bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification, Purification, and Characterization of the Molecular Forms of Aplysia californica Peptidylglycine α‐Amidating Enzyme
Authors: Boudreault Alain , Castellucci Vincent F. , Chrétien Michel , Lazure Claude
Published: July 2010

Abstract: Peptidylglycine α‐amidating enzyme (PAM; EC 1.14.17.3) is responsible for the conversion of peptides with a COOH‐terminal glycine into α‐amidated peptides, a posttranslational modification often required for biological activity and/or increased stability. Such an activity able to convert the model peptide d‐Tyr‐Val‐Gly into d‐Tyr‐Val‐amide was found to be present in the marine mollusk Aplysia californica. Examination of this amidating activity as well as its immunoreactivity demonstrates that (1) it can be found mainly in the atrial gland, heart, and CNS but is barely detectable in the hepatopancreas and gonads, (2) it requires as essential cofactors copper, molecular oxygen, and ascorbate, and (3) it exists in at least two molecular forms, a soluble and a membrane‐bound form. Purification of this activity from the atrial gland was accomplished using Cu2+‐chelating Sepharose, gel permeation, and hydroxyapatite chromatography. In addition, using polyclonal antibodies raised against various parts of the rat amidating enzyme, we demonstrate that numerous immunologically recognized regions are conserved in both the soluble and membrane‐bound Aplysia californica PAM.
NMR Studies of Pi‐Containing Extracellular and Cytoplasmic Compartments in Brain
Authors: Fitzpatrick James H. , Kintner Douglas , Anderson Mark , Westler William M. , Emoto Sherrie E. , Gilboe David D.
Published: July 2010

Abstract: The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt‐Levenberg (M‐L) nonlinear curve fit algorithm software (Peak‐Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal‐to‐noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M‐L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three‐compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10–35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high‐energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2‐deoxyglucose (2‐DG), 2‐DG‐6‐phosphate (2‐DG‐6‐P) would appear in the PME portion of the spectrum ionized according to pHi. Four 2‐DG‐6‐P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces.
Stimulation of an Insulin Receptor Activates and Down‐Regulates the Ca2+‐Independent Protein Kinase C, Apl II, Through a Wortmannin‐Sensitive Signaling Pathway in Aplysia
Authors: Sossin Wayne S. , Chen Chin‐Sih , Toker Alex
Published: July 2010

Abstract: Activation of tyrosine kinase‐linked receptors has been shown to stimulate Ca2+‐independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase‐linked receptor in these cells persistently activated and down‐regulated the Ca2+‐independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down‐regulate the Ca2+‐activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3‐kinase because (a) diC16phosphatidylinositol 3,4,5‐trisphosphate, a synthetic phosphoinositide 3‐kinase product, stimulated autophosphorylation of baculovirus‐expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3‐kinase, blocked the activation and down‐regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase‐linked receptors may mediate some of their effects in neurons through activation of Ca2+‐independent protein kinase C isoforms.
Regulation of Tryptophan Hydroxylase by Cyclic AMP, Calcium, Norepinephrine, and Light in Cultured Chick Pineal Cells
Authors: Florez Jose C. , Takahashi Joseph S.
Published: July 2010

Abstract: The level of 35S incorporation into tryptophan hydroxylase (TPH) shows a circadian rhythm in cultured chick pineal cells. The TPH oscillation peaks in the early subjective night, persists in constant darkness, and can be phase shifted by light, in parallel to the effect of these treatments on melatonin synthesis. Using quantitative two‐dimensional polyacrylamide gel electrophoresis, we have examined the regulation of TPH by agents known to affect melatonin synthesis in the chick pineal. We report here that 35S incorporation into TPH is induced by cyclic AMP and calcium, and partially inhibited by acute exposure to light. Cyclic AMP also causes a proportional increase in the radiolabeling of one of the TPH isoforms and a concomitant decrease in another isoform, possibly reflecting a change in the phosphorylation state of TPH. This effect is reversed by treatments known to reduce intracellular cyclic AMP levels in the chick pineal. Cyclic AMP thus appears to be involved in both translational and posttranslational processes regulating the expression of TPH in chick pineal cells.
Matrix Metalloproteinases in the Neocortex and Spinal Cord of Amyotrophic Lateral Sclerosis Patients
Authors: Lim Giselle P. , Backstrom Jon R. , Cullen Michael J. , Miller Carol A. , Atkinson Roscoe D. , Tökés Zoltán A.
Published: July 2010

Abstract: Matrix metalloproteinases (MMPs) were analyzed by immunohistochemistry and zymography in amyotrophic lateral sclerosis (ALS) and control brain and spinal cord specimens. Three major bands of enzyme activity (70, 100, and 130 kDa) were consistently observed and were subsequently identified as MMP‐2 (70 kDa; also known as EC 3.4.24.24 or gelatinase A) and MMP‐9 (100 and 130 kDa; also known as EC 3.4.24.35 or gelatinase B). Immunohistochemical studies established the presence of MMP‐2 in astrocytes and MMP‐9 in pyramidal neurons in the motor cortex and motor neurons in the spinal cord of ALS patients. Although a significant decrease in MMP‐2 activity was noticed in the ALS motor cortex, statistically significant increases in MMP‐9 (100‐kDa) activity were observed in ALS frontal and occipital cortices (BA10 and 17) and all three spinal cord regions when compared with control specimens. The highest MMP‐9 (100‐kDa) activities in ALS were found in the motor cortex and thoracic and lumbar cord specimens. The abnormally high amount of MMP‐9 and its possible release at the synapse may destroy the structural integrity of the surrounding matrix, thereby contributing to the pathogenesis of ALS.
High Levels of Glycine and Serine as a Cause of the Seizure Symptoms of Cavernous Angiomas?
Authors: Von Essen Claes , Rydenhag Bertil , Nyström Britta , Mozzi Rita , Van Gelder Nico , Hamberger Anders
Published: July 2010

Abstract: Cavernous angiomas are vascular malformations that cause neurodegeneration and symptoms including epileptiform seizures, headache, and motor deficits. Following neurosurgical removal of the angiomas, patients mostly recover well and become seizure‐free. This study reports on the levels of certain amino acids in angiomas, obtained from 13 patients. Distinct zones of the angiomas were analyzed, from the thrombotic core, via gliotic, hemosiderin‐infiltrated intermediate zones, to a periphery without macroscopic abnormalities. The neurotransmitter amino acids glutamate, aspartate, and GABA as well as phosphoethanolamine displayed decreasing levels from the periphery to the core, reflecting the gradual neuronal loss. Compared with normal brain tissue, there was a marked increase in the levels of serine (fivefold), glycine (10‐fold), and ethanolamine (20‐fold) in the peripheral zone of the cavernous angiomas. The results are discussed in relation to seizures and NMDA receptor activation, neuron‐glia interactions, membrane phospholipids, and blood‐brain barrier function.
N‐Acetylsuccinimidylglutamate, a Cyclic Imide Form of the Peptide N‐Acetylaspartylglutamate, Is Present in Low Micromolar Concentrations in Murine and Bovine CNS
Authors: Brovia Verónica , Costa Alicia , Barbeito Luis
Published: July 2010

Abstract: N‐Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N‐acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the α‐ and β‐carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 ± 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 ± 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amounts in the superior colliculus and lateral genicutale nucleus (1.17 ± 0.05 and 0.82 ± 0.13 pmol/mg wet weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions (r = 0.74, p < 0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)‐NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.
ATP‐Activated Nonselective Cation Current in NG108‐15 Cells
Authors: Kaiho Hiromi , Kimura Junko , Matsuoka Isao , Kumasaka Tadanori , Nakanishi Hironori
Published: July 2010

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108‐15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1‐[6‐[[17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino]hexyl]‐1H‐pyrrole‐2,5‐dione (U73122), whereas the sustained increase was abolished by removal of external Ca2+. We examined the mechanism of the ATP‐elicited sustained [Ca2+]i increase using the fura‐2 fluorescent method and the whole‐cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC50 of ATP was ∼0.75 mM. The permeability ratio of various cations carrying this current was Na+ (defined as 1) > Li+ (0.92 ± 0.01; n = 5) > K+ (0.89 ± 0.03; n = 6) > Rb+ (0.55 ± 0.02; n = 6) > Cs+ (0.51 ± 0.01; n = 5) > Ca2+ (0.22 ± 0.03; n = 3) > N‐methyl‐d‐glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP‐induced current was larger at lower concentrations of external Mg2+. ATP analogues that induced the current were 2‐methylthio‐ATP (2MeSATP), benzoylbenzoic‐ATP, adenosine 5′‐thiotriphosphate (ATPγS), and adenosine 5′‐O‐(2‐thiodiphosphate), but not adenosine, ADP, α,β‐methylene‐ATP (AMPCPP), β,γ‐methylene‐ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca2+]i increase. We conclude that ATP activates a class of Ca2+‐permeable nonselective cation channels via the P2z receptor in NG108‐15 cells.
Adrenergic Regulation of ICER (Inducible Cyclic AMP Early Repressor) and β1‐Adrenergic Receptor Gene Expression in C6 Glioma Cells
Authors: Fitzgerald Laura Rydelek , Li Zhongwei , Machida Curtis A. , Fishman Peter H. , Duman Ronald S.
Published: July 2010

Abstract: ICER (inducible cyclic AMP early repressor), a member of the cyclic AMP response element (CRE) modulator (CREM) family of transcription factors, is a powerful repressor of cyclic AMP‐mediated transactivation. Our studies characterize the regulation of ICER in C6 glioma cells and investigate its role in repressing transcription of the β1‐adrenergic receptor (β1AR) gene. Incubation with isoproterenol (100 nM) results in a rapid induction in levels of mRNA for ICER and its splice variant ICERγ, with maximal induction occurring after 2 h of treatment. Incubation with isoproterenol also increased levels of CREM isoforms within 1 h; this was unexpected given previous reports that these isoforms are not rapidly induced. Increased expression of ICER and CREM was accompanied by induction of two CRE‐binding complexes. The presence of ICER in these two CRE‐binding complexes is demonstrated by their disruption with CREM antibody and by their comigration with recombinant ICER. Because the time course for isoproterenol induction of ICER mRNA and CRE binding corresponds to that for down‐regulation of β1AR mRNA levels in C6 glioma cells, the influence of ICER on β1AR transcription was directly examined. Coexpression of ICER significantly decreased transcriptional activity of a rat β1AR promoter‐luciferase reporter construct that contains a CRE. In contrast, coexpression of ICER did not influence two truncated rat β1AR promoter constructs that lack the CRE site. These data demonstrate that ICER can interact at the β1AR promoter to repress transcription.
Rapid Stimulation of EAAC1‐Mediated Na+‐Dependent l‐Glutamate Transport Activity in C6 Glioma Cells by Phorbol Ester
Authors: Dowd Lisa A. , Robinson Michael B.
Published: July 2010

Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1‐mediated Na+‐dependent l‐[3H]glutamate transport. Although a 30‐min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12‐myristate 13‐acetate (PMA) increased transport activity two‐ to threefold. PMA caused a time‐dependent and concentration‐dependent increase in EAAC1‐mediated l‐[3H]glutamate transport activity. A 2‐min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5‐fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, d‐[3H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na+‐dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α‐phorbol 12,13‐didecanoate, did not stimulate l‐[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride‐sensitive Na+/H+ transport activity in C6 glioma. In the present study, pre‐ and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1‐mediated transport activity. This rapid increase in Na+‐dependent l‐[3H]‐glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.
Neuronal Release of Serotonin in the Cerebellum of Behaving Rats: An In Vivo Microdialysis Study
Authors: Mendlin Anna , Martín Francisco J. , Rueter Lynne E. , Jacobs Barry L.
Published: July 2010

Abstract: Release of endogenous serotonin [5‐hydroxy‐tryptamine (5‐HT)] in the cerebellum of awake rats was characterized using in vivo microdialysis. 5‐HT output was increased (∼70%) by local application of KCl (100 mM) and was reduced (∼60%) by both tetrodotoxin (0.5 µM) and omission of Ca2+ from the perfusion fluid. 5‐HT release was decreased (∼70%) by the selective 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (0.25 mg/kg, s.c.), and this effect was rapidly reversed by a selective 5‐HT1A antagonist, N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl)cyclohexane‐carboxamide trihydrochloride (WAY‐100635; 0.1 mg/kg, i.p.). These results indicate that a large portion of the measurable 5‐HT output in the cerebellum is of neuronal origin, is dependent on impulse flow, and is sensitive to 5‐HT1A autoreceptor activation. Further studies examined the relationship between 5‐HT levels and general activity of the animals across the light‐dark transition and during behavioral manipulations. Both 5‐HT levels and behavioral activity were significantly elevated during the dark period, with changes in 5‐HT efflux closely paralleling changes in activity. Similar increases (∼40%) in 5‐HT output were observed during both feeding and feeding in the presence of a stressor (tail pinch). These findings suggest that behavioral state is an important factor determining neuronal 5‐HT release in cerebellum under physiological conditions.
Serotonin Transporters in the Rat Frontal Cortex: Lack of Circadian Rhythmicity but Down‐Regulation by Food Restriction
Authors: Zhou Dan , Huether Gerald , Wiltfang Jens , Hajak Göran , Rüther Eckart
Published: July 2010

Abstract: Molecular biological findings have indicated that the affinity and the density of presynaptic serotonin transporters may be subject to adaptive regulation, but the physiological conditions that may act to trigger such changes are presently unknown. By means of [3H]paroxetine binding to rat cortical membranes, we studied the influence of two physiological variables that are clearly associated with altered serotonergic activity—circadian rhythm and semistarvation—on KD and Bmax values of the serotonin transporter of the rat frontal cortex. No circadian fluctuations of both parameters were observed. Also, semistarvation (50% reduction of normal voluntary food intake) for 2 days had no effect on either KD or Bmax values of cortical [3H]paroxetine binding. Food restriction for either 7 days or 2 weeks, however, resulted in a significant, ∼30%, reduction of the density of cortical serotonin transporters with unchanged transporter affinity. These findings indicate that long‐term changes in the density of cortical serotonin transporters can be induced by long‐lasting alterations of certain environmental variables. Because the duration and the radius of action of presynaptically released serotonin are governed by the efficiency of the reuptake mechanism, such adaptive changes of serotonin transporter density must be expected to cause long‐term alterations of the modulatory impact of the central serotonin system on certain brain functions.
Quantitative Analysis of Similarities and Differences in Neurotoxicities Caused by Adenosine and 2′‐Deoxyadenosine in Sympathetic Neurons
Authors: Kulkarni Jayant S. , Wakade Arun R.
Published: July 2010

Abstract: These experiments characterize the nucleoside transport and quantify the neurotoxicity of adenosine and 2′‐deoxyadenosine (dAdo) in chick sympathetic neurons. We show that [3H]adenosine transport was sensitive to low temperature, specific inhibitors of nucleoside transport, and an excess concentration of adenosine. However, many of these treatments had a marginal effect on [3H]dAdo transport. Total retention of [3H]dAdo over short and long periods was ∼10 times less than that of [3H]adenosine. These data suggest that adenosine and dAdo enter sympathetic neurons by different routes. Uptake of [3H]norepinephrine ([3H]NE) decreased in neurons damaged by nucleosides and increased to control levels when neurons were protected by various agents against adenosine or dAdo toxicity. These results indicate that [3H]NE uptake serves as a quantitative index of toxicity by the nucleosides. Using this approach we demonstrate that phosphorylation of both nucleosides is essential for their lethal action. For example, iodotubercidin prevented nucleoside‐induced neuronal death, but the effect was much more pronounced in the case of dAdo toxicity (IC50 of 0.83 ± 0.4 vs. 30 ± 1.6 nM). Another kinase inhibitor, 5′‐amino 5′‐deoxyadenosine, was effective in protecting neurons against dAdo but had no effect against adenosine toxicity. These results suggest that specific kinases are associated with the phosphorylation of adenosine and dAdo in sympathetic neurons to produce toxic metabolic products. Finally, neurons were susceptible to dAdo toxicity from the time of plating to 4 weeks in culture but were resistant to adenosine toxicity 8 h after plating. In conclusion, our results highlight major differences in the mechanism of neurotoxicity by adenosine and dAdo and provide insights for identification of biochemical pathways leading to neuronal death.
Expression and Deletion Mutagenesis of Tryptophan Hydroxylase Fusion Proteins: Delineation of the Enzyme Catalytic Core
Authors: D'Sa Carrol M. , Arthur Robert E. , Kuhn Donald M.
Published: July 2010

Abstract: cDNAs encoding the full‐length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1–98) or catalytic (amino acids 99–444) domains of the enzyme, were cloned and expressed as glutathione S‐transferase fusion proteins in E. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione‐agarose. The full‐length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full‐length enzyme and the catalytic core, while adsorbed to glutathione‐agarose beads, obeyed Michaelis‐Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S‐transferase‐tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full‐length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99–444 of the full‐length enzyme, contains the sequence motifs needed for subunit assembly. Both wild‐type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full‐length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.
Three Dynamin‐Encoding Genes Are Differentially Expressed in Developing Rat Brain
Authors: Cook T. , Mesa K. , Urrutia R.
Published: July 2010

Abstract: Dynamin proteins are members of a recently described family of GTPases involved in receptor‐mediated processes. To date, three different dynamin‐encoding genes have been identified in mammalian tissues. Dynamin I is expressed only in neurons, whereas dynamin II is ubiquitously expressed. A third isoform, dynamin III, was originally isolated from a rat testis cDNA library and shown to be testis‐specific. However, here we report the cloning and characterization of dynamin III from brain and lung, demonstrating a more extended pattern of expression for this isoform. In addition, we have investigated the temporal pattern of expression of these three genes during brain development. We find that both dynamin I and dynamin III mRNA levels are up‐regulated during embryogenesis, whereas dynamin II mRNA levels remain unchanged. From these results, we conclude that dynamin III is not a testis‐specific isoform and, furthermore, that rat brain expresses three different dynamin‐encoding genes that are differentially regulated during development. Therefore, this large isoform diversity of dynamin proteins in brain predicts a significant complexity in the understanding of dynamin‐based processes in this tissue.
Neurotrophin‐3 and Brain‐Derived Neurotrophic Factor Activate Multiple Signal Transduction Events but Are Not Survival Factors for Hippocampal Pyramidal Neurons
Authors: Marsh H. Nicholas , Palfrey H. Clive
Published: July 2010

Abstract: Expression of the neurotrophin‐3 (NT‐3) receptor (TrkC) and the effects of NT‐3 on signal transduction were investigated in highly enriched populations of embryonic rat hippocampal pyramidal neurons grown in bilaminar cultures. PCR analysis revealed that the predominant trkC isoform is K1, which lacks an insert in the kinase domain. Polyclonal TrkC‐specific antibodies stained >90% of the neurons and revealed a single ∼145‐kDa protein in immunoblots of extracts from adult hippocampus and pyramidal neuron cultures. Addition of NT‐3 (50 mg/ml) to these cultures induced the tyrosine phosphorylation of TrkC but not TrkB, as determined by anti‐phosphotyrosine staining of immunoprecipitates; thus, all the effects of NT‐3 are mediated through TrkC. NT‐3 also increased the tyrosine phosphorylation of 42‐, 44‐, 49‐, 55‐, 95‐, and 145‐kDa proteins; the pattern induced by brain‐derived neurotrophic factor (BDNF) was similar but not identical to that induced by NT‐3, suggesting that subtle differences may exist in signaling by TrkB and TrkC receptors. Immunoprecipitation of p21ras from 32P‐prelabeled cells showed that NT‐3 increased the level of the GTP‐bound form of the protein threefold over the control within 5 min. Mitogen‐activated protein (MAP) kinase activity was maximally elevated by NT‐3 within 2 min and then returned slowly toward baseline over the next 60 min. Tyrosine phosphorylation of phospholipase C‐γ increased rapidly after NT‐3, suggesting that this enzyme becomes activated. Consistent with this, the neurotrophin rapidly increased protein kinase C activity as well as intracellular Ca2+ levels. The effects of both NT‐3 and BDNF on Ca2+ levels were attenuated in Ca2+‐free medium, suggesting that both neurotrophins increase Ca2+ flux across the plasma membrane as well as release from internal stores. NT‐3 also increased c‐Fos expression in >80% of the cells; the effect peaked at 30 minand declined to baseline by 120 min. Despite the activation of ras‐MAP kinase and phosphoinositide signaling pathways, neither NT‐3 nor BDNF alone or in combination could sustain hippocampal pyramidal neurons deprived of glial support. We conclude that in this system NT‐3 and BDNF do not appear to be acting as classical “neurotrophic” factors and that activation of the MAP kinase pathway is insufficient for the promotion of neuronal survival.
Cultured Leptomeningeal Cells Secrete Cerebrospinal Fluid Proteins
Authors: Ohe Yoshihide , Ishikawa Koichi , Itoh Zen , Tatemoto Kazuhiko
Published: July 2010

Abstract: To extrapolate the function of the leptomeninges, we examined the profile of the proteins secreted from the cultured leptomeningeal cells prepared from 1–2‐day‐old rats. In sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the medium conditioned with the cultured cells, 20–25 differentially distinctive protein bands were noted. Through several chromatographic procedures (Sephadex G‐75, Mono Q, and 7C8‐300), altogether 18 proteins were purified to homogeneity, and the partial amino acid sequence of each protein was determined. Homology search revealed that the major proteins included prostaglandin‐d‐synthase or β‐trace protein, insulin‐like growth factor (IGF)‐II, IGF‐binding protein‐2, apolipoprotein E, β2‐microglobulin, cystatin C, transferrin, peptidyl‐prolyl cis‐trans isomerase or cyclophilin C, secreted protein acidic and rich in cysteine, ubiquitin, lysozyme C, extracellular superoxide dismutase, and collagen α‐1 (III). Most of these proteins are known to be the major brain‐derived protein constituents of CSF and are thought to play important roles in certain biological events in the brain. Considering the morphological features, the present findings suggest the importance of the leptomeninges as an origin of such proteins in CSF.
Characterization of Inhibitor‐Sensitive and ‐Resistant Adenosine Transporters in Cultured Human Fetal Astrocytes
Authors: Gu J. G. , Nath A. , Geiger J. D.
Published: July 2010

Abstract: The kinetic characteristics of [3H]adenosine uptake, the extent to which accumulated [3H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis‐Menten kinetic values of KT and Vmax, and the sensitivities with which nucleoside transport inhibitors blocked [3H]adenosine accumulations were determined in cultured human fetal astrocytes. KT and Vmax values for accumulations of [3H]‐labeled purines using 15‐s incubations in the absence of the adenosine deaminase inhibitor erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′‐iodotubercidin (ITU) were 6.2 µM and 0.15 nmol/min/mg of protein for the high‐affinity and 2.6 mM and 21 nmol/min/mg of protein for the low‐affinity components respectively. In the presence of EHNA and ITU, where <4% of accumulated [3H]adenosine was metabolized, transport per se was measured, and kinetic values for KT and Vmax were 179 µM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [3H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [3H]‐labeled purines (adenosine plus its metabolites) were 1.4‐fold higher than in the medium. No apparent concentrative accumulations of [3H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [3H]adenosine transport; for the inhibitor‐sensitive components the IC50 values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor‐resistant component the IC50 values were 2.5 µM for NBI, 5.1 µM for dilazep, and 39.0 µM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor‐sensitive and ‐resistant adenosine transporters in nontransformed human cells.
Physiologic Levels of β‐Amyloid Activate Phosphatidylinositol 3‐Kinase with the Involvement of Tyrosine Phosphorylation
Authors: Luo Y. , Sunderland T. , Wolozin B.
Published: July 2010

Abstract: The β‐amyloid protein (Aβ) peptide plays an important role in Alzheimer's disease, but the potential actions of physiologic levels of Aβ (225–625 pM) have not been explored. We recently showed that picomolar doses of Aβ can stimulate tyrosine phosphorylation of neuronal cells and now show that leads to the activation of the lipid kinase phosphatidylinositol 3‐kinase (PI3 kinase). Three independent lines of evidence support the hypothesis that Aβ is activating PI3 kinase through a tyrosine kinase‐mediated mechanism. Immunoblotting studies show that Aβ induces tyrosine phosphorylation of p85 as well as association of the p85 subunit of PI3 kinase with tyrosine‐phosphorylated proteins. Studies of membrane proteins show that Aβ induces a translocation of p85 to membrane‐bound glycoproteins, which are likely to be receptors. Finally, direct studies of PI3 kinase activity in both anti‐phosphotyrosine immunocomplexes and wheat germ agglutinin precipitates show that Aβ increases formation of the product of PI3 kinase. Wortmannin, a selective inhibitor of PI3 kinase, blocks this Aβ‐stimulated PI3 kinase activity. Thus, physiologic levels of Aβ stimulate tyrosine phosphorylation and PI3 kinase activity.
Extracellular Calcium Has Distinct Effects on Fast and Slow Components of the Depolarization‐Induced Secretory Response from Chromaffin Cells
Authors: Granja R. , Izaguirre V. , Calvo S. , González‐García C. , Ceña V.
Published: July 2010

Abstract: An increase in extracellular Ca2+ concentration from 0.25 to 10 mM enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K+ concentration (75 mM). The increment in extracellular Ca2+ concentration also increased the observed peak inward Ca2+ current in response to long (10‐s) depolarizing pulses from a holding potential of −55 mV to +5 mV, from about −26 to −400 pA. However, the total amount of Ca2+ influx into the cell only increased when the extracellular Ca2+ concentration was raised from 0.25 to 1 mM and then remained constant up to 10 mM extracellular Ca2+. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 mM extracellular Ca2+, of ∼4 and 41 s, being the fast component of secretion produced by the exocytosis of ∼220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca2+ concentration.
Inactivation of Brain Tryptophan Hydroxylase by Nitric Oxide
Authors: Kuhn Donald M. , Arthur Robert E.
Published: July 2010

Abstract: Tryptophan hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S‐nitroso‐N‐acetylpenicillamine, and S‐nitrosocysteine. The inactivation occurs in an oxygen‐free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO‐induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron‐sulfur groups in this enzyme serve as the substrate for NO‐induced nitrosylation of the protein, resulting in enzyme inactivation.
Mechanism of Catecholamine Synthesis Inhibition by Neuropeptide Y: Role of Ca2+ Channels and Protein Kinases
Authors: McCullough Laura A. , Westfall Thomas C.
Published: July 2010

Abstract: We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization‐stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca2+ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m‐hydroxybenzylhydrazine (NSD‐1015). The L‐type Ca2+ channel blocker nifedipine inhibited the depolarization‐induced stimulation of DOPA production by ∼90% and attenuated the inhibitory effect of NPY. In contrast, the N‐type Ca2+ channel blocker ω‐conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca2+/calmodulin‐dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP‐dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca2+/phospholipid‐dependent protein kinase (PKC) with phorbol 12‐myristate 13‐acetate (PMA) did not affect the basal rate of DOPA production in NGF‐differentiated PC12 cells but did produce a concentration‐dependent inhibition of depolarization‐stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca2+ influx through L‐type voltage‐gated Ca2+ channels, possibly through a PKC‐mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization‐stimulated catecholamine synthesis.
Alterations in Serotonin Parameters in Brain of Thiamine‐Deficient Rats Are Evident Prior to the Appearance of Neurological Symptoms
Authors: Mousseau D. D. , Raghavendra Rao V. L. , Butterworth R. F.
Published: July 2010

Abstract: Biochemical alterations of serotoninergic parameters have been demonstrated in experimental thiamine deficiency. In addition, hypophagia and hypothermia, two physiological processes associated with changes in the serotonin [5‐hydroxytryptamine (5‐HT)] system, are manifest early during the progression of thiamine deficiency. The binding of selected 5‐HT radioligands was therefore investigated in discrete brain regions of pyrithiamine‐induced thiamine‐deficient rats. Using quantitative receptor autoradiography, the binding of 8‐hydroxy‐2‐(di‐n‐[3H]propylamino)tetralin, a ligand used to label the somatodendritic 5‐HT1A autoreceptor of the dorsal raphe nucleus, was found to be unaffected in this region, suggesting that the structural integrity of the 5‐HT cell bodies is maintained throughout the course of pyrithiamine treatment. Increased binding of [3H]‐ketanserin was observed in regions considered vulnerable as well as in some considered to be nonvulnerable during the course of thiamine deficiency. These binding changes, which appear to represent changes in the density of the postsynaptic 5‐HT2A receptor population rather than the “tetrabenazine‐sensitive” vesicular monoamine transporter, are evident before the appearance of histopathologic lesions and coincide with altered tissue concentrations of 5‐HT. These data suggest that 5‐HT neurons, although structurally intact, are functionally affected early during the progression of thiamine deficiency. These alterations, which are likely a part of adaptive neuronal change consequent to thiamine dysfunction, may be important in the physiological manifestations and the learning deficits commonly encountered in experimental thiamine deficiency.
Polymerization of τ into Filaments in the Presence of Heparin: The Minimal Sequence Required for τ ‐ τ Interaction
Authors: Pérez Mar , Valpuesta José M. , Medina Miguel , Montejo de Garcini Esteban , Avila Jesús
Published: July 2010

Abstract: Paired helical filaments isolated from the brains of patients with Alzheimer's disease are composed of a major protein component, the microtubule‐associated protein termed τ, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of τ into filamentous structures, we have analyzed the ability of the whole τ protein or some of its fragments to self‐assemble in the presence of heparin. Different τ fragments, all of them containing some sequences of the tubulin‐binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18‐residue‐long peptide corresponding to the third tubulin‐binding motif of τ. This suggests that the ability of τ for self‐assembly could be localized in a short sequence of amino acids present in the tubulin‐binding repeats of the τ molecule.
Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System
Authors: Yoshimura Takeo , Satake Marie , Kobayashi Takuro
Published: July 2010

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady‐state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0‐fold with 100 µM forskolin, whereas that of P0 increased 7.0‐fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti‐Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P0 expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P0, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.
l‐[3H]Nitroarginine and l‐[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology
Authors: Raghavendra Rao V. L. , Butterworth R. F.
Published: July 2010

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l‐arginine and its inhibitor, NG‐nitro‐l‐arginine (l‐NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l‐arginine and l‐NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l‐NOARG (650 pmol/mg of protein) was three to four times higher than that of l‐arginine (170 pmol/mg of protein). l‐NOARG uptake showed biphasic kinetics (Km 1 = 0.72 mM, Vmax 1 = 0.98 nmol/min/mg of protein; Km 2 = 2.57 mM, Vmax 2 = 16.25 nmol/min/mg of protein). l‐Arginine uptake was monophasic with a Km of 106 µM and a Vmax of 0.33 nmol/min/mg of protein. l‐NOARG uptake was selectively inhibited by l‐NOARG, NG‐nitro‐l‐arginine methyl ester, and branched‐chain and aromatic amino acids. l‐Alanine and l‐serine also inhibited l‐NOARG uptake but with less potency. Uptake of l‐arginine was selectively inhibited by NG‐monomethyl‐l‐arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l‐NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l‐arginine is transported by the basic amino acid carrier system y+ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l‐NOARG and l‐arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.
Novel Neonicotinoid‐Agarose Affinity Column for Drosophila and Musca Nicotinic Acetylcholine Receptors
Authors: Tomizawa Motohiro , Latli Bachir , Casida John E.
Published: July 2010

Abstract: Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high‐affinity binding site for [3H]IMI with KD values of 1–2 nM and Bmax values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α‐bungarotoxin (α‐BGT)‐agarose affinity column are known to be α‐subunit homooligomers. This study uses 1 ‐ [N ‐ (6 ‐ chloro ‐ 3 ‐ pyridylmethyl) ‐ N ‐ ethyl]amino ‐ 1 ‐ amino‐2‐nitroethene (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2–3 nM) to develop a neonicotinoid‐agarose affinity column. The procedure—introduction of Triton‐solubilized Drosophila or Musca head membranes into this neonicotinoid‐based column, elution with IMI, and analysis by lithium dodecyl sulfate‐polyacrylamide gel electrophoresis—gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the α‐BGT‐agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I‐α‐BGT‐4‐azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.
Modulation of Major Histocompatibility Complex Class I Genes by Interferon‐γ and Ganglioside GT1b in Astrocytes: Involvement of Protein Tyrosine Phosphatases
Authors: Massa Paul T. , Wu Charlene
Published: July 2010

Abstract: We have previously reported that the polysialoganglioside GT1b suppresses the induction of major histocompatibility complex class I molecules by interferon‐γ in astrocytes. Suppression by GT1b depended on the presence of sialic acid moieties because asialo‐GM1 was not suppressive. In the present report, GT1b was found to act transcriptionally to suppress class I genes because both the interferon‐γ induction of RNA and the activity of class I promoter constructs were inhibited. Furthermore, GT1b suppressed promoter activity through interferon regulatory factor elements, indicating an effect on the transcription activation factor, interferon regulatory factor 1. Interferon‐γ induced interferon regulatory factor 1 within 8 h, and GT1b suppressed this induction. The suppression of interferon regulatory factor 1 by GT1b correlated with the suppression of γ‐activated factor binding at the promoter of the interferon regulatory factor 1 gene. The suppression of γ‐activated factor by GT1b appeared to involve increased protein tyrosine phosphatase activity because treatment of the cells with pervanadate reversed the effect of GT1b on the γ‐activated factor and, correspondingly, phosphotyrosine content. In sum, GT1b displays specific effects on interferon‐γ signaling and negative feedback regulatory molecules in astrocytes.
Creation and Characterization of Mitochondrial DNA‐Depleted Cell Lines with “Neuronal‐Like” Properties
Authors: Miller Scott W. , Trimmer Patricia A. , Parker W. Davis , Davis Robert E.
Published: July 2010

Abstract: Mitochondrial dysfunction and attendant bioenergetic defects are increasingly recognized as playing an important role in neurodegenerative disorders. The increased attention on mitochondrial involvement points to the need for developing cell lines that have neuron‐like characteristics for the genetic analysis and modeling of these diseases. We describe the creation of respiratory‐deficient SH‐SY5Y neuroblastoma cell lines (ρ064/5) by selectively depleting mitochondrial DNA through prolonged exposure to ethidium bromide. Oxygen consumption in these cells and activities of the electron transport chain enzyme complexes I and IV that contain subunits encoded by the mitochondrial genome are eliminated. In contrast, the function of complex II, a nuclear‐encoded electron transport chain component, is largely intact in these cells. The ρ064/5 cells retain the ability to differentiate into cells with neuron‐like phenotypes following treatment with phorbol ester or retinoic acid. Normal respiratory function is recovered by repopulation of ρ064/5 cells with exogenous human platelet mitochondria. The ρ064/5 cell line serves as a valuable model for the study of neurologic diseases suspected of involving mitochondrial dysfunction.