bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Cytotoxicity of Ethylcholine Mustard Aziridinium in Cerebral Microvascular Endothelial Cells
Authors: Gómez Carmen , Martín Carmen , Galea Elena , Estrada Carmen
Published: October 2006

Abstract: The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibitor of choline uptake in brain synaptosomes and is used as a neurotoxin to produce animal models of cholinergic hypofunction. However, previous studies have shown that intraocular administration of AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of lactate dehydrogenase activity in the culture medium, AF64A produced similar concentration‐dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human cholinergic neuroblastoma cell line SK‐N‐MC, but not in bovine cerebral smooth muscle cells. The toxic effect of AF64A correlated well with the affinity of the choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell choline carrier, as demonstrated by the following observations: (a) AF64A inhibited [3H]choline uptake in a concentration‐dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of choline or hemicholinium‐3 to the culture medium prevented the AF64A‐induced toxicity in endothelial cell cultures.
Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure
Authors: Lin Lie‐Huey , Whiting Paul , Harris R. Adron
Published: October 2006

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ‐aminobutyric acid (GABAA) receptor‐gated Cl− channel response is a common action of structurally diverse anesthetics, suggesting that the GABAA receptor plays an important role in anesthesia. To determine if GABAA receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA‐activated Cl− currents. Potentiation of GABA‐activated currents by enflurane was dependent on the composition of GABAA receptor protein subunits; the order of sensitivity was α1β1 > α1β1γ2s=α1β1γ2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABAA receptor protein complex to modulate the Cl− channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.
Synaptosomal Glutamate Uptake Declines Progressively in the Spinal Cord of a Mutant Mouse with Motor Neuron Disease
Authors: Battaglioli Gino , Martin David L. , Plummer Julie , Messer Anne
Published: October 2006

Abstract: It has been suggested that the degeneration of motor neurons in amyotrophic lateral sclerosis is a consequence of excitotoxicity resulting from a loss of synaptosomal glutamate uptake. The role of synaptosomal glutamate uptake in the pathogenesis of motor neuron disease was studied in the Mnd mouse. Glutamate uptake in spinal‐cord synaptosomes declined in parallel with the onset of behavioral deficits in Mnd mice but lagged considerably behind the appearance of pathology in motor neurons. Glutamate uptake did not decline significantly in corpus striatum, and GABA uptake did not change significantly in either spinal cord or striatum. The presence of pronounced histopathological changes before the loss of glutamate uptake suggests that the decline of glutamate uptake is a consequence rather than the primary cause of motor neuron disease in the Mnd mouse.
Brain Phospholipids as Dietary Source of (n‐3) Polyunsaturated Fatty Acids for Nervous Tissue in the Rat
Authors: Bourre Jean‐Marie , Dumont Odile , Durand Georges
Published: October 2006

Abstract: In a previous work, we calculated the dietary α‐linolenic requirements (from vegetable oil triglycerides) for obtaining and maintaining a physiological level of (n‐3) fatty acids in developing animal membranes as determined by the cervonic acid content [22:6(n‐3), docosahexaenoic acid]. The aim of the present study was to measure the phospholipid requirement, as these compounds directly provide the very long polyunsaturated fatty acids found in membranes. Two weeks before mating, eight groups of female rats (previously fed peanut oil deficient in α‐linolenic acid) were fed different semisynthetic diets containing 6% African peanut oil supplemented with different quantities of phospholipids obtained from bovine brain lipid extract, so as to add (n‐3) polyunsaturated fatty acids to the diet. An additional group was fed peanut oil with rapeseed oil, and served as control. Pups were fed the same diet as their respective mothers, and were killed at weaning. Forebrain, sciatic nerve, retina, nerve endings, myelin, and liver were analyzed. We conclude that during the combined maternal and perinatal period, the (n‐3) fatty acid requirement for adequate deposition of (n‐3) polyunsaturated fatty acids in the nervous tissue (and in liver) of pups is lower if animals are fed (n‐3) very long chain polyunsaturated fatty acids found in brain phospholipids [this study, ˜60 mg of (n‐3) fatty acids/100 g of diet, i.e., ˜130 mg/1,000 kcal] rather than α‐linolenic acid from vegetable oil triglycerides [200 mg of (n‐3) fatty acids/100 g of diet, i.e., ˜440 mg/1,000 kcal].
Brain Purines in a Genetic Mouse Model of Lesch‐Nyhan Disease
Authors: Jinnah H. A. , Page T. , Friedmann T.
Published: October 2006

Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine‐guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch‐Nyhan disease. The current‐studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT‐immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT‐). Despite the absence of HPRT‐mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [14C]formate incorporation into brain purines, is accelerated four‐ to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT‐ mice from potential depletion of brain purines despite complete impairment of HPRT‐mediated purine salvage.
Characteristics of [14C]Guanidinium Accumulation in NG 108‐15 Cell Exposed to Serotonin 5‐HT3 Receptor Ligands and Substance P
Authors: Emerit M. B. , Riad M. , Fattaccini C. M. , Hamon M.
Published: October 2006

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5‐HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108‐15 that could be assessed by measuring the cell capacity to accumulate [14C]guanidinium for 10‐15 min at 37°C. In addition to 5‐HT (EC50, 0.33 μM), the potent 5‐HT3 receptor agonists 2‐methyl‐serotonin, phenylbiguanide, and m‐chlorophenylbiguanide, and quipazine, markedly increased [14C]guanidinium uptake in NG 108‐15 cells exposed to 10 μM SP. In contrast, 5‐HT3 receptor antagonists prevented the effect of 5‐HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5‐HT on [14C]guanidinium uptake, on the one hand, and to displace [3H]zacopride specifically bound to 5‐HT3 receptors on NG 108‐15 cells, on the other hand, clearly demonstrated that [14C]guanidinium uptake was directly controlled by 5‐HT3 receptors. Various compounds such as inorganic cations (La3+, Mn2+, Ba2+, Ni2+, and Zn2+), D‐tubocurarine, and memantine inhibited [14C]guanidinium uptake in NG 108‐15 cells exposed to 5‐HT and SP, as expected from their noncompetitive antagonistic properties at 5‐HT3 receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5‐HT3 receptor stimulation, prevented the effects of 5‐HT plus SP on [14C]guanidinium uptake. The cooperative effect of SP on this 5‐HT3‐evoked response resulted neither from an interaction of the peptide with the 5‐HT3 receptor binding site nor from a possible direct activation of G proteins in NG 108‐15 cells. Among SP derivatives, [D‐Pro9]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [14C]guanidinium uptake in NG 108‐15 cells exposed to 5‐HT. Although the cellular mechanisms involved deserve further investigations, the 5‐HT‐evoked [14C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5‐HT3 receptors in NG 108‐15 cells.
Characterization of Two Distinct Monoclonal Antibodies to Paired Helical Filaments: Further Evidence for Fetal‐Type Phosphorylation of the τ in Paired Helical Filaments
Authors: Hasegawa Masato , Watanabe Atsushi , Takio Koji , Suzuki Masami , Arai Takao , Titani Koiti , Ihara Yasuo
Published: October 2006

Abstract: Two monoclonal antibodies C5 and M4 raised against Sarkosyl‐insoluble paired helical filaments (PHF) specifically labeled fetal τ, but hardly labeled normal adult τ. C5 immunoreactivity was eliminated by alkaline phosphatase treatment at 37°C, whereas M4 reactivity could be removed only by the treatment at 67°C. Epitope analysis showed that C5 and M4 recognition sites are in residues 386–406 and 198–250, respectively, according to the numbering of the longest human τ isoform. Thus, the phosphorylation sites are located in the amino‐ and carboxyl‐terminal portions of the microtubule‐binding region. These two well‐characterized monoclonals should be valuable in the identification of a protein kinase(s) that converts normal τ into PHF‐τ.
Presence of a Cyclic AMP Response Element‐Binding Protein in Oligodendrocytes
Authors: Sato‐Bigbee Carmen , Yu Robert K.
Published: October 2006

Abstract: Several lines of evidence indicate that cyclic AMP (cAMP) induces oligodendrocyte differentiation. However, the mechanism(s) of this stimulation remains unknown. Because in several cell types the transcriptional activity of various cAMP‐responsive genes is regulated through a cisacting DNA sequence known as cAMP response element (CRE), we investigated the possible presence of a CRE binding (CREB) protein in myelinating oligodendrocytes. A double‐stranded oligonucleotide containing a tandem repeat of the CRE sequence was labeled with T4 kinase in the presence of [32P]ATP and then incubated with a nuclear protein extract from 14‐day‐old rat brain oligodendrocytes. The reaction mixture was then electrophoresed on nondenaturing polyacrylamide gels. The results indicated the presence of a protein that specifically binds to the CRE sequence. The results were supported by southwestern blotting assays in which the CRE probe bound to a ˜45‐kDa protein species. In separate experiments, it was shown that the 45‐kDa protein can be phosphorylated in vitro by the catalytic subunit of protein kinase A. Developmental analysis of CREB protein expression indicated a peak at 14 days of age, preceding the peak of myelinogenesis.
Taurine‐Induced Regeneration of Goldfish Retina in Culture May Involve a Calcium‐Mediated Mechanism
Authors: Lima L. , Matus P. , Drujan B.
Published: October 2006

Abstract: A possible mechanism of action of taurine as a trophic substance was studied in goldfish retina by investigating the effect of extracellular and intracellular calcium chelators on in vitro outgrowth, and the effect of taurine on calcium influx into postcrush retinal cells in culture. The amino acid stimulated the outgrowth from goldfish retinal explants, an effect that was blocked by EGTA and 1,2‐bis (oaminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid methyl ester (BAPTA). The influx of calcium into cultured cells from postcrush retina was increased by taurine by day 5 in culture, but not by day 10, supporting previous results indicating a critical period in which taurine stimulates out‐growth. The present results suggest that taurine partially exerts its regenerative effect on postcrush retinal explants by increasing calcium influx.
Development of Polyclonal Anti‐D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction
Authors: Boundy Virginia A. , Luedtke Robert R. , Molinoff Perry B.
Published: October 2006

Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel‐purified and used to immunize rabbits for the production of polyclonal anti‐receptor antisera. The anti‐fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid‐phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [125I]NCQ 298 and digitonin‐solubilized extracts of canine and rat caudate. [125I]‐NCQ 298 bound reversibly and with high affinity (KD= 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin‐agarose. The anti‐UBI‐D2i3L and anti‐UBI‐D2i3s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [125I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP‐binding proteins in digitonin‐solubilized rat caudate. Both anti‐UBI‐D2i3L and anti‐UBI‐D2i3s antisera were able to inhibit the high‐affinity binding of the agonist N‐propylnorapomorphine to digitonin‐solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.
Regional Variation in γ‐Aminobutyric Acid Turnover: Effect of Castration on γ‐Aminobutyric Acid Turnover in Microdissected Brain Regions of the Male Rat
Authors: Grattan David R. , Selmanoff Michael
Published: October 2006

Abstract: This study compared the turnover of GABA neurons in different brain areas of the male rat and examined the effect of castration on GABA turnover in regions of the brain associated with the control of gonadotropin secretion. To estimate GABA turnover, GABA was quantified by HPLC in microdissected brain regions 0,30,60,90, and 120 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, i.p.). GABA accumulation was linear in all areas for 90 min (p < 0.01), and GABA turnover was estimated as the slope of the line formed by increased GABA concentration versus time, determined by linear regression. There was considerable regional variation both in the initial steady‐state concentrations of GABA and in the rates of GABA turnover. Of 10 discrete brain structures, GABA turnover was highest in the medial preoptic nucleus and lowest in the caudate nucleus. Turnover times in the terminal fields of known GABAergic projection neurons ranged sevenfold, from 2.6 h in the substantia nigra to 0.4 h in the lateral vestibular nucleus. The effect of castration on GABA turnover in 13 microdissected brain regions was investigated by measuring regional GABA concentrations before and 30 min after injection of aminooxyacetic acid in intact rats or 2 or 6 days postcastration. Following castration, steady‐state GABA concentrations were increased, and GABA turnover decreased in the diagonal band of Broca, the medial preoptic area, and the median eminence. GABA turnover increased in the medial septal nucleus and was unaffected in the cortex, striatum, and hindbrain. These results are consistent with the hypothesis that testosterone negative‐feedback control of luteinizing hormone‐releasing hormone involves steroid‐sensitive GABAergic neurons in the rostral and medial basal hypothalamus.
Regional Heterogeneity of Polyamine Effects on the N‐Methyl‐D‐Aspartate Receptor in Rat Brain
Authors: Subramaniam Swaminathan , McGonigle Paul
Published: October 2006

Abstract: Polyamines have pronounced effects on N‐methyl‐D‐aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [3H]MK‐801 binding in rat brain sections. For example, spermidine enhances the binding of [3H]MK‐801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [3H]MK‐801 binding were measured in well‐washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration‐response relationship for spermidine or the KD for [3H]MK‐801 in the presence of 75 μM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration‐response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the Emax value in the ventromedial caudate putamen without changing the EC50. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1, 10‐diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a regionspecific manner.
Glucose Deprivation Causes Posttranscriptional Enhancement of Brain Capillary Endothelial Glucose Transporter Gene Expression via GLUT1 mRNA Stabilization
Authors: Boado Ruben J. , Pardridge William M.
Published: October 2006

Abstract: The absence of neuroglucopenia symptoms in chronic hypoglycemia may be due to up‐regulation of the blood‐brain barrier glucose transporter type 1 (GLUT1). Therefore, we investigated the effect of glucose deprivation on the abundance of the GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture (ECL). Northern blot analysis performed under high stringency conditions with 4–5 μg of ECL poly(A)+ mRNA showed that glucose deprivation (5 mg% glucose) caused a 2.4 ± 0.2‐fold increase in the GLUT1/actin mRNA ratio versus control incubations (100 mg% glucose). This rise was dose and time dependent, and the maximum effect was observed 20–24 h after the hexose deprivation. Nuclear transcription run‐on assay showed no changes in either the GLUT1 or actin gene transcription rate 24 h after glucose deprivation. To determine whether the increase in the abundance of the GLUT1 mRNA induced by glucose deprivation was due to increased stability of this transcript, the GLUT1 mRNA half‐life was measured in ECL cells incubated with actinomycin D. The levels of the GLUT1 transcript continued to be augmented in glucose‐deprived cells compared with controls 2 and 4 h after the transcription inhibitor was added to the media. Glucose deprivation induced a 78% increase in the t1/2 of the GLUT1 mRNA (from 3.6 to 6.4 h). Incubation of ECL cells with the protein synthesis inhibitor, cycloheximide, for 4 h partially reversed the effect of glucose deprivation on the abundance of the GLUT1 transcript. On the other hand, incubation with cycloheximide for 24 h completely blocked the effect of glucose deprivation on the GLUT1 transcript. Desensitization of cellular protein kinase C was performed by incubation of ECL cells with 1 μM phorbol ester for 24 h. This also blocked the effect of glucose deprivation on the abundance of the GLUT1 mRNA. Overall, the present study demonstrates that glucose deprivation increases the abundance of GLUT1 mRNA in brain capillary endothelial culture cells. This increase is due to enhanced stability of the GLUT1 mRNA without changes in gene transcriptional rate. This appears to be mediated by an ongoing protein synthesis and by a protein kinase C‐dependent mechanism. The increase in the abundance of the GLUT1 gene transcript may represent the initial step in the up‐regulation of this gene in hypoglycemia.
31P NMR Relaxation Does Not Affect the Quantitation of Changes in Phosphocreatine, Inorganic Phosphate, and ATP Measured In Vivo During Complete Ischemia in Swine Brain
Authors: Corbett Ronald J. T. , Laptook Abbot R.
Published: October 2006

Abstract: Ischemia‐induced changes in 31P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T1 were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P1 levels had reached a plateau. The T1 for P1 did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25‐s) or short (1‐s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T1 relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P1 during ischemia, implying that the T*2 values remain constant. Our results suggest that the 31P T1 and T*2 for phosphocreatine, Pi, and ATP do not change during ischemia, and therefore changes in 31P NMR peak intensity accurately reflect changes in metabolite concentrations.
Protective Effect of Hypothermia on Hippocampal Injury After 30 Minutes of Forebrain Ischemia in Rats Is Mediated by Postischemic Recovery of Protein Synthesis
Authors: Widmann R. , Miyazawa T. , Hossmann K.‐A.
Published: October 2006

Abstract: Regional protein synthesis of brain was measured by quantitative autoradiography in normo‐ and hypothermic rats submitted to 30 min of four‐vessel occlusion. The tracer, [14C]leucine, was applied by controlled intravenous infusion to achieve constant plasma specific activity, and the admixture by proteolysis of unlabeled amino acids to the brain amino acid precursor pool was corrected by measuring the ratio of the labeled‐to‐unlabeled leucine distribution space in plasma and brain. In normothermic rats preischemic protein synthesis rate was 16.0 ± 3.2, 9.2 ± 3.4, 15.5 ± 2.8, and 15.5 ± 3.1 nmol of leucine/g/min (mean ± SD) in the frontal cortex, striatum, hippocampal CA1 sector, and thalamus, respectively. After 30 min of ischemia at a constant brain temperature of 36°C and a recirculation time of 1 h, protein synthesis was reduced in these regions to 6, 9, 8, and 36%, respectively. With ongoing recirculation, protein synthesis gradually returned to normal within 3 days in all areas except in the stratum pyramidale of the hippocampal CA1 sector where inhibition of neuronal protein synthesis was irreversible. Lowering of brain temperature to 30°C during ischemia did not prevent the early global postischemic depression of protein synthesis, but promoted recovery to or above normal within 6 h in all areas including the stratum pyramidale of the CA1 sector. Improvement of protein synthesis in the CA1 sector was associated with improved neuronal survival, which increased from 1% in the normothermic to 69% in the hypothermic animals. These observations suggest that the protective effect of mild hypothermia on ischemic injury of the hippocampal CA1 sector is mediated by the reversal of the postischemic inhibition of protein synthesis.
K‐252a and Staurosporine Promote Choline Acetyltransferase Activity in Rat Spinal Cord Cultures
Authors: Glicksman Marcie A. , Prantner J. Eric , Meyer Sheryl L. , Forbes M. Elizabeth , Dasgupta Malini , Lewis Michael E. , Neff Nicola
Published: October 2006

Abstract: The protein kinase inhibitor K‐252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose‐dependent manner (EC50 of ∼100 nM) with maximal stimulatory activity at 300 nM resulting in as much as a fourfold increase. A single application of K‐252a completely prevented the marked decline in ChAT activity occurring over a 5‐day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor Staurosporine also increased ChAT activity (EC50 of ∼0.5 nM). Effective concentrations of K‐252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin‐like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose‐dependent increases in ChAT activity in spinal cord cultures. The combination of K‐252a with insulin‐like growth factor‐l or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K‐252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K‐252a and Staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.
The Triplet of Lysine Residues (Lys724‐Lys725‐Lys726) of Alzheimer's Amyloid Precursor Protein Plays an Important Role in Membrane Anchorage and Processing
Authors: Usami Mihoko , Yamao‐Harigaya Wakako , Maruyama Kei
Published: October 2006

Abstract: One of the pathological changes of Alzheimer's disease is the deposit of β/A4 protein, which is derived from Alzheimer amyloid precursor protein (APR). In the secretory pathway, APR is cleaved at an internal region of β/A4 protein by a hypothetical enzyme “secretase.” Our previous study showed that the site of cleavage of APR by secretase is determined by the length from the membrane‐spanning region. To investigate the role of the transmem‐ brane region in APR secretion, we constructed the mutations of triplet lysine residues (Lys724‐Lys725‐Lys726), which are located just in the carboxyl region after the proposed membrane domain. The mutations were as follows: VVK, Val724‐Val725‐Lys726; LLI, Leu724‐Leu725‐lle726; and EEE, Glu724‐Glu725‐Glu726. Wild‐type APR and mutant APPs were expressed transiently in COS‐1 cells by cDNA trans‐fection. The hydrophobic mutant VVK and LLI were processed and secreted in a way similar to that of the wild‐ type APR, although the rate of secretion was decreased. The acidic mutant EEE was not secreted into medium. Proteinase K treatment and cell surface biotinylation of the COS‐1 cells expressing APR revealed that APR was located in the plasma membrane with a short intracellular carboxyl region. However, EEE was completely digested by proteinase K treatment, which suggested that the whole residues of this mutant are located at the outer surface of the cell, including its proposed membrane domain and carboxyl region. This mutant was not cleaved at all by secretase. These findings suggested that the triplet lysine residues of APR after the predicted membrane spanning domain play an important role in the membrane anchorage. In addition, the membrane anchorage was also important for the normal processing by secretase.
Ouabain Releases Striatal Polyamines In Vivo Independently of N‐Methyl‐d‐Aspartate Receptor Activation
Authors: Page D. , Voltz C. , Carter C.
Published: October 2006

Abstract: Intrastriatally infused ouabain (200 or 1,000 μM) markedly increased the extracellular levels of striatal spermidine and spermine in dialysis experiments in halothane‐anesthetized rats. The effects of ouabain (1 mM) on sper‐ midine release were rapid and unaffected by local infusion of the competitive N‐methyl‐d‐aspartate (NMDA) antagonist 3‐(2‐carboxypiperazin‐4‐yl)propyl‐1 ‐phosphonic acid (CPP; 100 μM) or by systemically administered MK‐801 (0.3 mg/kg i.p.), both of which treatments markedly inhibit the effects of intrastriatally administered NMDA. The peak effects of ouabain (1 mM) on spermine release were delayed with respect to those on spermidine release, or to the effects of NMDA, and were also insensitive to locally administered CPP (100 μM). However, systemically administered MK‐801 (0.3 mg/kg i.p., 30 min before the striatal infusion of drugs), which totally inhibits the effects of NMDA, or CPP (10 mg/kg i.p.; 30 min before the striatal infusion of drugs) partially inhibited the effects of ouabain on spermine release, suggesting partial mediation of the delayed effects of ouabain on spermine release by indirect NMDA‐receptor activation. Despite partial sensitivity of ouabain‐induced spermine release to systemically administered NMDA antagonists, both spermidine and spermine can be released in vivo by sodium‐pump inhibition, independently of NMDA‐receptor activation.
Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D‐[1‐13C]Glucose Metabolism
Authors: Shank Richard P. , Leo Gregory C. , Zielke H. Ronald
Published: October 2006

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ‐aminobutyrate (GABA), glutamine, and aspartate. d‐[1‐13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for 13C‐NMR analysis and amino acid analysis. The absolute amount of 13C present within each carbon‐atom pool was determined for C‐2, C‐3, and C‐4 of glutamate, glutamine, and GABA, for C‐2 and C‐3 of aspartate, and for C‐3 of lactate. The natural abundance 13C present in extracts from control rats was also determined for each of these compounds and for N‐acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl‐CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia‐specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from 13C‐13C coupling.
Protein S, an Antithrombotic Factor, Is Synthesized and Released by Neural Tumor Cells
Authors: Phillips Donald J. , Greengard Judith S. , Fernandez Jose A. , Ribeiro Maria , Evatt Bruce L. , Griffin John H. , Hooper W. Craig
Published: October 2006

Abstract: Protein S, an anticoagulant factor in the protein C antithrombotic pathway, was found to be synthesized and released by six tumor cell lines of neural origin by western blotting and ELISA. The rate of synthesis ranged from three‐to 11‐fold higher than that of a microvascular endothelial cell line and 36–144% that of a hepatoma cell line. The secreted protein S displayed specific anticoagulant activity similar to that of purified plasma protein S, implying that it was fully γ‐carboxylated. Ten primary brain tumor tissues also expressed protein S antigen, as shown by western blot analysis. Expression of anticoagulantly active protein S by neural cells raises important questions concerning possible physiologic roles for this multidomain protein beyond its function in control of thrombosis.
Anandamide, a Brain Endogenous Compound, Interacts Specifically with Cannabinoid Receptors and Inhibits Adenylate Cyclase
Authors: Vogel Zvi , Barg Jacob , Levy Rivka , Saya Danielle , Heldman Eliahu , Mechoulam Raphael
Published: October 2006

Abstract: A putative endogenous cannabinoid ligand, arachidonylethanolamide (termed “anandamide”), was isolated recently from porcine brain. Here we demonstrate that this compound is a specific cannabinoid agonist and exerts its action directly via the cannabinoid receptors. Anandamide specifically binds to membranes from cells transiently (COS) or stably (Chinese hamster ovary) transfected with an expression plasmid carrying the cannabinoid receptor DNA but not to membranes from control non‐transfected cells. Moreover, anandamide inhibited the forskolin‐stimulated adenylate cyclase in the transfected cells and in cells that naturally express cannabinoid receptors (N18TG2 neuroblastoma) but not in control nontransfected cells. As with exogenous cannabinoids, the inhibition by anandamide of the forskolin‐stimulated adenylate cyclase was blocked by treatment with pertussis toxin. These data indicate that anandamide is an endogenous agonist that may serve as a genuine neurotransmitter for the cannabinoid receptor.
Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry
Authors: Benecke Silke , Ostermann‐Latif Christel , Mader Michael , Schmidt Bernhard , Unger Jürgen W. , Westarp Martin E. , Felgenhauer Klaus
Published: October 2006

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity‐purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity‐purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168‐ 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.
Dopamine Agonist‐Mediated Inhibition of Acetylcholine Release in Rat Striatum Is Modified by Thyroid Hormone Status
Authors: Foley P. B. , Crocker A. D.
Published: October 2006

Abstract: K+‐evoked acetyl[3H]choline ([3H]ACh) release was inhibited in a concentration‐dependent manner by apomorphine and the D2 agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D2 agonist sensitivity reported for stereotyped behavior. The D2 antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [3H]ACh release by apomorphine, confirming an inhibitory role for D2 receptors and an opposing role for D1 receptors. Because there is no difference in D1 or D2 receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D2 receptor sensitivity affects a receptor‐mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [3H]ACh release. In fact, increased [3H]‐ ACh release was observed, an effect reduced by SCH 23390, providing evidence that D1 receptors enhance [3H]‐ ACh release, and confirming that a PTX‐sensitive G protein mediates the D2 response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist‐ mediated inhibition of ACh.
Evaluation of RNAs Present in Synaptodendrosomes: Dendritic, Glial, and Neuronal Cell Body Contribution
Authors: Rao Anuradha , Steward Oswald
Published: October 2006

Abstract: “Synaptodendrosomes” are subcellular fractions that contain pinched‐off axon terminals and dendrites. These fractions are a potential source of RNAs that are localized in dendrites. However, these fractions may also contain RNAs that are seen in situ in neuronal cell bodies and glia. To evaluate whether Synaptodendrosomes could be used as a source of dendritic RNA, we studied the RNA content of this fraction as compared with RNA isolated from total forebrain and a cell body‐enriched fraction. RNA was analyzed by gel electrophoresis, oligo‐dT chromatography, and northern blot hybridization. RNA from Synaptodendrosomes contained a greater proportion of low‐molecular‐weight nonpolyadenylated RNAs. RNAs known to be present in dendrites (mRNA for the a subunit of the calcium/calmodulin‐dependent protein kinase II and the polymerase III transcript BC1) were detected in Synaptodendrosomes; RNAs that are restricted to neuronal and glial cell bodies (mRNAs for the 68‐kDa neurofilament protein, 43‐kDa growth‐associated protein, β‐tubulin, and β‐ actin) were present only at low levels. However, the mRNA for glial fibrillary acidic protein (seen in situ in glial cell bodies and processes) was present at high levels in the synap‐ todendrosomes. These results support and extend previous studies indicating that a limited subset of mRNAs is present in neuronal and astrocyte processes and reveal that both of these types of mRNAs are present in Synaptodendrosomes. Thus, Synaptodendrosomes may be useful as a source of dendritic RNAs, but it will be necessary to develop strategies to subtract mRNAs present in astroglial processes.
The Presence of (+)‐S‐Adenosyl‐l‐Methionine in the Rat Brain and Its Lack of Effect on Phenylethanolamine N‐Methyltransferase Activity
Authors: Beaudouin Corinne , Haurat Gisèle , Laffitte Jean Alex , Renaud Bernard
Published: October 2006

Abstract: (+)‐S‐Adenosyl‐l‐methionine [(+)‐SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by 1H‐NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)‐SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse‐phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N‐methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)‐SAM has no effect on phenylethanolamine N‐methyltransferase activity; therefore, it is unlikely that (+)‐SAM plays any possible role in regulation of adrenaline synthesis in the brain.
Homologous Desensitization of Serotonin 5‐HT2 Receptor‐Stimulated Intracellular Calcium Mobilization in C6BU‐1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway
Authors: Kagaya Ariyuki , Mikuni Masahiko , Muraoka Shin‐ichiro , Saitoh Kazuko , Ogawa Tetsuo , Shinno Hideto , Yamawaki Shigeto , Takahashi Kiyohisa
Published: October 2006

Abstract: Serotonin 5‐HT2 receptor‐mediated intracellular Ca2+ mobilization was investigated in rat glioma C6BU‐1 cells. The receptors became desensitized after previous exposure to 5‐HT in a time‐and concentration‐dependent manner. The desensitization of 5‐HT2 receptor‐mediated intracellular signaling appeared to be homologous because previous exposure to 5‐HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5‐HT. The desensitization induced by pretreatment with 5‐HT was potently prevented by the naphthalenesulfonamide derivative W‐7, a calmodulin antagonist, when it was cosupplied with 5‐HT. Furthermore, the preventive effect of W‐7 was greater than that of W‐5, a weak analogue of W‐7, and than that of H‐7, a nonselective inhibitor of protein kinases. These results suggest that 5‐HT2 receptor‐mediated Ca2+ mobilization can be desensitized homologously after prolonged exposure to 5‐HT in a calmodulin‐dependent manner in rat glioma C6BU‐1 cells.
Activity and Distribution of Tissue Transglutaminase in Association with Nerve‐Muscle Synapses
Authors: Hand David , Campoy F. Javier , Clark Sandra , Fisher Anna , Haynes Laurence W.
Published: October 2006

Abstract: We have measured, characterized, and localized calcium‐dependent protein cross‐linking activity in rat skeletal muscle, and in myotubes cultured independently or In coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate‐free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross‐linking of endogenous proteins, as measured by the content of ɛ‐(γ‐glutamyl)lysine isppeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and paniculate (B) forms of tTG were separated by ion‐exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B1) particulate form. Km values for calcium activation of the three forms of tTG were in the range of 5–15 μM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B1 transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.
High and Low Molecular Weight r Proteins Are Differentially Expressed from a Single Gene
Authors: Mavilia C. , Couchie D. , Mattel M. G. , Nivez M. P. , Nunez J.
Published: October 2006

Abstract: Both high and low molecular weight (HMW and LMW) T proteins are expressed in the immature and adult mouse spinal cord. Northern blot analysis, performed with probes complementary to domains common and uncommon to the LMW and HMW entities, suggested that HMW τ proteins found in the immature mouse spinal cord are not translated from the single transcript of 6 kb expressed at these stages, but are transported within this nervous structure by axons arising in the periphery. In contrast, another minor transcript of 8 kb was detected in the adult mouse spinal cord by a HMW τ specific probe, suggesting that a small fraction of the HMW τ forms present in adulthood are translated within mouse spinal cord neurons. LMW spinal cord T forms are encoded by mRNAs of 6 kb that contain three and four homologous repeats at immature and mature stages, respectively, whereas adult HMW entities contain four repeats. PCR analysis performed with mouse genomic DMA also showed that the nonhomologous region specific for HMW τ is a single exon. Southern blot and gene mapping showed that the same gene, located on the murine chromosome 11, encodes all the LMW and HMW τ variants. All these τ forms, therefore, are produced by an alternative splicing mechanism that is neuron‐specific and developmentally regulated.
Effects of Hypoxia on Oligodendrocyte Signal Transduction
Authors: Qi Yan , Dawson Glyn
Published: October 2006

Abstract: We have previously established that 21‐day‐old postnatal rat oligodendrocytes, maintained in monolayer culture and subjected to 6 h of hypoxia, show reversible inhibition of synthesis of α‐hydroxy fatty acid and myelin basic protein but a dramatic induction of a 22‐kDa protein, suggesting that this is a good model to study the mechanism of CNS demyelination caused by hypoxic injury. We now report that hypoxia also dramatically inhibits the basal protein kinase C‐mediated phosphorylation of myelin basic protein and myelin 2′,3′‐cyclic nucleotide phosphohydrolase by 80%, but that the inhibition of phosphorylation can be reversed by addition of a protein kinase C activator, phorbol 12‐myristate 13‐acetate. The mechanism of action appears to involve the uncoupling of signal transduction at a site before phospholipase C, because hypoxia did not affect protein kinase C activity or its translocation to the membrane fraction. The most potent activator of phospholipase C (as measured by inositol phosphate release) was carbachol (muscarinic M1 receptor agonist), followed by L‐phenylephrine (α1‐adrenergic receptor agonist) in normal oligodendrocytes. Excitatory amino acids and histamine were ineffective. Hypoxia for 6 h completely inhibited both muscarinic and α1‐adrenergic receptor‐mediated inositol monophosphate release but did not affect phospholipase D‐coupled phosphatidylethanolamine production in response to carbachol. We therefore conclude from this and earlier work that early, reversible changes in oligodendrocytes metabolism result not simply from ATP depletion, but may specifically target GTP binding protein‐mediated processes.
Nitric Oxide Synthase in Bovine Superior Cervical Ganglion
Authors: Sheng Hong , Gagne Gerard D. , Matsumoto Takahiro , Miller Mahlon F. , Förstermann Ulrich , Murad Ferid
Published: October 2006

Abstract: We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by NG‐methyl‐L‐arginine (NMA), and the inhibition was reversed by L‐arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2′,5′‐ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca2+/calmodulin dependent and required NADPH and tetrahydrobiopterin as co‐factors. Superoxide dismutase protected and oxyhemo‐globin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155‐kDa protein band. Immunohisto chemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine‐β‐hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine‐induced stimulation of soluble guanylyl cyclase.