bluuebunny/crossref_metadata_2025 huggingface dataset:
β‐Amyloid‐Induced Cell Toxicity: Enhancement of 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide‐Dependent Cell Death
Authors: Hertel C. , Hauser N. , Schubenel R. , Seilheimer B. , Kemp J. A.
Published: July 2010

Abstract: In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of β‐amyloid (Aβ) peptides have been widely studied. At high micromolar concentrations Aβ peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Aβ peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Aβ peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Aβ render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.
σ Receptor Modulation of Noradrenergic‐Stimulated Pineal Melatonin Biosynthesis in Rats
Authors: Steardo L. , Monteleone P. , D'Istria M. , Serino I. , Maj M. , Cuomo V.
Published: July 2010

Abstract: Because σ receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the σ‐receptor ligands 1,3‐di(2‐tolyl)guanidine and (+)‐N‐allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3‐di(2‐tolyl)guanidine and (+)‐N‐allylnormetazocine potentiated the enhancement of N‐acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3‐di(2‐tolyl)guanidine and (+)‐N‐allylnormetazocine enhanced significantly the physiological increases in both pineal N‐acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific σ‐receptor antagonist. These findings suggest that, in rats, the activation of pineal σ‐receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it pontentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.
Tetanus Toxin‐Induced Effects on Extracellular Amino Acid Levels in Rat Hippocampus: An In Vivo Microdialysis Study
Authors: Britton P. , Whitton P. S. , Fowler L. J. , Bowery N. G.
Published: July 2010

Abstract: Tetanus toxin is a potent neurotoxin that is widely considered to produce its effect through impairment of inhibitory neurotransmission. We report the effect of a single unilateral intrahippocampal injection of tetanus toxin on extracellular levels of neuroactive amino acids in freely moving rats, at times ranging between 1 and 7 days posttreatment. Tetanus toxin treatment did not alter extracellular levels of aspartate, glutamate, and taurine at any time during the study. However, although extracellular GABA levels were unaffected by toxin injection 1, 2, and 3 days after treatment, they were reduced (45 ± 8% of contralateral vehicle‐injected level) at day 7. Challenge with a high K+ concentration, 7 days after treatment, produced elevations in extracellular levels of taurine and GABA in both vehicle‐ and toxin‐injected hippocampi, with evoked levels of GABA being lower in the toxin‐treated side (39 ± 16% of contralateral vehicle‐injected level). Aspartate and glutamate levels were not increased by high‐K+ infusion. These findings are discussed in relation to the possible role that an imbalance in excitatory/inhibitory tone may play in the production of tetanus toxin‐induced neurodegeneration.
Evidence for the Induction of Repetitive Action Potentials in Synaptosomes by K+‐Channel Inhibitors: An Analysis of Plasma Membrane Ion Fluxes
Authors: Tibbs Gareth R. , Dolly J. Oliver , Nicholls David G.
Published: July 2010

Abstract: The effects of four K+‐channel inhibitors on synaptosomal free Ca2+ concentrations and 86Rb+ fluxes are analysed. 4‐Aminopyridine, α‐dendrotoxin, charybdotoxin, and tetraethylammonium all increase the free Ca2+ concentration, although their potencies differ widely. In each case, the elevation in free Ca2+ concentration is reversed by the subsequent addition of tetrodotoxin. The transient 86Rb+ efflux from preequilibrated synaptosomes induced with high concentrations of veratridine is partially inhibited by 4‐aminopyridine and α‐dendrotoxin. In contrast, when 4‐aminopyridine or α‐dendrotoxin is added to polarized synaptosomes, an enhanced86Rb+ flux is seen, both for uptake and for efflux with no change in the total 86Rb+/K+ content of the synaptosomes and with only a slight time‐averaged plasma membrane depolarization (6.4 and 3.3 mV, respectively). The enhancements of flux by 4‐aminopyridine or α‐dendrotoxin are sensitive to ouabain and/or to tetrodotoxin. Furthermore, these flux changes show the same concentration dependencies as the blocked component of veratridine‐stimulated 86Rb+ efflux, the elevation of free Ca2+ concentration, and the facilitation of glutamate exocytosis that are elicited by 4‐aminopyridine or α‐dendrotoxin. It is concluded that these findings support the proposal of spontaneous, repetitive firing of synaptosomes evoked by K+‐channel inhibitors and that the enhanced 86Rb+ flux is a consequence of the activity of 4‐aminopyridine‐ and α‐dendrotoxin‐insensitive K+ channels during these action potentials.
Kinetics of tert‐[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization
Authors: Giorgi Osvaldo , Orlandi Marzia , Lecca Daniele , Serra Giuliana P. , Zhang Lei , Corda Maria G.
Published: July 2010

Abstract: The effects of GABA on the kinetics of tert‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the convulsant site of GABAA receptors were studied in membrane suspensions from the cerebral cortex of newborn (1‐day‐old) and adult (90‐day‐old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [35S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast (t1/2, 11 min) and slow (t1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µM GABA, however, was more robust in neonates than in adults (six‐ to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µM GABA (dissociation started by adding 40 µM picrotoxin) were two‐ to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µM picrotoxin plus 2 µM GABA). Taken together, these results suggest that (1) the closed state of GABAA receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.
p‐Chlorophenylalanine Changes Serotonin Transporter mRNA Levels and Expression of the Gene Product
Authors: Rattray Marcus , Baldessari Sonia , Gobbi Marco , Mennini Tiziana , Samanin Rosario , Bendotti Caterina
Published: July 2010

Abstract: After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p‐chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down‐regulation of serotonin (5‐HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5‐HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [3H]‐citalopram binding and synaptosomal [3H]5‐HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by ∼20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [3H]citalopram binding to 5‐HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5‐HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5‐HT transporter mRNA are not temporally related to changes in 5‐HT transporter protein levels. In addition, our results suggest that the 5‐HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5‐HT neurons are differentially regulated by drugs, depending on their location.
Characterization of the Human Protease Nexin‐1 Promoter and Its Regulation by Sp1 Through a G/C‐Rich Activation Domain
Authors: Guttridge Denis C. , Cunningham Dennis D.
Published: July 2010

Abstract: Protease nexin‐1 (PN‐1) is a potent inhibitor of serine proteases in the extracellular environment. It is abundantly expressed in the nervous system, where it is thought to participate in local injury and repair processes. Although some information has been obtained regarding PN‐1 gene structure, relatively little is known about the cis‐ and trans‐acting factors that regulate its expression. Elucidation of these factors should provide a better understanding of PN‐1 function during development and wound repair. In this report we describe the characterization of the human PN‐1 promoter and identify regulatory domains and a transactivator mediating its transcriptional activity. The promoter is highly G/C rich proximal to the transcriptional start site. It exhibits tissue specificity and is negatively regulated by a silencer element upstream of position −480. A positive regulatory element was mapped between −199 and −45, which contains multiple putative Sp1 consensus binding sites. Electrophoretic mobility shift analysis confirmed that Sp1 specifically binds this region of the PN‐1 promoter. DNase I foot‐printing revealed six potential Sp1 binding sites between −103 and −56 that were protected by recombinant Sp1. Cotransfection experiments into the Sp1‐deficient Drosophila SL2 cell line also showed that Sp1 activates PN‐1 promoter activity in a dose‐dependent fashion. Thus, our analysis demonstrates that activation of PN‐1 transcription is regulated by Sp1 through G/C‐rich cis‐acting elements in the 5′ proximal promoter region.
Ciliary Neurotrophic Factor (CNTF) Genotypes and CNTF Contents in Human Sciatic Nerves as Measured by a Sensitive Enzyme‐Linked Immunoassay
Authors: Takahashi Ryousuke , Kawamura Katsumi , Hu Jianguo , Hayashi Michiyuki , Deguchi Takeo
Published: July 2010

Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme‐linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.
Age‐Dependent Sensitivity of Cultured Peripheral Sympathetic Neurons to 1‐Methyl‐4‐Phenylpyridinium: Role of Glutathione
Authors: Bhave Sanjiv V. , Johannessen Jan N. , Lash Lawrence H. , Wakade Taruna D. , Wakade Arun R.
Published: July 2010

Abstract: We demonstrate that 1‐methyl‐4‐phenylpyridinium (MPP+) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP+ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 µM. Toxicity was blocked by brief preincubation with the norepinephrine (NE)‐reuptake blocker desipramine (DMI; 10 µM for 30 min). MPP+ blocked the uptake of [3H]NE by sympathetic neurons in a dose‐dependent manner with a potency roughly equal to DMI. At concentrations up to 10 µM, MPP+ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP+ diminished gradually with increasing lengths of time in culture. When MPP+ was added to the culture medium 48 h after plating, concentrations up to 100 µM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP+‐induced cell death could not be explained by an increasing capacity for sequestration of MPP+ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompanied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP+ (1 µM) to cultures previously maintained for 2 days in the presence of the GSH‐synthesis inhibitor l‐buthionine‐[S,R]‐sulfoximine (1 µM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP+ in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sympathetic neurons in vivo from the toxicity of MPP+.
Regulation of Ryanodine Receptor Calcium Release Channels by Diadenosine Polyphosphates
Authors: Holden C. P. , Padua R. A. , Geiger J. D.
Published: July 2010

Abstract: [3H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca2+ release channel complexes are modulated by several adenosine‐based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (ApnAs; n = 2–6 phosphate groups) on [3H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [3H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP1,P5‐di(adenosine‐5′) pentaphosphate (Ap5A) and by 209% with the same concentration of the metabolism‐resistant ATP analogue βγ‐methyleneadenosine 5′‐triphosphate (AMP‐PCP) compared with control values for [3H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose‐related increases in [3H]ryanodine binding were observed for all five ApnAs tested [P1,P2‐di(adenosine‐5′) pyrophosphate (Ap2A), P1,P3‐di(adenosine‐5′) triphosphate (Ap3A), P1,P4‐di(adenosine‐5′) tetraphosphate (Ap4A), Ap5A, and P1,P6‐di(adenosine‐5′) hexaphosphate (Ap6A)] as well as AMP‐PCP; oxidized salts of ApnAs stimulated [3H]ryanodine binding to a greater degree than did nonoxidized ApnAs. The apparent rank order for the capacity of these agents to increase [3H]‐ryanodine binding was oxidized Ap4A = oxidized Ap5A > oxidized Ap3A > Ap6A > AMP‐PCP > Ap5A > Ap2A. Addition of the approximate EC50 dose of oxidized Ap4A (37 µM) increased the affinity (KD) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (Bmax) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [3H]‐ryanodine binding by either oxidized Ap4A or nonoxidized Ap5A were not further enhanced by coincubation with AMP‐PCP, which suggests a similar site of action for the ApnAs and AMP‐PCP. [3H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap4A, Ap5A, and AMP‐PCP. Oxidized Ap4A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that ApnAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor‐gated Ca2+ release channels.
Direct Observation of Serotonin 5‐HT3 Receptor‐Induced Increases in Calcium Levels in Individual Brain Nerve Terminals
Authors: Nichols Robert A. , Mollard Patrice
Published: July 2010

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5‐HT3 receptor, a ligand‐gated ion channel. The 5‐HT3 receptor agonist‐induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo‐3 to measure relative changes in internal free Ca2+ concentration ([Ca2+]i), K+‐induced depolarization resulted in variable but rapid increases in apparent [Ca2+]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+]i peaks of varying size (two‐ to 12‐fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+]i (two‐ to 2.5‐fold over basal levels). Agonist activation of 5‐HT3 receptors induced slow increases in [Ca2+]i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2‐fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+. We conclude that significant increases in brain nerve terminal Ca2+, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand‐gated ion channels.
N‐Linked Oligosaccharides Are Required for Cell Surface Expression of the Norepinephrine Transporter but Do Not Influence Substrate or Inhibitor Recognition
Authors: Nguyen Tien T. , Amara Susan G.
Published: July 2010

Abstract: The contribution of N‐linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site‐directed mutagenesis to inactivate the two most carboxy‐terminal (NQQ mutant) or all three (QQQ mutant) sites for N‐glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50‐kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order Vmax of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild‐type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N‐linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.
Regional Reductions of Transketolase in Thiamine‐Deficient Rat Brain
Authors: Sheu Kwan‐Fu Rex , Calingasan Noel Y. , Dienel Gerald A. , Baker Harriet , Jung Eun‐Hee , Kim Kwang‐Soo , Paoletti Francesco , Gibson Gary E.
Published: July 2010

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine‐induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.
Activator Protein‐1 Binding Activities in Discrete Regions of Rat Brain After Acute and Chronic Administration of Methamphetamine
Authors: Ishihara Takeshi , Akiyama Kazufumi , Kashihara Kenichi , Ujike Hiroshi , Hamamura Takashi , Okada Shoshiro , Kuroda Shigetoshi
Published: July 2010

Abstract: The activator protein‐1 (AP‐1) binding activities increased in three brain regions (striatum, nucleus accumbens, and cingulate cortex) after a single methamphetamine (METH) injection to rats. Pretreatment with SCH 23390, but not (−)‐eticlopride, significantly inhibited the enhanced AP‐1 binding activities induced by acute METH administration. The magnitude of enhancement of AP‐1 binding activities 3 h after the last dose of chronic METH administration (4 mg/kg once daily for 14 days) was significantly attenuated as compared with those 3 h after a single METH administration. The AP‐1 binding activities after a 1‐, but not 4‐, week abstinence from chronic administration of METH were still significantly higher than those of the saline‐treated controls. A METH challenge after a 4‐week abstinence period induced significantly lower AP‐1 binding activities in rats chronically injected with METH than in rats chronically injected with saline. The supershift assay revealed that the levels of Jun family protein, but not Fos‐related antigen, increased significantly in the striatum and nucleus accumbens of chronically METH‐treated rats after a 1‐week abstinence. These results suggest that chronic METH administration leads to delayed decay of the induced AP‐1 binding activities and Jun component levels after abstinence for up to 1 week but results in no change in or decreases these activities and attenuates METH challenge‐induced AP‐1 binding activities after abstinence for 4 weeks.
Tetrahydrobiopterin and Biogenic Amine Metabolism in the hph‐1 Mouse
Authors: Hyland K. , Gunasekera R. S. , Engle T. , Arnold L. A.
Published: July 2010

Abstract: hph‐1 mice, which have defective tetrahydrobiopterin biosynthesis due to decreased GTP cyclohydrolase I activity, have been used to investigate the effects of tetrahydrobiopterin deficiency on aromatic l‐amino acid monooxygenases and brain monoamine metabolism. Liver tetrahydrobiopterin levels were decreased, and tetrahydrobiopterin deficiency and reduced levels of dopamine, norepinephrine, serotonin, and their metabolites in the brain occurred both pre‐ and postnatally. Chronic subcutaneous tetrahydrobiopterin elevated brain levels to values higher than those seen in controls but had no effect on monoamine metabolism. In vivo activities of tyrosine hydroxylase and tryptophan hydroxylase were significantly decreased. There was a 30% decrease in the in vitro activity of striatal tyrosine hydroxylase and 50% decrease in liver phenylalanine hydroxylase. Western blotting demonstrated that the lower monooxygenase activities resulted from a reduced absolute amount of tyrosine hydroxylase and phenylalanine hydroxylase protein. The findings suggest involvement of tetrahydrobiopterin in the control of the steady‐state concentration of the aromatic l‐amino acid monooxygenases. In addition, demonstration of central monoamine changes in the hph‐1 mouse make it a possible model system for the investigation of the neuropathological mechanisms in Dopa‐responsive dystonia, which has recently been linked with mutations in the gene for GTP cyclohydrolase I.
Phospholipase D Activity of Rat Brain Neuronal Nuclei
Authors: Kanfer Julian N. , McCartney Douglas , Singh Indrapal N. , Freysz Louis
Published: July 2010

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36‐fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 mM in the presence of 0.75 mM phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF2, AIF3, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.
Isolation, Localization, and Bioactivity of the FMRFamide‐Related Neuropeptides GYIRFamide and YIRFamide from the Marine Turbellarian Bdelloura candida
Authors: Johnston Richard N. , Shaw Chris , Halton David W. , Verhaert Peter , Blair Kevin L. , Brennan Gerard P. , Price David A. , Anderson Peter A. V.
Published: July 2010

Abstract: Two FMRFamide‐related neuropeptides, GYIRFamide and YIRFamide, were isolated from the marine turbellarian Bdelloura candida. The peptides elicited a dose‐dependent contraction of isolated turbellarian muscle fibers, and both were more potent than FMRFamide. Structure‐activity studies, using a range of analogues of the tetrapeptide amide, indicated that the structure of the endogenous peptides was optimal for peak activity. Immunocytochemistry, using an autologous antiserum, revealed a widespread distribution of peptide immunoreactivity within central and peripheral neurons and their processes. This study indicates an important role for GYIRFamide and YIRFamide in the control of neuromuscular function in turbellarians.
Demonstration and Characterization of the Iron Regulatory Protein in Human Brain
Authors: Hu Jing , Connor James R.
Published: July 2010

Abstract: Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that regulate expression of ferritin, erythroid 5‐aminolevulinic acid synthase, and transferrin receptor through interaction with conserved RNA stem‐loop structures called iron‐responsive elements (IREs). Two IRPs (IRP1 and IRP2) have been reported. In the present study we provide evidence for and initial characterization of the IRPs in human brain. Two RNA‐protein complexes were obtained by RNA band shift assay on cytoplasmic extracts from human brain. Competition studies indicate that the formations of the RNA‐protein complexes are specific to the IRE structure. UV cross‐linking of brain cytoplasmic extracts with ferritin IRE RNA transcripts revealed a single RNA‐protein complex with a molecular mass of 110 kDa. A single band at 100 kDa was obtained with IRP1 antiserum on western blot analysis of brain cytoplasmic extracts, and a supershift in the RNA‐protein complexes was observed with an IRP1 antiserum. Two cDNA clones were isolated from a human brain cDNA library with IRP1 cDNA probes, and both of these cDNA probes recognized a single mRNA species (4.0 kb) from human astrocytoma cells. Purified human brain IRP protein has a molecular mass of ∼100 kDa and is capable of forming two RNA‐protein complexes with ferritin IRE RNA and reacts strongly with IRP1 antiserum. These data indicate that IRP1 is predominant in the adult human brain and, in this tissue, is capable of forming a double IRE/IRP complex. This latter observation suggests the brain IRP undergoes posttranslational modification, the result of which may influence the stability of the IRE/IRP complex.
Involvement of Protein Kinase C in γ‐Aminobutyric Acid Release from Xenopus Oocytes Injected with Rat Brain mRNA
Authors: Kan S. , Mameya S. , Kataoka Y. , Kaibara M. , Yamashita K. , Taniyama K.
Published: July 2010

Abstract: Involvement of protein kinase C (PKC) in the release of γ‐aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA‐injected oocytes preloaded with [3H]GABA showed spontaneous release of [3H]GABA, ∼0.5% of GABA content per 1 min. Stimulation with either Ca2+ ionophore (A23187) or a high K+ concentration increased the release of [3H]GABA from slices of rat deep cerebellar nucleus and mRNA‐injected oocytes but not from noninjected and water‐injected oocytes. 12‐O‐Tetradecanoylphorbol 13‐acetate (10–300 nM) but not 4α‐phorbol 12,13‐didecanoate (300 nM) potentiated the A23187‐stimulated release of [3H]GABA from slices and from mRNA‐injected oocytes, in a concentration‐dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.
In Vivo Imaging of Human Cerebral Acetylcholinesterase
Authors: Pappata S. , Tavitian B. , Traykov L. , Jobert A. , Dalger A. , Mangin J. F. , Crouzel C. , Di Giamberardino L.
Published: July 2010

Abstract: We report here the first positron emission tomography (PET) images showing the in vivo regional distribution of acetylcholinesterase (AChE) in human brain. The study was carried out in eight healthy human volunteers using as a tracer [11C]physostigmine ([11C]PHY), an inhibitor of AChE. After intravenous injection of [11C]PHY, radioactivity was rapidly taken up in brain tissue and reached maximal uptake within a few minutes, following a regional pattern mostly related to cerebral perfusion. After the peak, the cerebral radioactivity gradually decreased with a half‐life varying from 20 to 35 min, depending on the brain structure. [11C]PHY retention was higher in regions rich in AChE, such as the striatum (half‐life, 35 min), than in regions poor in AChE, such as the cerebral cortex (half‐life, 20 min). At later times (25–35 min postinjection), the cerebral distribution of [11C]PHY was typical of AChE activity: putamen‐caudate > cerebellum > brainstem > thalamus > cerebral cortex, with a striatal to cortex ratio of 2. These results suggest that PET studies with [11C]PHY can provide in vivo brain mapping of human AChE and are promising for the study of changes in AChE levels associated with neurodegenerative diseases.
Characterization of a Cerebellar Granule Cell‐Specific Gene Encoding the γ‐Aminobutyric Acid Type A Receptor α6 Subunit
Authors: Jones Alison , Bahn Sabine , Grant Andrea L. , Köhler Martin , Wisden William
Published: July 2010

Abstract: The α6 subunit of γ‐aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell‐specific gene expression in the brain. The mouse α6 subunit gene has nine exons and spans ∼14 kb. The largest intron (intron 8) is ∼7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA‐less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat α6 gene upstream region confers expression on a β‐galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell‐specific expression, although expression varied with lobule region. Other founders had ectopic but neuron‐specific expression, with β‐galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate‐putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the α6 subunit gene and that confers neuron‐specific expression.
Functional Expression of P‐Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4
Authors: Begley David J. , Lechardeur Delphine , Chen Zheng‐Duan , Rollinson Christopher , Bardoul Michèle , Roux Françoise , Scherman Daniel , Abbott N. Joan
Published: July 2010

Abstract: The presence of P‐glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P‐glycoprotein is expressed as part of the normal blood‐brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P‐glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P‐glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [3H]colchicine and [3H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3′‐keto‐Bmt1]‐[Val2]‐cyclosporin) at 50 or 100 µM concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P‐glycoprotein activity both in the blood‐brain barrier and in multidrug resistance in general.
Characterization of Pituitary Adenylate Cyclase‐Activating Polypeptide 38 (PACAP38)‐, PACAP27‐, and Vasoactive Intestinal Peptide‐Stimulated Responses in N1E‐115 Neuroblastoma Cells
Authors: Chik Constance L. , Inukai Toshihiko , Ogiwara Takayuki , Boyd Heather , Li Bing , Karpinski Edward , Ho Anthony K.
Published: July 2010

Abstract: In this study, the effects of three related peptides, pituitary adenylate cyclase‐activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca2+ concentration ([Ca2+]i) were compared in N1E‐115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60‐fold with EC50 values of 0.54 and 0.067 nM, respectively. The effect of VIP on cAMP accumulation was less potent. The binding of 125I‐PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC50 values of 0.44 and 0.55 nM, respectively) but not by VIP. In fura‐2‐loaded cells, both PACAP38 and PACAP27 increased [Ca2+]i with EC50 values around 10 nM. The interactions of these three peptides with ionomycin, a Ca2+ ionophore, and 4β‐phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (>1 nM), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca2+]i through the type I PACAP receptors in N1E‐115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose‐dependent stimulatory or inhibitory effect on the PACAP38‐ or PACAP27‐stimulated cAMP accumulation.
A Novel Transmembrane Protein with Epidermal Growth Factor and Follistatin Domains Expressed in the Hypothalamo‐Hypophysial Axis of Xenopus laevis
Authors: Eib Douglas W. , Martens Gerard J. M.
Published: July 2010

Abstract: Through the use of a screening strategy designed to isolate novel cDNAs encoding proteins concerned with pituitary secretion in Xenopus laevis, we discovered clone X7365, which encodes a transmembrane protein with a signal peptide, two follistatin modules, a unique epidermal growth factor domain, and a short cytoplasmic region. RNA expression analyses indicated that the X7365 transcript is enriched in neuroendocrine tissues. Immunohistochemical studies demonstrated that, in addition to being expressed in the optic tectum and in astrocytes in the optic nerve, the X7365 protein is concentrated in a discrete population of hypothalamic magnocellular neurons. Axons projecting to the median eminence and the neurointermediate lobe of the pituitary were also immunopositive, suggesting that X7365 functions in the regulation of magnocellular neurosecretion.
Development of NADPH‐Diaphorase in the Avian Retina: Regulation by Calcium Ions and Relation to Nitric Oxide Synthase
Authors: De Carvalho R. Paes , De Faria M. H. , Do Nascimento J. L. M. , Hokoç J. N.
Published: July 2010

Abstract: Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH‐diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8‐day‐old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit ∼65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 mM calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17‐day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH‐diaphorase activity by NG‐nitro‐l‐arginine or l‐NG‐(iminoethyl)ornithine is concentration dependent with IC50 values of ∼1 mM at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC50 values in the low micromolar range. NADPH‐diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium‐dependent isoform is predominant in early periods of development.
Neurochemical Evidence that Postsynaptic Nucleus Accumbens D3 Receptor Stimulation Enhances Cocaine Reinforcement
Authors: Parsons L. H. , Caine S. B. , Sokoloff P. , Schwartz J.‐C. , Koob G. F. , Weiss F.
Published: July 2010

Abstract: The mechanism by which two D3 receptor‐preferring agonists, 7‐hydroxydipropylaminotetralin (7‐OH‐DPAT) and quinelorane, modulate cocaine reinforcement was examined by monitoring nucleus accumbens dopamine levels with in vivo microdialysis while rats intravenously self‐administered the following four different drug solutions consecutively: (1) cocaine; (2) a combination of cocaine plus a low dose of either agonist; (3) either agonist alone; and finally, (4) a physiological saline solution. Both 7‐OH‐DPAT (4 µg/infusion) and quinelorane (0.25 µg/infusion) decreased cocaine (0.25 mg/infusion) intake in a manner indicating an enhancement of cocaine reinforcement and simultaneously decreased the cocaine‐induced elevations in nucleus accumbens dopamine levels by >50%. Subsequent self‐administration of either 7‐OH‐DPAT (4 µg/infusion) or quinelorane (0.25 µg/infusion) alone resulted in significant, but stable, increases in drug intake, with a concurrent decrease in nucleus accumbens dopamine levels to ∼50% below nondrug baseline levels. These findings indicate that postsynaptic D3 receptor stimulation in the nucleus accumbens enhances the reinforcing properties of cocaine. In a second experiment, local application of 7‐OH‐DPAT via reverse dialysis (30 and 100 nM perfusate concentrations) dose‐dependently decreased nucleus accumbens dopamine efflux to 76 ± 3.9 and 61 ± 6.3% of baseline, respectively, whereas there was no effect of this agonist on dopamine efflux in the ipsilateral striatum of these same animals. Coperfusion with the D3 receptor‐preferring antagonist nafadotride dose‐dependently blocked the effect of 7‐OH‐DPAT on nucleus accumbens dopamine efflux. These results suggest that, at low concentrations, 7‐OH‐DPAT selectively activates D3 receptors in vivo.
Neurotoxicity of a Carboxyl‐Terminal Fragment of the Alzheimer's Amyloid Precursor Protein
Authors: Kim Seong‐Hun , Suh Yoo‐Hun
Published: July 2010

Abstract: We have previously shown that a recombinant carboxyl‐terminal 105‐amino‐acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non‐selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 µM)‐ and time (from 48 h)‐dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid β protein (Aβ). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, Aβ increased LDH release only slightly even at 50 µM but significantly inhibited 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self‐aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas Aβ impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and Aβ, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.
Investigations into the Mechanism of Action of a Novel Nitric Oxide Generator on Cellular Respiration
Authors: Hurst R. D. , Chowdhury R. , Clark J. B.
Published: July 2010

Abstract: Nitric oxide may regulate cellular respiration by competition with oxygen at mitochondrial cytochrome oxidase. Using an astrocyte‐derived cell line, we have compared the mechanism of action of the nitric oxide‐generating compound Roussin's black salt with that of sodium nitroprusside on cellular oxygen consumption. Intense light exposure induced the release of large quantities of nitric oxide from both of the donor compounds. However, in room light only Roussin's black salt generated low levels of the radical. Simultaneous measurement of oxygen consumption and of nitric oxide production demonstrated that sodium nitroprusside only had inhibitory actions when exposed to intense light (nitric oxide release), whereas Roussin's black salt had inhibitory actions in room light. Extracellular haemoglobin did not prevent the inhibition of respiration rate induced by Roussin's black salt even though stimulation of nitric oxide release on light exposure was markedly reduced. Preincubation of cells with Roussin's black salt and subsequent measurement of levels of light‐liberated nitric oxide demonstrated that the compound was rapidly internalised. The uptake of sodium nitroprusside was minimal. These data suggest that, in contrast to sodium nitroprusside, the cellular internalisation of Roussin's black salt allows site‐directed nitric oxide release and very effective inhibition of cellular respiration.
Lazaroid Treatment Prevents Death of Cultured Rat Embryonic Mesencephalic Neurons Following Glutathione Depletion
Authors: Grasbon‐Frodl Eva M. , Andersson Anders , Brundin Patrik
Published: July 2010

Abstract: Reactive oxygen species are believed to play a crucial role in situations where dopamine neurons die, such as in Parkinson's disease or during intracerebral transplantation of embryonic mesencephalic tissue. The present study was designed to address the question whether, and to what extent, the glutathione redox system is important for the viability of rat embryonic dopamine neurons in vitro. Furthermore, we studied whether the lazaroid U‐83836E, a 2‐methylaminochroman that inhibits lipid peroxidation, affects the survival of cultured mesencephalic neurons subjected to experimentally induced glutathione depletion. Glutathione depletion was achieved by exposing dissociated mesencephalic cell cultures to l‐buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, at four different concentrations (1, 10, 100, and 1,000 µM). Dopamine neuron survival was significantly reduced by 65–94% in a concentration‐dependent manner by 10–1,000 µM BSO. The neurotoxic effects of BSO were almost completely prevented by supplementing the culture medium with 0.3 µM U‐83836E. As assessed by HPLC analysis, BSO treatment was associated with a marked reduction of cellular glutathione content, and this depletion was not altered by the presence of U‐83836E. We conclude that in the present insult model of severe glutathione depletion, the lazaroid can afford efficient neuroprotection that does not seem to be mediated by a direct interaction with BSO or glutathione, but rather via an independent pathway.