bluuebunny/crossref_metadata_2025 huggingface dataset:
Inorganic Phosphate Compartmentation in the Normal Isolated Canine Brain
Authors: Gilboe David D. , Kintner Douglas , Anderson Mark E. , Fitzpatrick James H. , Emoto Sherrie E. , Markley John L.
Published: October 2006

Abstract: In vivo 31P magnetic resonance spectra of 16 isolated dog brains were studied by using a 9.4‐T wide‐bore superconducting magnet. The observed Pi peak had an irregular shape, which implied that it represented more than one single homogeneous pool of Pi. To evaluate our ability to discriminate between single and multiple peaks and determine peak areas, we designed studies of simulated 31Pi spectra with the signal‐to‐noise (S/N) ratios ranging from ∞ to 4.4 with reference to the simulated Pi peak. For the analysis we used computer programs with a linear prediction algorithm (NMR‐Fit) and a Marquardt–Levenberg nonlinear curve‐fit algorithm (Peak‐Fit). When the simulated data had very high S/N levels, both methods located the peak centers precisely; however, the Marquardt‐Levenberg algorithm (M‐L algorithm) was the more reliable at low S/N levels. The linear prediction method was poor at determining peak areas; at comparable S/N levels, the M‐L algorithm determined all peak areas relatively accurately. Application of the M‐L algorithm to the individual experimental in vivo dog brain data resolved the Pi peak into seven or more separate components. A composite spectrum obtained by averaging all spectral data from six of the brains with normal O2 utilization was fitted using the M‐L algorithm. The results suggested that there were eight significant peaks with the following chemical shifts: 4.07, 4.29, 4.45, 4.62, 4.75, 4.84, 4.99, and 5.17 parts per million (ppm). Although linear prediction demonstrated the presence of only three peaks, all corresponded to values obtained using the M‐L algorithm. The peak indicating a compartment at 5.17 ppm (pH 7.34) was assigned to venous pH on the basis of direct simultaneous electrode‐based measurements. On the basis of earlier electrode studies of brain compartmental pH, the peaks at 4.99 ppm (pH 7.16) and 4.84 ppm (pH 7.04) were thought to represent interstitial fluid and the astrocyte cytoplasm, respectively.
Adrenocorticotropin/α‐Melanocyte‐Stimulating Hormone (ACTH/MSH)‐Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor‐Coupled Mechanism
Authors: Florijn Wouter J. , Mulder Arie H. , Versteeg Dirk H. G. , Gispen Willem Hendrik
Published: October 2006

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α‐melanocyte–stimulating hormone (ACTH/MSH)‐like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration‐dependently increased by ACTH‐(1–24), α‐MSH (EC50 values 16 and 6 nM, respectively), and [Nle4,D‐Phe7]α‐MSH (EC50 value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1‐9‐Dideoxy‐forskolin did not augment the response of adenylate cyclase to ACTH‐(1–24). Various peptide fragments were tested for their ability to enhance [3H]cyclic AMP production. [Nle4,D‐Phe7]α‐MSH increased [3H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH‐(1–24), ACTH‐(1–16)‐NH2, α‐MSH, ACTH‐(1–13)‐NH2, [MetO4]α‐MSH, [MetO24,D‐Lys8,Phe9]ACTH‐(4–9), ACTH‐(7–16)‐NH2, ACTH‐(1–10), and ACTH‐(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide‐induced display of excessive grooming. ACTH‐(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ˜20%) and septal slices (maximal effect, ˜40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle4,D‐Phe7]α‐MSH on [3H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH‐(1–24) did not affect the dopamine D1 or D2 receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α‐MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.
Synthesis of Deoxyglucose‐1‐Phosphate, Deoxyglucose‐1,6‐Bisphosphate, and Other Metabolites of 2‐Deoxy‐D‐[14C]Glucose in Rat Brain In Vivo: Influence of Time and Tissue Glucose Level
Authors: Dienel Gerald A. , Cruz Nancy F.
Published: October 2006

Abstract: Abstract: When the kinetics of interconversion of deoxy[14C]glucose ([14C]DG) and [14C]DG‐6‐phosphate ([14C]DG‐6‐P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [14C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the 14C‐labeled compounds in ethanol extracts of brains of rats given [14C]DG. Six 14C‐labeled metabolites, in addition to [14C]DG‐6‐P, were detected and separated. The major acid‐labile derivatives, DG‐1‐phosphate (DG‐1‐P) and DG‐1,6‐bisphosphate (DG‐1,6‐P2), comprised ˜5 and ˜10–15%, respectively, of the total 14C in the brain 45 min after a pulse or square‐wave infusion of [14C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid‐labile compounds could be synthesized from DG‐6‐P by phosphoglucomutase in vitro. DG‐6‐P, DG‐1‐P, DG‐1,6‐P2, and ethanol‐insoluble compounds were rapidly labeled after a pulse of [14C]DG, whereas there was a 10–30‐min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [14C]DG‐6‐P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [14C]DG‐6‐P into other ethanol‐soluble and ethanol‐insoluble 14C‐labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [14C]DG‐1‐P can explain the overestimation of the [14C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.
[3H]Adenosine Transport in Synaptoneurosomes of Postmortem Human Brain
Authors: Gu J. G. , Kala G. , Geiger J. D.
Published: October 2006

Abstract: Abstract: [3H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor‐stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [3H]adenosine. For 5‐s incubations, two kinetically distinguishable processes were identified, i.e., a high‐affinity adenosine transport system with Kt and Vmax values of 89 μM and 0.98 nmol/min/mg of protein, respectively, and a low‐affinity adenosine transport system that did not appear to be saturable. For incubations with 1 μM [3H]adenosine as substrate, intrasynaptoneurosomal concentrations of [3H]adenosine were 0.26 μM at 5 s and 1 μM at 600 s. Metabolism of accumulated [3H]adenosine to adenine nucleotides was 15% for 5‐s, 23% for 15‐s, 34% for 30‐s, 43% for 60‐s, and 80% for 600‐s incubations. The concentrations (μM) of total accumulated 3H‐purines ([3H]‐adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of 3H‐purines was higher than the initial concentration of 1 μM [3H]adenosine in the reaction medium. For 5‐, 15‐, 30‐, 60‐, and 600‐s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5′‐iodotubercidin, metabolism of the transported [3H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total 3H‐purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 μM, respectively. Thus, the apparently “concentrative'’accumulation of 3H‐purines can be prevented by inhibition of adenosine metabolism and, taken together, these results suggest that adenosine transport in at least synaptoneurosomes prepared from postmortem human brain is via a nonconcentrative and equilibrative system.
Effects of Substance P on the Binding of Agonists to the Nicotinic Acetylcholine Receptor of Torpedo Electroplaque
Authors: Min Churl K. , Weiland Gregory A.
Published: October 2006

Abstract: Abstract: The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 mM). Substance P had no effect on the initial rate of 125I‐α‐bungarotoxin binding at concentrations of <100 μM. The peptide did not bind to the high‐affinity local anesthetic site but allosterically modulated [3H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [3H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine‐induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low‐affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.
Early Signals in Serum‐Induced Increases in Ouabain‐Sensitive Na+‐K+ Pump Activity and in Glucose Transport in Rat Skeletal Muscle Are Amiloride‐Sensitive
Authors: Brodie Chaya , Sampson S. R.
Published: October 2006

Abstract: The acute effects of serum on sodium‐potassium (Na+‐K+) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo‐tubes in phosphate‐buffered saline caused Na+‐K+ pump activity (as measured by changes in the ouabain‐sensitive component of both membrane potential and 86Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage‐dependent Na+ channels. On transfer of myotubes to Na+‐free, choline buffer, resting Na+‐K+ pump activity decreased to about 10% of that in phosphate‐buffered saline. Addition of regular serum, but not Na+‐free serum, caused Na+‐K+ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na+ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na+‐H+ exchange for serum‐induced increases in Na+‐K+ pump activity and glucose transport. The lack of complete blockade of serum‐induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.
β‐Amyloid (25‐35) or Substance P Stimulates [3H]MK‐801. Binding to Rat Cortical Membranes in the Presence of Glutamate and Glycine
Authors: Calligaro David O. , O'Malley Patrick J. , Monn James A.
Published: October 2006

Abstract: Micromolar concentrations of β‐amyloid (25–35) or substance P stimulated [3H] MK‐801 binding in the presence of low concentrations of glutamate (1 γM) and glycine (0.02 γM). Unlike polyamines spermine and spermidine, neither β‐amyloid (25–35) nor substance P increased [3H] MK‐801 binding in the presence of maximally stimulating concentrations of glutamate and glycine. 5,7‐Dichloro‐kynurenic acid, CGS‐19755, and arcaine completely inhibited the stimulated [3H] MK‐801 binding. There was an apparent decreased potency of the [3H] MK‐801 binding inhibition curve for 5,7‐dichlorokynurenic acid, but not CGS‐19755 or arcaine, in the presence of either β‐amyloid (25–35) or substance P. The compounds do not appear to act through the strychnine‐insensitive glycine binding site because neither β‐amyloid (25–35) nor substance P displaced [3H] glycine binding. Full‐length β‐amyloid (1‐40), up to 10 γM, did not stimulate [3H] MK‐801 binding. Concentrations >10 γM could not be tested because they formed large aggregate precipitates in the assay. The data indicate that β‐amyloid (25–35) or substance P does not stimulate [3H] MK‐801 binding at either the N‐methyl‐D‐aspartate, glycine, or polyamine binding sites. Furthermore, the nonpeptide substance P receptor (NK,) antagonist, CP‐96,345, did not block β‐amyloid (25–35)‐ or substance P‐stimulated [3H] MK‐801 binding. Therefore, the effect is not due to an interaction between the substance P receptors and the N‐methyl‐D‐aspartate receptor‐operated ionophore. Finally, if these observations can be verified using single‐channel recording techniques, they may have implications in the pattern of selective neuronal loss observed in patients with neurodegenerative processes such as Alzheimer's, Parkinson's, and Huntington's diseases.
(+)‐Anatoxin‐a Is a Potent Agonist at Neuronal Nicotinic Acetylcholine Receptors
Authors: Thomas P. , Stephens M. , Wilkie G. , Amar M. , Lunt G. G. , Whiting P. , Gallagher T. , Pereira E. , Alkondon M. , Albuquerque E. X. , Wonnacott S.
Published: October 2006

Abstract: The effects of the nicotinic agonist (+)‐anatoxin‐a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)‐Anatoxin‐a was most potent (EC50= 48 nM) in stimulating 86Rb+ influx into M10 cells, which express the nicotinic receptor subtype comprising α4 and β2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)‐anatoxin‐a (EC50= 140 nM). α‐Bungarotoxin‐sensitive neuronal nicotinic receptors, studied using patch‐clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)‐anatoxin‐a with an EC50 of 3.9 γM, whereas α7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 γM for (+)‐anatoxin‐a. In these diverse preparations, (+)‐anatoxin‐a was between three and 50 times more potent than (–)‐nicotine and ˜20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.
Secretogranin II Is Synthesized and Secreted in Astrocyte Cultures
Authors: Fischer‐Colbrie Reiner , Kirchmair Rudolf , Schobert Angelika , Olenik Claudia , Meyer Dieter K. , Winkler Hans
Published: October 2006

Abstract: Astrocyte cultures from rat brain were analyzed for their ability to synthesize and secrete secretogranin II (chromogranin C). Northern blot analysis of polyA‐selected RNA established the presence of secretogranin II mRNA in these cells. By radioimmunoassay, 11.6 fmol/106 astrocytes of secretogranin II was found in these cells. About twice the amount was released into the medium within 3 days. Secretogranin II within the astrocytes was practically unprocessed, as shown by HPLC. These results establish for the first time that astrocytes in vitro synthesize and sec rete a protein of the acidic chromogranin family.
In Vivo Imaging of Vesicular Monoamine Transporters in Human Brain Using [11C]Tetrabenazine and Positron Emission Tomography
Authors: Kilbourn Michael R. , DaSilva Jean N. , Frey Kirk A. , Koeppe Robert A. , Kuhl David E.
Published: October 2006

Abstract: The pharmacokinetics of [11CJtetrabenazine, a high‐affinity radioligand for the monoamine vesicular transporter, were determined in living human brain using in vivo imaging by positron emission tomography (PET). The radiotracer showed high brain uptake and rapid washout from all brain regions with relatively slower clearance from regions of highest concentrations of monoamine vesicular transporters (striatum), resulting in clear differential visualization of these structures at short intervals after injection (10–20 min). As the first human PET imaging study of a vesicular neurotransmitter transporter, these experiments demonstrate that external imaging of vesicular transporters forms a new and valuable approach to the in vivo quantification of monoaminergic neurons, with potential application to the in vivo study of neurodegenerative disorders such as Parkinson's disease.
Drosophilaγ‐Aminobutyric Acid Receptor Gene Rdl Shows Extensive Alternative Splicing
Authors: Ffrench‐Constant Richard H. , Rocheleau Thomas A.
Published: October 2006

Abstract: The Drosophilaγ‐aminobutyric acid (GABA) receptor subunit gene Rdl was isolated on the basis of a mutant phenotype showing high levels of insensitivity to picrotoxinin and cyclodiene insecticides. Following analysis of two dissimilar cDNAs isolated from the locus, we report that Rdl undergoes extensive alternative splicing at two locations in the putative extracellular domain. At each location a choice is made between exons of the same size: “a'’or “b'’(23 amino acids long with two substitutions) and “c'’or “D'’(46 residues long with 10 substitutions). The function of these alternative exons remains unclear; however, exon d contains a putative site for casein kinase II phosphorylation. AH possible combinations of exons (a with c or d and b with c or d) were found in RNA isolated from early embryos. This is the first demonstration of alternative splicing in a GABA receptor gene from invertebrates.
Identification of Chaperonin Particles in Mammalian Brain Cytosol and of T‐Complex Polypeptide 1 as One of Their Components
Authors: Roobol Anne , Carden Martin J.
Published: October 2006

Abstract: An ˜950‐kDa heteromeric particle was purified from guinea‐pig and rat brain by sucrose gradient fractionation of post‐mitochondrial supernatants. Further purification, by affinity chro‐matography on ATP‐Sepharose and anion exchange FPLC on MonoQ, yielded a particle with typical chaperonin ultrastructure. One of the component polypeptides was recognized by a monoclonal antibody to murine T‐complex polypeptide 1. Brain cytosolic chaperonin particles formed a binary complex with unfolded tubulin subunits. The polypeptide compositions of the cytosolic chaperonin particles appeared very similar between brain and testicular tissues of the same animal, but differed subtly between the guineapig and rat.
Adenosine A2a Receptor Modulation of Electrically Evoked Endogenous GABA Release from Slices of Rat Globus Pallidus
Authors: Mayfield R. Dayne , Suzuki Fumio , Zahniser Nancy R.
Published: October 2006

Abstract: Adenosine A2a receptors have been localized to GABAergic striatopallidal neurons, but their functional role is unknown. To address this question, the modulation of endogenous GABA release by adenosine A2a receptors was examined in slices of rat globus pallidus. The selective adenosine A2a receptor agonist CGS‐21680 (3.0–10 nM) significantly increased electrically stimulated release (overflow) of GABA, with 10 nM CGS‐21680 resulting in a 44% increase compared with the control. Both the nonselective adenosine receptor antagonist 8‐phenyltheophylline (10 μM) and the selective A2a receptor antagonist KF‐17837 (100 nM) abolished the CGS‐21680‐induced increase in GABA overflow. Higher concentrations of CGS‐21680 (0.10–1.0 μM) decreased GABA overflow by ˜25%.8‐Phenyltheophylline (10 μM) antagonized these effects, whereas KF‐17837 (100 nM) did not, suggesting actions of CGS‐21680 on other adenosine receptors at these concentrations. These results demonstrate that activation of adenosine A2a receptors augments electrically stimulated release of GABA from globus pallidus slices and suggest a mechanism by which adenosine may modulate GABAergic output from the striatopallidal efferent system.
Distribution of 125I‐Ferrotransferrin Binding Sites in the Mesencephalon of Control Subjects and Patients with Parkinson's Disease
Authors: Faucheux B. A. , Hirsch E. C. , Villares J. , Selimi F. , Mouatt‐Prigent A. , Javoy‐Agid F. , Hauw J. J. , Agid Y.
Published: October 2006

Abstract: Iron is abnormally accumulated in the substantia nigra pars compacta of patients with Parkinson's disease (PD). Because neuronal and glial iron uptake seems to be mediated by the binding of ferrotransferrin to a specific high‐affinity receptor on the cell surface, the number of transferrin receptors could be altered in this disease. The regional distribution of specific binding sites for human 125I‐diferric transferrin has been studied in the mesencephalon, on cryostat‐cut sections from autopsy brains of control subjects and parkinsonian patients by in vitro autoradiography. Densities of binding sites were highest in the central gray substance (˜10 fmol/mg of tissue equivalent), intermediate in the catecholaminergic cell group A8, superior colliculus, and ventral tegmental area, and almost nonexistent in the substantia nigra. The density of 125I‐transferrin binding sites was not significantly different between parkinsonian and control brains in any region analyzed. These results show that in the mesencephalon the regional density of transferrin binding sites is lowest in the dopaminergic cell groups, which are the most vulnerable to PD, and suggest that iron does not accumulate through an increased density of transferrin receptors at the level of the substantia nigra.
Pre‐and Postmortem Influences on Brain RNA
Authors: Barton A. J. L. , Pearson R. C. A. , Najlerahim A. , Harrison P. J.
Published: October 2006

Abstract: Many potentially valuable techniques for the understanding of human neurobiological and neuropathological processes require the use of RNA obtained from postmortem tissue. As with earlier neurochemical studies, there are two particular problems posed by such tissue in comparison with tissue from experimental animals. These are the postmortem interval and the condition of the patient prior to death, referred to as the agonal state. We review the nature and extent of the effects of postmortem interval and agonal state on RNA in brain tissue, with particular reference to the study of neuropsychiatric disorders. Perhaps surprisingly, postmortem interval has at most a modest effect on RNA. Abundant intact and biologically active RNA is present in tissue frozen 36 h or more after death. Postmortem interval does not account for the marked variability observed among human brains in all RNA parameters. Despite the overall stability of RNA after death, some evidence suggests that individual RNAs may undergo postmortem decay. Less attention has been paid to the effects of agonal state. The existing data indicate that events in the premortem period such as hypoxia and coma can affect the amount of some messenger RNAs. The nature of agonal state influences depends on the messenger RNA in question, though the basis for this selective vulnerability is unknown. No agonal state effect on overall RNA level or activity has been found. The data show that postmortem brain tissue can be used for RNA research. However, considerable attention must be paid to controlling for the influences of pre‐and postmortem factors, especially when quantitative analyses are performed.
Classical Noncholinergic Neurotransmitters and the Vesicular Transport System for Acetylcholine
Authors: Clarkson Edward D. , Bahr Ben A. , Parsons Stanley M.
Published: October 2006

Abstract: The acetylcholine transporter exhibits such low affinity and specificity for acetylchoiine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [3H]serotonin, [3H]noradrenaline, or [3H]glutamate occurred. Serotonin, noradrenaline, and N‐acetylaspartyl glutamate inhibited active transport of [3H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and γ‐aminobutyric acid were shown here not to inhibit active transport of [3H]‐acetylcholine. Noradrenaline was competitive with respect to [3H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [3H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.
The SV2 Protein of Synaptic Vesicles Is a Keratan Sulfate Proteoglycan
Authors: Scranton Todd W. , Iwata Mineo , Carlson Steven S.
Published: October 2006

Abstract: We have determined that synaptic vesicles contain a vesicle‐specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an Mr of 100, 000, and the H form, with a lower mobility that migrates with an Mr of ∼250, 000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.
Corticosterone Exacerbates Kainate‐Induced Alterations in Hippocampal Tau Immunoreactivity and Spectrin Proteolysis In Vivo
Authors: Elliott Elicia M. , Mattson Mark P. , Vanderklish Peter , Lynch Gary , Chang Ingrid , Sapolsky Robert M.
Published: October 2006

Abstract: Aberrant elevations in intracellular calcium levels, promoted by the excitatory amino acid glutamate, may be a final common mediator of the neuronal damage that occurs in hypoxic‐ischemic and seizure disorders. Glutamate and altered neuronal calcium homeostasis have also been proposed to play roles in more chronic neurodegenerative disorders, including Alzheimer's disease. Any extrinsic factors that may augment calcium levels during such disorders may significantly exacerbate the resulting damage. Glucocorticoids (GCs), the adrenal steroid hormones released during stress, may represent one such extrinsic factor. GCs can exacerbate hippocampal damage induced by excitotoxic seizures and hypoxia‐ischemia, and we have observed recently that GCs elevate intracellular calcium levels in hippocampal neurons. We now report that the excitotoxin kainic acid (KA) can elicit antigenic changes in the microtubule‐associated protein tau similar to those seen in the neurofibrillary tangles of Alzheimer's disease. KA induced a transient increase in the immunoreactivity of hippocampal CA3 neurons towards antibodies that recognize aberrant forms of tau (5E2 and Alz‐50). The tau immunoreactivity appeared within 3h of KA injection, preceded extensive neuronal damage, and subsequently disappeared as neurons degenerated. KA also caused spectrin breakdown, indicating the involvement of calcium‐dependent proteases. Physiological concentrations of corticosterone (the species‐typical GC of rats) enhanced the neuronal damage induced by KA and, critically, enhanced the intensity of tau immunoreactivity and spectrin breakdown. Moreover, the GC enhancement of spectrin proteolysis was prevented by energy supplementation, supporting the hypothesis that GC disruption of calcium homeostasis in the hippocampus is energetic in nature. Taken together, these findings demonstrate that neurofibrillary tangle‐like alterations in tau, and spectrin breakdown, can be induced by excitatory amino acids and exacerbated by GCs in vivo.
Mechanisms of α‐Sialosyl Cholesterol Action to Suppress Both Cyclic AMP Production and DNA Synthesis of Rat Glial Cells
Authors: Ito Jin‐ichi , Kato Taiji , Tanaka Ryo
Published: October 2006

Abstract: α‐Sialosyl cholesterol (α‐SC) that elicited morphological differentiation of rat astrocytes not only lowered intracellular cyclic AMP (cAMP) levels but also inhibited cAMP production induced by either α‐isoproterenol, cholera toxin, or forskolin. The targets of α‐SC in the cAMP production system of rat astrocytes were investigated to understand the mechanism of the a‐SC action on cAMP production. cAMP production evoked by a‐isoproterenol (1 μM) was entirely canceled by β blockers such as propranolol and timolol (1 μM), but not by α‐SC. Concentrations of α‐SC greater than 15 μM were required for 50% inhibition of the activation by a β agonist. Although α‐SC inhibited in a dose‐dependent manner the activities of membrane‐associated adenylate cyclase that had been stimulated by either GTPγS or forskolin, α‐SC inhibited neither GTP‐binding activities nor GTPase activities of the membrane‐associated G proteins. These findings suggest that α‐SC suppresses adenylate cyclase directly, but not β receptors or G proteins, and that it promotes the morphological differentiation of rat astrocytes through a mechanism regulating directly the cytoskeletal organization, regardless of intracellular cAMP level. α‐SC (30 μM) suppressed 40% of DNA synthesis in the cell‐free system, which contained the cytosolic extracts and the nucleus fraction prepared from rat astrocytoma C6 cells. Approximately 25% of α‐SC incorporated in the astrocyte cytoplasm was transferred to the nuclei by 10 min after the addition. Thus, it is likely that α‐SC that had been incorporated in the cytosol suppressed adenylate cyclase by acting from the cytosolic side of the plasma membrane, and separately suppressed nuclear DNA synthesis.
125I‐Ifenprodil: Synthesis and Characterization of Binding to a Polyamine‐Sensitive Site in Cerebral Cortical Membranes
Authors: Mercer L. D. , Jarrott B. , Beart P. M.
Published: October 2006

Abstract: The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by 125I‐ifenprodil, a noncompetitive NMDA receptor antagonist, are described. 125I‐ifenprodil was synthesized using Na125I in the presence of chloramine‐T and purified by paper chromatography. Binding of the 125I‐ligand was optimal at pH 7.7 in 5 mM Tris · HCl buffer. Equilibrium binding of 125I‐ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 μM). Zn2+, Ca2+, and Mg2+ inhibited specific binding of 125I‐ifenprodil in a concentration‐dependent manner, with IC50 values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (KD) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single‐binding‐site model (Hill coefficient = 0.95), with a density of binding sites (Bmax) of 141 pmol/mg of protein. Binding of 125I‐ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3‐diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine‐sensitive binding of 125I‐ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that σ/antitussive agents partially inhibited specific binding, but inclusion of the σ drug GBR 12909 had little effect on the binding of 125I‐ifenprodil, suggesting this site was not involved. The binding site labeled by 125I‐ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the σ site.
Changes in Sympathetic Nerve Terminals in the Heart of Cold‐Exposed Rats
Authors: Fillenz M. , Stanford S. C. , Coles B. G.
Published: October 2006

Abstract: Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4°C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine β‐hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [3H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [3H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.
Reduction of Dopamine Synthesis Inhibition by Dopamine Autoreceptor Activation in Striatal Synaptosomes with In Vivo Reserpine Administration
Authors: Tissari Anja H. , Lillgäls Monica S.
Published: October 2006

Abstract: In an attempt to clarify the mechanisms by which dopamine (DA) autoreceptor activation inhibits DA synthesis, the efficacy and potency of the D2 DA agonists bromocriptine, lisuride, and pergolide, and the D1,‐D2 DA agonist apomorphine were studied in rat striatal synapto‐ somes, in which the rate of DA synthesis (formation of 14CO2 from l‐[1–14C]tyrosine) was increased 103% by treating the animals from which the synaptosomes were obtained with reserpine (5 mg/kg i.p. twice, 24 and 2 h before they were killed), using the striatal total homogenate as the standard synaptosomal preparation. The increase in DA synthesis evoked by reserpine was additive with that produced by treatment of the synaptosomes with dibutyryl cyclic AMP, suggesting that, not a cyclic AMP‐dependent, but possibly a Ca2+‐dependent mechanism was involved. The DA agonists showed a concentration‐dependent inhibition of DA synthesis in the control synaptosomes, which was antagonized by the selective D2 DA antagonist (‐)‐sulpiride. In the synaptosomes with increased rate of DA synthesis obtained from the rats treated with reserpine, the concentration‐response curves of DA synthesis inhibition for the other DA agonists were shifted to the right, and the effect of bromocriptine was completely eliminated, whereas bromocriptine antagonized the effect of apomorphine. The increased rate of DA synthesis was not preserved in the striatal P1+ P2 fraction obtained from the reserpine‐treated rats, but the effects of the DA agonists were still reduced to the same degree as those in the total homogenate. (‐)‐Sulpiride did not enhance DA synthesis in synaptosomes from the reserpine‐ treated rats. The results presented indicate that the reduced effect of the DA agonists in synaptosomes from the reserpine‐treated rats was not due to endogenous DA occupying the DA autoreceptors. Because it is known from the literature that reserpine in vivo increases impulse activity in DA neurons and, as a result, increases the Ca2+ concentration, these results suggest that the effect of DA agonists was reduced because DA autoreceptors may normally control DA synthesis by decreasing the free intraneuronal Ca2+ concentration, and consequently, the Ca2+‐dependent phosphorylation of tyrosine hydroxylase.
Nefiracetam (DM‐9384) Preserves Hippocampal Neural Cell Adhesion Molecule‐Mediated Memory Consolidation Processes During Scopolamine Disruption of Passive Avoidance Training in the Rat
Authors: Doyle E. , Regan C. M. , Shiotani T.
Published: October 2006

Abstract: Scopolamine (0.15 mg/kg), a muscarinic antagonist, when administered during training or at a discrete 6‐h posttraining time point, is demonstrated to inhibit the recall of a step‐down passive avoidance response when tested at 24 and 48 h after task acquisition. Nefiracetam (3 mg/ kg), a piracetam‐related nootropic, when given with scopolamine during training tended to improve task recall, and this effect was more pronounced when given at the 6‐h posttraining time. Co‐administration of nefiracetam with scopolamine was not necessary to achieve the antiamnesic action, as nefiracetam given during training significantly improved the memory deficits produced by scopolamine at the 6‐h posttraining time. The paradigm‐specific increase in hippocampal neural cell adhesion molecule sialylation, which is observed during consolidation of a passive avoidance response, was attenuated by the presence of scopolamine during training and at the 6‐h posttraining time, and this effect was reversed by co‐administration of nefiracetam, albeit in a paradigm‐independent manner. These results suggest nefiracetam exerts a neurotrophic action that protects memory consolidation from drug inter‐ ventive insults.
α1‐Antichymotrypsin Binding to Alzheimer Aβ Peptides Is Sequence Specific and Induces Fibril Disaggregation In Vitro
Authors: Fraser Paul E. , Nguyen Jack T. , McLachlan Donald R. , Abraham Carmela R. , Kirschner Daniel A.
Published: October 2006

Abstract: The serine protease inhibitor α1‐antichymotrypsin (ACT) consistently colocalizes with amyloid deposits of Alzheimer's disease (AD) and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. AD amyloid fibrils are composed primarily of Aβ, which is a proteolytic fragment of the larger β‐amyloid precursor protein. Using negative‐stain and immunochemical electron microscopy, we have investigated the binding of ACT to the fibrils formed by four synthetic Aβ analogues corresponding to the wild‐type human 1–40 sequence [HWt(1–40)], a 1–40 peptide [HDu(1–40)] containing the Glu22→ Gln mutation found in hereditary cerebral hemorrhage with amyloidosis of the Dutch type, the N‐terminal 1–28 residues [β(1–28)], and an internal fragment of Aβ containing residues 11–28 [β(11–28)]. Each of these peptide analogues assembled into 70–90‐Å‐diameter fibrils resembling native amyloid and, except for β(11–28), bound ACT, as indicated by the appearance of 80–100‐Å globular particles that adhered to preformed fibrils and that could be decorated with anti‐ACT antibodies. Under the conditions used, ACT binding destabilized the in vitro fibrils and produced a gradual dissolution of the macromolecular assemblies into constituent filaments and shorter fragments. The internal fragment (11–28) did not exhibit ACT binding or any structural changes. These results suggest that a specific sequence likely contained within the N‐terminal 10 residues of Aβ is responsible for the formation of the ACT‐amyloid complex. Although the observed fibril disassembly is surprising in view of the notion that ACT contributes directly to the physical process involved in amyloid fibril formation, the induced structural changes may expose new domains in Aβ for additional proteolysis or for interactions with cell‐surface receptors.
Comparison of the Promoter Region of the Human and Porcine Choline Acetyltransferase Genes: Localization of an Important Enhancer Region
Authors: Hersh Louis B. , Kong Chuang Fong , Sampson Craig , Mues Gabriele , Li Yi‐Ping , Fisher Ann , Hilt Dana , Baetge E. Edward
Published: October 2006

Abstract: Genomic clones of human and porcine choline acetyltransferase were obtained by screening genomic libraries with synthetic oligonucleotides. The human and porcine genes exhibit significant conservation of both their intron/exon structure and the nucleotide sequence in their 5’flanking regions. However, the two genes differ in several respects, including the absence of a “TATA” box in the human gene and differences in the position of the methionine start codon. Analysis of the promoter region of the two genes has led to the localization of an enhancer element that appears necessary for efficient transcription of the gene.
Entorhinal Cortex Lesions Transiently Alter Glucocorticoid but Not Mineralocorticoid Receptor Gene Expression in the Rat Hippocampus
Authors: O'Donnell Dajan , Baccichet Annie , Seckl Jonathan R. , Meaney Michael J. , Poirier Judes
Published: October 2006

Abstract: Entorhinal cortex lesions destroy an important hippocampal input and lead to axonal sprouting in the dentate gyrus. Glucocorticoids are known to inhibit this reinnervation process. In the present study, we examined changes in hippocampal glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression using in situ hybridization following unilateral entorhinal cortex lesioning (ECL) in the rat. As early as 1 day postlesioning, a 33% bilateral decrease in GR mRNA expression was observed in the dentate gyrus. By contrast, a 36% bilateral increase in GR mRNA expression was detected in the CA1 cell field. GR mRNA levels in both regions returned to those of control animals 2 days postlesioning, indicating that these effects were transient. Adjacent sections hybridized with probes to MR mRNA revealed no changes in hippocampal MR gene expression as a result of ECL. The selective decrease in GR mRNA expression observed in the dentate gyrus following ECL is specific to the hippocampal subregion targeted for reactive synaptogenesis and thus may serve to attenuate the inhibitory actions of circulating glucocorticoids.
Transport of l‐[3H]Arginine in Cultured Neurons: Characteristics and Inhibition by Nitric Oxide Synthase Inhibitors
Authors: Westergaard Niels , Beart Philip M. , Schousboe Arne
Published: October 2006

Abstract: To investigate whether transport of l‐arginine into neurons plays an integral role in the metabolism of the novel neuromodulator nitric oxide, the uptake of l‐[3H]arginine was characterized in cultured neurons of mouse cerebellum and cerebral cortex (neocortex). The transport of l‐[3H]arginine was saturable and monophasic into both types of neurons with an apparent Km of 100 μM and Vmax of 2.6 nmol/min/mg of protein. The transport process was stereospecific, and the pattern of inhibition by basic l‐amino acids was consistent with a “y+” transport system. The potencies with regard to inhibition of l‐arginine uptake of the two inhibitors of nitric oxide synthase, NG‐monomethyl‐l‐arginine and NG‐amino‐l‐arginine (IC50 values of 120 and 65 μM, respectively), suggest that these nitric oxide synthase inhibitors may also reduce formation of nitric oxide and cyclic GMP via a block of arginine uptake. Overall, these data indicate that uptake of l‐arginine could regulate its intracellular concentration and, consequently, its availability for synthesis of nitric oxide.
Gangliosides Inhibit Platelet‐Derived Growth Factor‐Stimulated Receptor Dimerization in Human Glioma U‐1242MG and Swiss 3T3 Cells
Authors: Van Brooklyn James , Bremer Eric G. , Yates Allan J.
Published: October 2006

Abstract: We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet‐derived growth factor (PDGF) in a dose‐responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [35S]Methionine‐labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross‐linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti‐PDGFR antibody. Using both Swiss 3T3 and human glioma U‐1242MG cells, western blots with anti‐PDGFR and anti‐phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350‐kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF‐mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor‐mediated cell functions.
Ca2+/Calmodulin‐Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor
Authors: Machu Tina K. , Firestone Jordan A. , Browning Michael D.
Published: October 2006

Abstract: The γ2 subunit of the GABAA receptor (GABAA‐R) is alternatively spliced. The long variant (γ2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP‐dependent protein kinase A (PKA)‐stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin‐dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC‐and CAM kinase II‐stimulated phosphorylation of this peptide were 102 and 35 μM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight‐amino‐acid insert of the γ2L subunit has been shown to be necessary for ethanol potentiation of the GABAA‐R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.
Aurintricarboxylic Acid Prevents NMDA‐Mediated Excitotoxicity: Evidence for Its Action as an NMDA Receptor Antagonist
Authors: Zeevalk Gail D. , Schoepp Darryle , Nicklas William J.
Published: October 2006

Abstract: The effect of the endonuclease inhibitor aurintricarboxylic acid (ATA) versus NMDA‐mediated delayed cell death was examined in an ex vivo chick retinal preparation. Transient exposure to 100 μM NMDA for 60 min followed by a 24‐h recovery period resulted in a sevenfold increase in lactate dehydrogenase (LDH) release into the medium. ATA at 100 μM significantly reduced NMDA‐mediated LDH release by 60%. In clarifying the mechanism of protection versus NMDA, ATA was found to inhibit several acute NMDA‐mediated effects: ATA attenuated NMDA‐mediated GABA release in a dose‐dependent manner (IC50= 29.5 μM), prevented NMDA‐stimulated cyclic GMP formation, and blocked NMDA‐mediated 22Na+ influx. These acute inhibitory effects of ATA were overcome by increasing the NMDA concentration, which suggested a competitive interaction between NMDA and ATA. In a binding assay using membranes prepared from adult rat forebrain, ATA displaced the competitive NMDA receptor ligand [3H]CGS 19755 with an IC50 of 26.9 μM. Maximal displacement was 88% with 100 μM ATA. These studies demonstrate that ATA protected neurons from NMDA‐mediated cell death upstream of endonuclease inhibition, i.e., by antagonizing NMDA receptor activity in a manner consistent with competitive antagonism.