bluuebunny/crossref_metadata_2025 huggingface dataset:
Bovine Chromaffin Cells Release a Transforming Growth Factor‐β‐Like Molecule Contained Within Chromaffin Granules
Authors: Krieglstein Kerstin , Unsicker Klaus
Published: July 2010

Abstract: Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)‐β in terms of its biological activity. The assay used to assess the activity of TGF‐β is based on cells transfected with a plasminogen activator inhibitor‐1 promoter‐luciferase construct. The assay is highly specific in detecting TGF‐β1, ‐β2, and ‐β3 but does not detect several cytokines and growth factors, such as fibroblast growth factor‐2, transforming growth factor‐α, platelet‐derived growth factor‐AB, insulin‐like growth factor‐I, or neurotrophin‐3 or ‐4. Moreover, we show that this assay does not detect a wide range of TGF‐β superfamily members (activin A, bone morphogenetic protein‐2, ‐4, ‐6, and ‐7, growth/differentiation factor‐5, and glial cell line‐derived neurotrophic factor). Chromaffin granules contain ∼1 ng of TGF‐β/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF‐β1/β2/β3. TGF‐β is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10−5M). These data suggest that TGF‐β is stored in chromaffin granules and can be released by exocytosis.
Sphingolipid Biosynthesis Is Necessary for Dendrite Growth and Survival of Cerebellar Purkinje Cells in Culture
Authors: Furuya Shigeki , Ono Kenichi , Hirabayashi Yoshio
Published: July 2010

Abstract: The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well‐differentiated dendrites and axons after 2 weeks in a serum‐free nutrient condition. Addition of 2 µM fumonisin B1, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [3H]galactose/glucosamine and [14C]serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell‐enriched sphingolipids, including GD1α, 9‐O‐acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B1, dose‐dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin‐treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less‐branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B1 were reversed, but not completely, by the addition of 6‐[[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐amino]caproyl]sphingosine (C6‐NBD‐ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.
Low Level Lead Exposure Decreases In Vivo Release of Dopamine in the Rat Nucleus Accumbens: A Microdialysis Study
Authors: Kala Subbarao V. , Jadhav Arun L.
Published: July 2010

Abstract: The basal and K+‐induced release of dopamine and its metabolites, 3,4‐dihydroxyphenylacetic acid and homovanillic acid, were measured in microdialysate samples obtained in vivo from the nucleus accumbens region of rats subchronically exposed to 50 ppm lead for 90 days. The basal and stimulus‐induced release of dopamine and the metabolites were significantly reduced in the lead‐exposed rats as compared with the controls. These reductions in dopamine and its metabolites are consistent with the reports of decreased dopamine availability associated with lead‐induced changes in certain behavioral indices (fixed‐interval performance) in rats. Furthermore, these changes were observed at blood lead levels similar to those considered to cause impairment in cognitive functions in children.
Neuropeptide Y in Frontal Cortex Is Not Altered in Major Depression
Authors: Ordway Gregory A. , Stockmeier Craig A. , Meltzer Herbert Y. , Overholser James C. , Jaconetta Susan , Widdowson Peter S.
Published: July 2010

Abstract: Previously, we reported a modest but significant reduction in the concentration of neuropeptide Y in frontal cortices from victims of suicide relative to age‐matched natural or accidental death control subjects. The reduction in neuropeptide Y appeared to be greatest in a subgroup of victims of suicide for which there was indirect evidence of histories of depression. We pursued these initial findings in the present study by measuring neuropeptide Y concentrations in frontal cortices from natural or accidental death control subjects and from suicide victims in whom a firm diagnosis of major depression was established by psychiatric autopsy. Because several subjects with major depression had a comorbid diagnosis of alcoholism, a group of victims of suicide that had an Axis I diagnosis of alcohol dependence was also studied. No significant differences in neuropeptide Y concentrations were observed between control subjects and victims of suicide with major depression or victims of suicide with alcohol dependence. These findings do not support a role for neuropeptide Y in major depression.
Neurotrophic Factors Attenuate Glutamate‐Induced Accumulation of Peroxides, Elevation of Intracellular Ca2+ Concentration, and Neurotoxicity and Increase Antioxidant Enzyme Activities in Hippocampal Neurons
Authors: Mattson Mark P. , Lovell Mark A. , Furukawa Katsutoshi , Markesbery William R.
Published: July 2010

Abstract: Exposure of cultured rat hippocampal neurons to glutamate resulted in accumulation of cellular peroxides (measured using the dye 2,7‐dichlorofluorescein). Peroxide accumulation was prevented by an N‐methyl‐d‐aspartate (NMDA) receptor antagonist and by removal of extracellular Ca2+, indicating the involvement of NMDA receptor‐induced Ca2+ influx in peroxide accumulation. Glutamate‐induced reactive oxygen species contributed to loss of Ca2+ homeostasis and excitotoxic injury because antioxidants (vitamin E, propyl gallate, and N‐tert‐butyl‐α‐phenylnitrone) suppressed glutamate‐induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and cell death. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain‐derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor, each suppressed accumulation of peroxides induced by glutamate and protected neurons against excitotoxicity. bFGF, NGF, and BDNF each increased (to varying degrees) activity levels of superoxide dismutases and glutathione reductase. NGF increased catalase activity, and BDNF increased glutathione peroxidase activity. The ability of the neurotrophic factors to suppress glutamate toxicity and glutamate‐induced peroxide accumulation was attenuated by the tyrosine kinase inhibitor genistein, indicating the requirement for tyrosine phosphorylation in the neuroprotective signal transduction mechanism. The data suggest that glutamate toxicity involves peroxide production, which contributes to loss of Ca2+ homeostasis, and that induction of antioxidant defense systems is a mechanism underlying the [Ca2+]i‐stabilizing and excitoprotective actions of neurotrophic factors.
Action of β‐N‐Oxalylamino‐l‐Alanine on Mouse Brain NADH‐Dehydrogenase Activity
Authors: Sabri Mohammad I. , Lystrup Barbara , Roy Dwijendra N. , Spencer Peter S.
Published: July 2010

Abstract: β‐N‐Oxalylamino‐l‐alanine (l‐BOAA), a non‐protein neuroexcitatory amino acid present in the seeds of Lathyrus sativus (chickling or grass pea), is known to produce its neurotoxic effects by overstimulation of non‐N‐methyl‐d‐aspartate receptors, especially α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) receptors, at micromolar concentrations. It has recently been reported that l‐BOAA selectively inhibits mitochondrial enzyme NADH‐dehydrogenase (NADH‐DH) in brain slices at subpicomolar concentrations. The present study finds that up to 4 mM concentrations of pure l‐BOAA fail to inhibit NADH‐DH activity in mouse brain homogenate and isolated brain mitochondria. Two known inhibitors (rotenone and 1‐methyl‐4‐phenylpyridinium ion, MPP+) of this mitochondrial enzyme produced significant inhibition under identical conditions. NADH‐DH inhibition was also not observed in the homogenate or mitochondria from the brains of animals systemically treated with convulsive doses of l‐BOAA. Some inhibition (20–37%) of NADH‐DH activity was observed in mouse brain slices incubated with 100–1,000 µM concentrations of l‐BOAA for 1 h at 37°C in an atmosphere of 95% O2 and 5% CO2, but the inhibition was nonselective, because the activity of another mitochondrial enzyme, succinic dehydrogenase, was similarly inhibited by l‐BOAA. These results are in contrast with the report that l‐BOAA inhibits mitochondrial NADH‐DH selectively at subpicomolar concentrations. We suggest the observed nonselective NADH‐DH inhibition in mouse brain slices treated with l‐BOAA is caused by neuronal damage through an excitotoxic mechanism.
Effects of Brefeldin A on Galactosphingolipid Synthesis in an Immortalized Schwann Cell Line: Evidence for Different Intracellular Locations of Galactosylceramide Sulfotransferase and Ceramide Galactosyltransferase Activities
Authors: Farrer Robert G. , Warden Matthew P. , Quarles Richard H.
Published: July 2010

Abstract: Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6‐h labeling period, a dose‐dependent inhibition of [35S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 µg/ml BFA. Labeling of newly synthesized galactosphingolipids with [3H]‐palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA‐GalCer) to 162% of control values, whereas labeling of GalCer containing 2‐hydroxy fatty acids (HFA‐GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA‐GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA‐GalCer synthesis. Examination of [3H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2‐hydroxylation or the formation of HFA ceramide. Incorporation of [3H]palmitic acid into glucosylceramide and GM3 was increased over control levels whereas labeling of GM2 was inhibited, consistent with what has been reported previously for the effect of BFA on these glycolipids in other cell types. These results suggest that there are at least two separate intracellular sites for the galactosylation of HFA and NFA ceramide, respectively, which can be distinguished by their sensitivity to BFA. Our results also indicate that the site of GalCer sulfation is not redistributed to the endoplasmic reticulum in the presence of BFA and therefore may be localized to the distal Golgi or trans‐Golgi network.
Enhancement of β‐Amyloid Peptide Aβ(1–40)‐Mediated Neurotoxicity by Glutamine Synthetase
Authors: Aksenov M. Yu. , Aksenova M. V. , Harris M. E. , Hensley K. , Butterfield D. A. , Carney J. M.
Published: July 2010

Abstract: The β‐amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C‐end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation‐sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ‐derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ‐mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.
Characterisation of Recombinant Serotonin 5‐HT1A Receptors Expressed in Chinese Hamster Ovary Cells: The Agonist [3H]Lisuride Labels Free Receptor and Receptor Coupled to G Protein
Authors: Sundaram Hardy , Turner Jon D. , Strange Philip G.
Published: July 2010

Abstract: Serotonin 5‐HT1A receptors expressed stably in recombinant Chinese hamster ovary cells have been studied using radioligand binding with the radiolabelled agonist [3H]lisuride. Competition studies with a range of antagonists versus [3H]lisuride confirmed that all of the specific [3H]lisuride binding was to 5‐HT1A receptors on the cells. Competition studies with the antagonist spiperone and several agonists gave data that fitted best to two‐binding‐site models. The affinities of these competing ligands at the two classes of sites were generally in agreement with their corresponding affinities determined in previous work with either 8‐[3H]hydroxydipropylaminotetralin ([3H]8‐OH‐DPAT; labels receptor coupled to G protein) or [3H]spiperone (labels free receptor). Saturation analyses with [3H]lisuride showed that this radioligand labels a single class of binding sites, but the level of radioligand binding was approximately twice that seen when either [3H]8‐OH‐DPAT or [3H]spiperone was used. [3H]Lisuride binding was partially inhibited by addition of guanine nucleotides, and the extent of inhibition decreased as the [3H]lisuride concentration was increased. This inhibition was due to the effect of guanine nucleotide to decrease slightly the affinity of [3H]lisuride for binding to the 5‐HT1A receptors on the cells. It is concluded that [3H]lisuride can label both the free receptor and the receptor coupled to G proteins but with slightly different affinities and that these two states of the receptor exist in roughly equal amounts in the cells. Agonists generally have a higher affinity for the receptor coupled to G protein, whereas antagonists, with the exception of spiperone (which has a higher affinity for the free receptor), have roughly equal affinities for the free receptor and the receptor coupled to G proteins.
Modulation of a Recombinant Glycine Transporter (GLYT1b) by Activation of Protein Kinase C
Authors: Sato Kohji , Adams Ralf , Betz Heinrich , Schloss Patrick
Published: July 2010

Abstract: Treatment of human embryonic kidney cells (HEK 293 cells) expressing the mouse glycine transporter 1 (GLYT1b) with the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) decreased specific [3H]glycine uptake. This down‐regulation resulted from a reduction of the maximal transport rate and was blocked by the PKC inhibitors 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7) and staurosporine. The inhibitory effect of PMA treatment was also observed after removing all five predicted phosphorylation sites for PKC in GLYT1b by site‐directed mutagenesis. These data indicate that glycine transport by GLYT1b is modulated by PKC activation; however, this regulation may involve indirect phosphorylation mechanisms.
The Expression of Syntaxin1B/GR33 mRNA Is Enhanced in the Hippocampal Kindling Model of Epileptogenesis
Authors: Kamphuis W. , Smirnova T. , Hicks A. , Hendriksen H. , Mallet J. , Lopes da Silva F. H.
Published: July 2010

Abstract: Syntaxin, a protein required for the docking of synaptic vesicles, may be involved in the manifestation of synaptic plasticity. The possible involvement of syntaxin in epileptogenesis was investigated by assessing the expression levels of syntaxin1B/GR33 mRNA by in situ hybridization at different stages of hippocampal kindling epileptogenesis and after the induction of generalized seizures. Densitometric analysis of the autoradiograms revealed that the expression was not changed in pyramidal and granular neurons of the hippocampal formation 24 h after the first kindling stimulation. However, the mRNA levels in CA1, CA3, and fascia dentata neurons were bilaterally enhanced after six afterdischarges and remained at this elevated level during the whole period along which afterdischarges were elicited. An immunoassay was unable to reveal a clear significant increase of syntaxin1B/GR33 protein levels in hippocampus homogenates of fully kindled animals. The use of syntaxin1B‐specific antibodies is necessary to draw definite conclusions on the changes at the protein level. At long term, 4 weeks after the last kindling‐elicited generalized seizure, no significant alterations in transcript levels could be detected. The results suggest that the induction of kindling epileptogenesis is associated with an enhanced expression of syntaxin1B/GR33, but this enhanced expression is not necessary for persistence of kindling‐induced synaptic plasticity.
Involvement of Nitric Oxide in Dopaminergic Transmission in Rat Striatum: An In Vivo Electrochemical Study
Authors: Lin Anya M.‐Y. , Kao L.‐S. , Chai C. Y.
Published: July 2010

Abstract: In vivo electrochemical detection with a Nafion‐coated carbon fiber working electrode, which provides information on the spatial and temporal dynamics of dopamine overflow, was used to investigate the involvement of nitric oxide (NO) in the dopaminergic transmission in the striatum of urethane‐anesthetized Sprague‐Dawley rats. A mixture of N‐methyl‐d‐aspartate (NMDA) and nomifensine, a dopamine uptake blocker, was locally pressure‐ejected to elicit a transient dopamine overflow from the dopamine‐containing nerve terminals in the striatum. Local application of Nω‐nitro‐l‐arginine methyl ester (l‐NAME), which blocks endogenous NO formation, increased the magnitude of dopamine release evoked by a subsequent NMDA and nomifensine application but resulted in no significant alteration in the time course. Furthermore, microejection of l‐arginine, an NO precursor, or sodium nitroprusside (SNP), an NO generator, did not cause detectable changes in dopamine level in the striatal extracellular space. However, NMDA‐induced dopamine release was profoundly inhibited with l‐arginine or SNP pretreatment. In addition, NO affects dopamine uptake in rat striatum. Exogenous dopamine applied through a micropipette, reversibly and reproducibly, elicited an electrochemical signal. The time course of these signals was significantly prolonged by l‐NAME treatment. These data suggest that NO is diversely involved in regulating dopaminergic transmission in rat striatum.
Increased Acetylcholine Content Induced by Antidromic Stimulation of a Sympathetic Ganglion: A Possible Retrograde Action of Adenosine
Authors: Tandon A. , Collier B.
Published: July 2010

Abstract: Prolonged high‐frequency orthodromic stimulation of superior cervical ganglia is known to result in increased acetylcholine (ACh) synthesis and ACh content after the period of stimulation. In a previous study, we provided evidence to suggest that adenosine acts as an extracellular signal to activate this increased ACh synthesis and we proposed that the source of that adenosine might be postsynaptic. Thus, the purpose of the present study was to test whether direct stimulation of the postganglionic nerves could affect ganglionic ACh content. Antidromic conditioning of ganglia (15 Hz, 45 min) did not affect significantly their ACh content. However, if ganglia were allowed a 15‐min rest period after this antidromic conditioning, their ACh stores were increased by 20%; a similar increase was induced by 4‐Hz stimulation before the rest period. During the 15‐Hz antidromic stimulation, ACh release was not clearly increased above the basal level, suggesting that preganglionic nerve endings were not stimulated to an extent that could explain the increased ACh content. Orthodromic stimulation (5 Hz) of ganglia 15 min after they had been subjected to antidromic conditioning (15 Hz, 45 min) showed increased ACh release in comparison with that from control unconditioned ganglia. Moreover, the extra ACh released by the conditioned ganglia was quantitatively similar to the increase in the ACh stores, as if most, or all, of the additional ACh was released by preganglionic stimulation. If the antidromic conditioning and the rest period were done during perfusion with Ca2+‐free medium, the ganglia did not accumulate extra ACh. The ACh content was also not changed if ganglia were conditioned in the absence of Ca2+ but rested with normal Ca2+. However, ACh content was increased by 23% when the antidromic stimulation was done with normal Ca2+ but the rest period was without Ca2+. To test the role of adenosine in this retrograde effect, the effect of nucleoside transport inhibitors was tested. Dipyridamole blocked the antidromic stimulation‐induced increase, but nitrobenzylthioinosine did not. Overall, these results are consistent with the idea that a diffusible retrograde messenger activates ACh synthesis. The sensitivity to blockade by dipyridamole suggests that adenosine might be that signal.
Phosphorylation of the Conditioning‐Associated GTP‐Binding Protein cp20 by Protein Kinase C
Authors: Nelson T. J. , Alkon D. L.
Published: July 2010

Abstract: The phosphorylation state of cp20, a low molecular weight membrane‐associated GTP‐binding protein, was previously shown to increase two‐ to threefold 24 h after associative conditioning. Here, cp20 is shown to be phosphorylated by protein kinase C (PKC) in vitro. Pronounced differences in activity were observed with the three major isoforms of PKC, whereas casein kinase, calcium/calmodulin‐dependent protein kinase II, and cyclic AMP‐dependent protein kinase produced no detectable phosphorylation of cp20. Phosphorylation of cp20 had no effect on its GTPase or GTP‐binding activity but caused a translocation of cp20 from cytosol to the nuclei/mitochondrial particulate fraction. These results suggest that the increase in phosphorylation of cp20 after conditioning may be due to PKC.
Diverse Expression of β1,4‐N‐Acetylgalactosaminyltransferase Gene in the Adult Mouse Brain
Authors: Yamamoto Akihito , Yamashiro Shuji , Takamiya Kogo , Atsuta Mitsuru , Shiku Hiroshi , Furukawa Koichi
Published: July 2010

Abstract: Among various tissues of mouse, β1,4‐N‐acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.
Activation of G Proteins Bidirectionally Affects Apoptosis of Cultured Cerebellar Granule Neurons
Authors: Yan Guang‐Mei , Lin Sui‐Zhen , Irwin Robert P. , Paul Steven M.
Published: July 2010

Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K+ (25 mM) and then switched to physiological concentrations of K+ (5 mM) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of Gs with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K+, whereas stimulation of Go/Gi with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K+. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell‐permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.
Bcl‐2 Protects Neural Cells from Cyanide/Aglycemia‐Induced Lipid Oxidation, Mitochondrial Injury, and Loss of Viability
Authors: Myers Kristin M. , Fiskum Gary , Liu Yuanbin , Simmens Samuel J. , Bredesen Dale E. , Murphy Anne N.
Published: July 2010

Abstract: The protooncogene bcl‐2 rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl‐2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl‐2 in a model of delayed neural cell death. We have examined the survival of control and bcl‐2 transfectants of a hypothalamic tumor cell line, GT1‐7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control or bcl‐2 transfectants; however, Bcl‐2‐expressing cells were protected from delayed cell death measured following 24–72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl‐2‐expressing cells. Bcl‐2‐expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl‐2‐expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl‐2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl‐2 or a Bcl‐2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including focal and global cerebral ischemia.
Nerve Growth Factor Regulates Transforming Growth Factor‐β1 Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms in PC12 Cells
Authors: Cosgaya Jose Miguel , Aranda Ana
Published: July 2010

Abstract: PC12 cells express two transforming growth factor (TGF)‐β1 transcripts, 1.7 and 2.5 kb in size. Other TGF‐βs are expressed at much lower levels. Incubation with nerve growth factor (NGF) produced a time‐ and dose‐dependent increase in content of TGF‐β1 transcripts. The level of the smaller mRNA increased little, whereas that of the 2.5‐kb transcript increased more, with the latter becoming the predominant TGF‐β1 message. NGF differentially regulated the stability of both mRNAs. The half‐life of the 2.5‐kb transcript was not altered by NGF; however, the half‐life of the 1.7‐kb mRNA was ∼6 h and increased to >30 h on incubation with NGF. In addition, induction of the 2.5‐kb TGF‐β1 mRNA by NGF required de novo protein synthesis, whereas induction of the 1.7‐kb TGF‐β1 mRNA was independent of protein synthesis. The NGF effect was independent of protein kinase C activation, which also preferentially induced the larger transcript. PC12 cells release a significant amount of TGF‐β1, and incubation with NGF further increases TGF‐β1 production. This factor is released in a latent form. These results indicate that an increase in expression and secretion of TGF‐β1 accompanies neuronal differentiation in PC12 cells. Regulation of TGF‐β1 gene expression by NGF is complex, involving both increased transcription of the TGF‐β1 gene and stabilization of the smaller TGF‐β1 transcript.
Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor‐Differentiated PC12 Cells
Authors: Li Xiaohua , Jope Richard S.
Published: July 2010

Abstract: This investigation examined if lithium, the primary therapeutic treatment for bipolar affective disorder, modulated the levels of selected signal transduction proteins in PC12 cells. Nerve growth factor (NGF) induced differentiation of PC12 cells, and after 12 days of NGF treatment there were large increases in the levels of the heterotrimeric G protein subunits αo1, αi1, β, and αs, small increases in those of αi2 and αq, and a slight decrease in that of αo2. Lithium (1 mM, equivalent to the therapeutic concentration) selectively reduced NGF‐induced increases in levels of G protein subunits, generally having the greatest inhibition on those that were increased the most by NGF. Lithium at 5 mM had greater inhibitory effects than 1 mM lithium on NGF‐induced increases in levels of G proteins, but neither concentration of lithium affected the induction of the cytoskeletal protein β‐tubulin. Examination of other proteins involved in signal transduction revealed that 12 days of NGF treatment increased the level of protein kinase C‐α, but not those of the β, ε, or ζ subtypes, and did not alter the levels of β, γ, or δ phospholipase C. Pretreatment with lithium inhibited the increase in content of protein kinase C‐α induced by NGF but had little effect on the proteins not responsive to NGF except for decreasing the levels of protein kinase C‐ε. The inhibitory effect of lithium was found not to be due to inhibition of NGF‐induced tyrosine phosphorylation, which was unaffected by 5 mM lithium, or to inositol depletion. In summary, use of the dynamic system of NGF‐induced PC12 cell differentiation provided a sensitive model in which to identify signal transduction proteins that were influenced by lithium treatment. The large changes caused by a therapeutically equivalent concentration of lithium lend support to the proposal that the selective inhibitory effects of lithium on subtypes of G proteins and protein kinase C may be important therapeutic targets.
Involvement of Presynaptic Histamine H3 Receptors in the Modulation of Somatostatin Binding and Its Effects on Adenylyl Cyclase Activity in the Rat Frontoparietal Cortex
Authors: Puebla Lilian , Arilla Eduardo
Published: July 2010

Abstract: Thioperamide (2 mg/kg, i.p.), a histamine H3‐receptor antagonist, increased the number of somatostatin (SS) receptors, with no change in the affinity constant, in the rat frontoparietal cortex. This effect was prevented by treatment with (R)‐α‐methylhistamine (3.2 mg/kg, i.p.), a histamine H3‐receptor agonist. Thioperamide also induced an increase in SS binding in rats pretreated with mepyramine, a histamine H1‐receptor antagonist, or cimetidine, a histamine H2‐receptor antagonist. Pretreatment with mepyramine plus cimetidine administered simultaneously antagonized the thioperamide effect on SS binding. The increase in the number of SS receptors was accompanied by a greater SS‐mediated inhibition of basal and forskolin‐stimulated adenylyl cyclase (AC) activity in frontoparietal cortical membranes in the thioperamide group. Furthermore, the functional activity of the guanine nucleotide‐binding inhibitory protein (Gi protein) was not altered by thioperamide or (R)‐α‐methylhistamine administration in frontoparietal cortical membranes. In rats treated with mepyramine plus thioperamide or cimetidine plus thioperamide, the increase in the number of SS receptors was also accompanied by an increased SS inhibition of AC activity. Thioperamide induced a significant increase in SS‐like immunoreactivity content in the frontoparietal cortex. Altogether, these results suggest that frontoparietal cortical histamine may play, at least in part, a role in the regulation of the somatostatinergic system.
[3H]LY303870, a Novel Nonpeptide Radioligand for the NK‐1 Receptor
Authors: Gehlert D. R. , Schober D. A. , Hipskind P. A. , Gitter B. D. , Howbert J. J.
Published: July 2010

Abstract: We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)‐1 receptor and characterized its binding to guinea pig striatal membranes. (R) ‐ N ‐ [2 ‐ [Acetyl[3H3][(2 ‐ methoxyphenyl) ‐ methyl]amino] ‐ 1 ‐ (1H ‐ indol ‐ 3 ‐ ylmethyl)ethyl][1,4′ ‐ bipiperidine]‐1′‐acetamide ([3H]LY303870) binds to a single class of sites with an equilibrium KD of 0.22 nM and a Bmax of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC50 of 0.56 nM, whereas the less active (S)‐enantiomer (LY306155) was substantially less potent. The nonpeptide NK‐1 antagonists (±)‐CP96,345 and (±)‐RP 67580 had IC50 values of 0.74 and 49 nM, respectively. Substance P (SP) was also a potent inhibitor with with an IC50 of 3.1 nM. The inhibition by SP could be separated into two components: a high‐affinity component with a Ki of 0.53 nM and a lower‐affinity component with a Ki of 155 nM. Addition of 100 µM guanylyl 5′‐imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low‐affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [3H]LY303870 was not changed by the presence of 100 µM Gpp(NH)p. The distribution of [3H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK‐1 receptors labeled by [3H]SP. Therefore, [3H]LY303870 is a potent and selective antagonist radioligand for NK‐1 receptors in guinea pig brain. In addition, regulation of NK‐1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.
Dysfunction of Cholinergic and Dopaminergic Neuronal Systems in β‐Amyloid Protein‐Infused Rats
Authors: Itoh Akio , Nitta Atsumi , Nadai Masayuki , Nishimura Kyoko , Hirose Mitsuhiko , Hasegawa Takaaki , Nabeshima Toshitaka
Published: July 2010

Abstract: Accumulations of β‐amyloid protein are characteristic and diagnostic features of the brain of Alzheimer's disease patients; however, the physiological role of this protein in CNS is unknown. We have previously reported that continuous infusion of β‐amyloid protein into rat cerebral ventricle impairs learning ability and decreases choline acetyltransferase activity, a marker enzyme of cholinergic neuron. In this study, the effects of β‐amyloid protein infusion on the release of neurotransmitters in cholinergic and dopaminergic neuronal systems were investigated by using an in vivo brain microdialysis method. Nicotine‐stimulated release of acetylcholine and dopamine in these animals was significantly lower than that in vehicle‐infused rats. Further, dopamine release induced by high‐K stimulation was decreased in β‐amyloid protein‐infused rats compared with vehicle‐infused rats. These results suggest that the release of the two transmitters, acetylcholine and dopamine, was decreased by β‐amyloid protein and that learning deficits observed in the β‐amyloid protein‐infused rats are partly due to the impairment of neurotransmitter release. Furthermore, continuous infusion of β‐amyloid protein may be a useful method to produce the animal model of Alzheimer's disease.
Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse, a Model of Krabbe's Disease
Authors: Sakai Norio , Inui Koji , Tatsumi Naoko , Fukushima Hisao , Nishigaki Toshinori , Taniike Masako , Nishimoto Junji , Tsukamoto Hiroko , Yanagihara Itaru , Ozono Keiichi , Okada Shintaro
Published: July 2010

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278‐nucleotide sequence including a 2,004‐nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6‐kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′‐untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.
The State of Phosphorylation of Normal Adult Brain τ, Fetal τ, and τ from Alzheimer Paired Helical Filaments at Amino Acid Residue Ser262
Authors: Liu Wan‐Kyng , Yen Shu‐Hui
Published: July 2010

Abstract: Antibody Ab262 was raised against a synthetic τ peptide (SKIGSTENLK, amino acids 258–267 of τ, termed Ser262 peptide). The antibody was more reactive with Ser262 peptide and unphosphorylated τ than a related phosphopeptide [SKIGS(P)TENLK, termed P‐Ser262 peptide] and τ phosphorylated by a partially purified kinase, glycogen synthase kinase (GSK) 3β. Ab262 reacted poorly with a peptide having the sequence DRVQSKIGSLD (amino acids 348–358). Treatment of P‐Ser262 peptide or GSK 3β phosphorylated τ with alkaline phosphatase increased Ab262 immunoreactivity, indicating that Ab262 is a reagent useful for studying τ phosphorylation at the Ser262 residue. The Ab262 immunoreactivity was detected in τ from normal brains and Alzheimer paired helical filament (PHF‐τ) and in PHFs. Alkaline phosphatase treatment had no effect on the Ab262 immunoreactivity of normal τ and PHF‐τ but altered the Tau‐1 and PHF‐1 immunoreactivities. τ proteins from rat brains at 3 and 8 h postmortem exhibited 5 and 19%, respectively, more Ab262 immunoreactivity than τ from fresh tissues. In comparison, rat τ at 8 h postmortem was 40% more immunoreactive with Tau‐1. The results suggest that Ser262 is not a major phosphorylation site in vivo. Moreover, there is little or no difference between PHF‐τ and normal τ in the extent of phosphorylation at Ser262.
Mast Cell Activation Causes Delayed Neurodegeneration in Mixed Hippocampal Cultures via the Nitric Oxide Pathway
Authors: Skaper Stephen D. , Facci Laura , Romanello Stefano , Leon Alberta
Published: July 2010

Abstract: Mast cells are pleiotropic bone marrow‐derived cells found in mucosal and connective tissues and in close apposition to neurons, where they play important roles in tissue inflammation and in neuroimmune interactions. Connective tissue mast cells, with which intracranial mast cells share many characteristics, contain cytokines that can cause inflammation. Here, we report that myelin basic protein, a major suspected immunogen in multiple sclerosis, as well as an antigenic stimulus, provokes mast cells to trigger a delayed cytotoxicity for neurons in mixed neuron‐glia cultures from hippocampus. Neurotoxicity required a prolonged period (12 h) of mast cell incubation, and appeared to depend largely on elaboration of the free radical nitric oxide by astrocytes. Activation of astrocytes was mediated, in part, by mast cell‐secreted tumor necrosis factor‐α. Myelin basic protein and 17β‐estradiol had a synergistic action on the induction of mast cell‐associated neuronal injury. The cognate mast cell line RBL‐2H3, when subjected to an antigenic stimulus, released tumor necrosis factor‐α which, together with exogenous interleukin‐1β (or interferon‐γ), induced astroglia to produce neurotoxic quantities of nitric oxide. A small but significant proportion of mast cell‐derived neurotoxicity under the above conditions occurred independently of glial nitric oxide synthase induction. Further, palmitoylethanolamide, which has been reported to reduce mast cell activation by a local autacoid mechanism, decreased neuron loss resulting from mast cell stimulation in the mixed cultures but not that caused by direct cytokine induction of astrocytic nitric oxide synthase. These results support the notion that brain mast cells could participate in the pathophysiology of chronic neurodegenerative and inflammatory diseases of the nervous system, and suggest that down‐modulation of mast cell activation in such conditions could be of therapeutic benefit.
Activation of Cyclic AMP‐Dependent Protein Kinase in Okadaic Acid‐Treated Neurons Potentiates Neurofilament Fragmentation and Stimulates Phosphorylation of Ser2 in the Low‐Molecular‐Mass Neurofilament Subunit
Authors: Giasson Benoit I. , Cromlish James A. , Athlan Eric S. , Mushynski Walter E.
Published: July 2010

Abstract: The activation of cyclic AMP‐dependent protein kinase (PKA) in rat dorsal root ganglion (DRG) cultures increased phosphorylation of the low‐molecular‐mass neurofilament subunit (NFL) at a site previously identified as Ser55 but had no effect on neurofilament integrity. When PKA was activated in DRG cultures treated with 20–250 nM okadaic acid, neurofilament fragmentation was enhanced, and there was a corresponding increase in phosphorylation of NFL at a novel site. This site was also phosphorylated by PKA in vitro and was determined to be Ser2 by mass spectrometric analysis of the purified chymotryptic phosphopeptide. The PKA sites in NFL were dephosphorylated by the purified catalytic subunit of protein phosphatase‐2A but not that of protein phosphatase‐1, and phosphoserine‐2 was a better substrate than phosphoserine‐55. The phosphorylation and dephosphorylation of Ser2 and Ser55 in NFL may therefore be involved in the modulation of neurofilament dynamics through the antagonistic effects of PKA and protein phosphatase‐2A.
Phosphorylation of Neurofilament Proteins in Isolated Goldfish Mauthner Axoplasm
Authors: Raabe Tim D. , Bittner George D.
Published: July 2010

Abstract: The six neurofilament proteins (NFPs) in the goldfish Mauthner axon (M‐axon) have molecular sizes of 235, 145, 123, 105, 80, and 60 kDa. To determine if NFPs in the M‐axon are phosphorylated, isolated Mauthner axoplasm (M‐axoplasm) and a neurofilament‐enriched extract (NFE) prepared from M‐axoplasm were incubated with 32P, which resulted in the radiolabeling of NFPs as determined by their detection on autoradiograms. Kinase inhibitors directed against cyclic AMP‐dependent kinases (PKAs) or cofactor‐independent kinases significantly reduced the in vitro phosphorylation of NFPs in NFE, whereas inhibitors directed against protein kinase C did not significantly reduce the in vitro phosphorylation of NFPs in NFE. Experiments using two kinase inhibitors directed against different kinases significantly reduced the in vitro phosphorylation of NFPs in NFE to a greater extent than the reduction produced using any single kinase inhibitor. These data suggest that NFPs in the M‐axon are phosphorylated and that the in vitro (and perhaps the in vivo) phosphorylation of NFPs is mediated by PKA and/or cofactor‐independent kinases that copurify with NFPs.
Proton Nuclear Magnetic Resonance Spectroscopy of Primary Cells Derived from Nervous Tissue
Authors: Bhakoo Kishore K. , Williams Ian T. , Williams Steve R. , Gadian David G. , Noble Mark D.
Published: July 2010

Abstract: Cell culture techniques, high‐resolution in vitro 1H nuclear magnetic resonance (NMR) spectroscopy, and chromatographic analyses were used to compare the properties of purified cell populations derived from the PNS and cortical neurones. Cell cultures were immunocytochemically characterised with specific antibodies to ensure purity of the individual cultures. Spectra of perchloric acid extracts of cultured Schwann cells, perineural fibroblasts, dorsal root ganglion neurones, and cortical neurones displayed several common features. However, statistically significant differences were found by 1H NMR spectroscopy in most metabolites among the cell types studied. In addition, cells could be distinguished by the presence or absence of certain amino acids. For example, N‐acetylaspartate was present in dorsal root ganglion neurones and cortical neurones, γ‐aminobutyric acid was present in large amounts in cortical neurones, and Schwann cell spectra displayed a large signal from glycine. These results extend our earlier findings that different cell types of the CNS exhibit highly characteristic metabolite profiles to now include the major cell types of the PNS. These latter cell types also exhibit characteristic metabolite compositions, such that even Schwann cells and oligodendrocyte type 2 astrocyte (O‐2A) progenitor cells—precursors of the myelinating cells of the CNS and PNS, respectively—can be readily distinguished from each other.
Assembly Properties of Neurofilament Light Chain Ser55 Mutants in Transfected Mammalian Cells
Authors: Gibb Barry J. M. , Robertson Janice , Miller Christopher C. J.
Published: July 2010

Abstract: Ser55 within the head domain of neurofilament light chain (NF‐L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser55 phosphorylation influences NF‐L assembly, wild‐type and mutant NF‐L genes in which Ser55 was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild‐type and mutant NF‐Ls localised to the Triton X‐100‐insoluble fraction, which suggests that phosphorylation of Ser55 does not inhibit assembly of NF‐L and NF‐L/vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild‐type and mutant NF‐Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild‐type or alanine mutant. These results suggest that phosphorylation of NF‐L Ser55 is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.