miriam , Monti Daniela , Salvioli Stefano , Lescai Francesco , Pierini Michela , Altilia Serena , Sevini Federica , Valensin Silvana , Ostan Rita , Bucci Laura , Franceschi Claudio
Published: October 2006

Abstract:  Immunosenescence is characterized by three main aspects: (i) the shrinkage of the T cell repertoire and the accumulation of oligoclonal expansions (megaclones) of memory/effector cells directed toward ubiquitary infectious agents; (ii) the involution of the thymus and the exhaustion of naïve T cells; and (iii) a chronic inflammatory status called inflamm‐aging. We present here possible strategies to counteract these main aspects of immunosenescence in humans with particular attention to the reduction of antigenic load by pathogens, such as CMV, and the normalization of intestinal microflora, the possible utilization of IL‐7 to reverse thymic involution, the purging of megaclones, the forced expression of CD28 on T lymphocytes, the reduction of inflamm‐aging and the administration of nutrients such as vitamin D. Possible drawbacks of all these strategies are discussed. Finally, the complexity of a rejuvenation approach is stressed, with particular attention to the inhibitory role played by the “old microenvironment” on the performance of progenitor cells, the best candidate to counteract the decline in regenerative potential characteristic of organs and tissues from old organisms.
Reversibility of the Differentiated State: Regeneration in Amphibians
Authors: Straube Werner L. , Tanaka Elly M.
Published: October 2006

Abstract:  In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, tail, eye lens, or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, dermis, and cartilage. In the case of multinucleated skeletal muscle fibers, cell cycle reentry into S‐phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo.