bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Sequence of Partial cDNA Clones of Human L1: Homology of Human and Rodent L1 in the Cytoplasmic Region
Authors: Harper John R. , Prince John T. , Healy Patricia A. , Stuart Jill K. , Nauman Susan J. , Stallcup William B.
Published: October 2006

Abstract: We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequence identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.
Developmental Expression of Myelin Protein Genes in Dysmyelinating Mutant Mice: Analysis by Nuclear Run‐Off Transcription Assay, In Situ Hybridization, and Immunohistochemistry
Authors: Shiota Chiyo , Ikenaka Kazuhiro , Mikoshiba Katsuhiko
Published: October 2006

Abstract: Gene expression for myelin proteolipid protein (PLP) and myelin basic protein (MBP) in the dysmyelinating mutant mice shiverer and jimpy was analyzed by nuclear run‐off transcription assay, in situ hybridization, and immunohistochemistry. The level of PLP transcription in shiverer brains was lower than that in controls at postnatal day 18 but relatively higher at later stages. In spite of the considerable amount of hybridization with PLP cDNA, immunoreaction for PLP was greatly reduced in shiverer mice throughout their lives, probably owing to a defect in the assembly of PLP into myelin. Abnormal deposition of PLP in oligodendroglial cell bodies suggested that transport of PLP to myelin is delayed in shiverer brains. The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mice. MBP mRNA in jimpy mice is localized preferentially in oligodendroglial cell bodies, a result suggesting that oligodendrocytes in jimpy are mostly the immature type. Although transcriptional activity of the MBP gene in jimpy was greatly reduced, a finding reflecting the decrease in the number of mature oligodendrocytes, that of the PLP gene remained high at early stages. The discrepancy of the two gene expressions is discussed relative to the role of PLP transcripts at early stages of myelination.
Developmental Changes in Distribution of Acidic Fibroblast Growth Factor in Rat Brain Evaluated by a Sensitive Two‐Site Enzyme Immunoassay
Authors: Ishikawa Rika , Nishikori Koji , Furukawa Shoei
Published: October 2006

Abstract: We developed a sensitive two‐site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti‐aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti‐aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin‐conjugated β‐D‐galactosidase (β‐D‐galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross‐reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two‐site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (<10 ng/g tissue). However, at the young adult stage (21‐ to 49‐day‐old animals), an extremely high level of aFGF (75–90 ng/g tissue) was found in the pons‐medulla; relatively high levels (30–40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5–15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.
Further Demonstration that [Pro9]‐Substance P Is a Potent and Selective Ligand of NK‐1 Tachykinin Receptors
Authors: Petitet F. , Beaujouan J.‐C. , Saffroy M. , Torrens Y. , Chassaing G. , Lavielle S. , Besseyre J. , Garret C. , Carruette A. , Glowinski J.
Published: October 2006

Abstract: Previous studies have indicated that [Pro9]‐substance P ([Pro9]‐SP) possesses very good affinity for NK‐1 binding sites and that, in contrast to substance P, it interacts selectively with these sites. Therefore, [3H][Pro9]‐SP (75 Ci/mmol) was synthesized in order to study its binding to membranes of the rat brain. Specific binding of [3H][Pro9]‐SP (75% of total binding) was temperature‐dependent, saturable, and reversible. Scatchard analysis and Hill plots revealed the existence of a single population of noninteracting binding sites (KD and Bmax values: 1.48 nM and 29.7 fmol/mg of protein, respectively). Competition studies with several tachykinins and analogues indicated that the pharmacological profile of [3H][Pro9]‐SP binding sites is identical to that of NK‐1 binding sites. Rat brain sections labeled with either [3H][Pro9]‐SP or [3H]SP, revealed a close similarity in the topographical distribution of [3H][Pro9]‐SP and [3H]SP binding sites. Biochemical, pharmacological, and autoradiographic data obtained with [3H][Pro9]‐SP did not provide any evidence for the existence of subtypes of NK‐1 binding sites. [Pro9]‐SP had neither agonist nor antagonist properties on NK‐2 and NK‐3 receptors. Indeed, it did not stimulate phosphoinositide turnover on the hamster urinary bladder (NK‐2 assay) and was devoid of activity on the contraction of the rabbit pulmonary artery (NK‐2 assay) and of the rat portal vein (NK‐3 assay). As a result of its high selectivity, [Pro9]‐SP thus appears an excellent tool for investigating the functional properties of NK‐1 receptors.
Effects of Pseudorabies Virus on the Neuronal Properties of PC 12 Cells
Authors: Schilter B. , Marchand C. M.‐F.
Published: October 2006

Abstract: The effect of pseudorabies virus on neuronal functions was investigated in PC12 cells. During the period investigated, choline acetyltransferase was not affected, while the acetylcholinesterase activity declined steadily starting at 12 h post infection (p.i.), reaching its minimal level of 40% of the control value at 24 h p.i. In contrast, the activity of tyrosine hydroxylase, the key enzyme in catecholamine synthesis, increased to 150% of the control level by 15 h p.i., dropping off slowly with the appearance of viral cytopathology. In parallel, the infection induced, by a process independent of the extracellular Ca2+, an increased release of dopamine at 11 h p.i., followed by noradrenaline at 20 h p.i. In the infected cells, the intracellular content of catecholamine was maintained only in the presence of a high amount of catecholamine precursors in the culture medium. Three plaque‐forming units per cell was the minimal multiplicity of infection required to obtain the maximal changes in enzyme activities; higher multiplicities induced more rapidly the maximal effects on tyrosine hydroxylase and acetylcholinesterase. Inhibition of DNA synthesis did not prevent the increase in tyrosine hydroxylase activity; however, protein synthesis was required. In conclusion, infection of the PC12 cells with pseudorabies virus induced significant changes in catecholaminergic and cholinergic metabolism, indicating the ability of this virus to interfere selectively with specialized neuronal functions.
Neurochemical Interactions of Competitive N‐Methyl‐D‐Aspartate Antagonists with Dopaminergic Neurotransmission and the Cerebellar Cyclic GMP System: Functional Evidence for a Phasic Glutamatergic Control of the Nigrostriatal Dopaminergic Pathway
Authors: Rao Tadimeti S. , Cler Julie A. , Mick Steve J. , Emmett Mark R. , Farah John M. , Contreras Patricia C. , Iyengar Smriti , Wood Paul L.
Published: October 2006

Abstract: Direct intrastriatal injection of N‐methyl‐D‐aspartate (NMDA; 100 μg/rat) increased striatal dopamine (DA) release in vivo. However, parenteral administration of (±)‐3‐(2‐carboxypiperizin‐4‐yl)propyl‐1‐phosphonic acid (CPP) and cis‐4‐phosphonomethyl‐2‐piperidine carboxylic acid (CGS‐19755) did not alter DA metabolism and release in several brain regions in the rat and mouse. Intracerebroventricular administration of the competitive NMDA antagonists CPP, CGS‐19755, 2‐amino‐5‐phosphonopentanoate, and 2‐amino‐7‐phosphonoheptanoate did not alter rat striatal DA metabolism and release but profoundly reduced cerebellar cyclic GMP (cGMP) levels in the same animals. CPP and CGS‐19755 decreased basal cerebellar cGMP levels in the mouse with ED50 values of 6 and 1 mg/kg, i.p., respectively. CPP antagonized the harmaline‐induced increases in cGMP levels with an ED50 value of 5.0 mg/kg, i.p. CPP (25 mg/kg, i.p.) also decreased basal cGMP levels in mouse cerebellum for up to 3 h, a result suggesting brain bioavailability and a long duration of NMDA receptor antagonism in vivo. These contrasting patterns suggest that NMDA receptors exert a tonic excitatory tone on the guanine nucleotide signal transduction pathway in the cerebellum while exerting a phasic control over nigrostriatal dopaminergic neurotransmission. These results also indicate that competitive NMDA antagonists, unlike phencyclidine receptor agonists, may not mediate biochemical and behavioral effects via dopaminergic mechanisms.
Enhanced NAD(P)H:Quinone Reductase Activity Prevents Glutamate Toxicity Produced by Oxidative Stress
Authors: Murphy Timothy H. , De Long Mary J. , Coyle Joseph T.
Published: October 2006

Abstract: Glutamate toxicity in the N18‐RE‐105 neuronal cell line results from the inhibition of high‐affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one‐electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT‐diaphorase activity. Using glutamate toxicity in N18‐RE‐105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t‐butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t‐butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.
Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides
Authors: Langan Thomas J. , Slater Mary C.
Published: October 2006

Abstract: The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum‐depleted (0.1%, vol/vol) for 48 h on days 4–6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70–80% of the cells were glial fibrillary acidic protein (GFAP)‐negative by indirect immunofluorescence; 79 ± 7% were GFAP‐positive after an additional 3 days. Serum shift‐up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 ± 6% to 75 ± 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 ± 6 versus 380 ± 32 cpm/μg of protein/h of [3H]thymidine uptake). Addition of mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 μM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine‐labeled nuclei increased from 0% to 75 ± 9%, and DNA synthesis increased 10‐fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.
Glycosphingolipid Glycosyltransferases in Human Fetal Brain
Authors: Percy Alan K. , Gottfries Johan , Vilbergsson Gudjon , Månsson Jan‐Eric , Svennerholm Lars
Published: October 2006

Abstract: The developmental pattern of gangliosides in human fetal brain should reflect the activities of the respective glycosyltransferases. LA2‐synthase activity, along with that of GM3‐, GD3‐, GM2‐, and GM1‐synthases, was determined in human fetal brain at 10–22 weeks of gestation. LA2‐syn‐thase is the pivotal enzyme in lacto series ganglioside formation. LA2‐synthase activity decreased during the study period, mirroring a similar temporal decline in levels of the lacto series, gangliosides, particularly 3′‐isoLM1. The developmental profiles of the ganglio series glycosyltransferase activities demonstrate distinct changes that correspond to the ganglioside pattern between fetal weeks 10 and 22. In particular, the marked increase in GM2‐synthase activity at 20 and 22 weeks of gestation and the decline in GD3‐synthase activity after 15 weeks could explain the prominent expression of the a series gangliosides in this period of rapid neuronal outgrowth. However, a similar decline (two‐ to 2.5‐fold) in GM3‐synthase activity suggests a more likely conclusion, namely, that the two sialyltransferase activities are derived mainly from astroglial cells, which show a marked proliferation during the 10–15th fetal weeks. The data do not negate the hypothesis that GM3‐ and GD3‐synthase are the critical enzymes in the regulation of ganglioside biosynthesis but do indicate a need to reevaluate the significance of GM2‐synthase in expression of the a series gangliosides.
Phorbol Myristate Acetate Inhibition of Phospholipase C Activation by Carbachol in Slices of Rat Brain Cortex Is a Delayed and Indirect Effect
Authors: Lee Horng‐Mo , Fain John N.
Published: October 2006

Abstract: We examined the effect of phorbol esters on phos‐pholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12‐myristate 13‐acetate (PMA) with carbachol on phos‐phoinositide breakdown due to carbachol over a 1‐h incubation of brain slices. However, if slices were preincu‐bated for 3 h with 1 μM. PMA. or 200 μM/sphingosine before addition of carbachol, there was a 35–50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3‐h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol‐stimulated phospholipase C activation using isolated membranes. However, the decrease in car‐bachol‐induced phosphoinositide breakdown seen in slices after a 3‐h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55 mM Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3‐h incubation with PMA. Because apamin, an inhibitor of Ca2+‐dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.
Regulation of the Expression of Acetylcholinesterase by Muscular Activity in Avian Primary Cultures
Authors: Vallett François‐Marie , Massoulié Jean
Published: October 2006

Abstract: Primary cultures of avian muscle cells express both globular and asymmetric molecular forms of acetylchoiinesterase (AChE) when grown in a simple defined culture medium. Under these conditions, we analyzed the role of various agents interfering with muscular activity: tetrodotoxin (TTX) and veratridine, as well as a depolarizing concentration of KC1. These treatments caused the complete cessation of contractions in mature myotubes. We observed no influence on cellular AChE activity. The paralyzing treatments induced different effects on AChE secretion: TTX increased the secretion by ∼25%, whereas KC1 and veratridine reduced it by ∼30%. The proportions of secreted molecular forms (mostly hydrophilic G4 and G2) were not modified significantly. TTX did not affect the pattern of molecular forms of cellular AChE (in particular, the proportion of A forms was not changed). Depolarization by veratridine or KC1 induced an increase in the proportion of A forms in mature myotubes by a factor of 2–3. Similar results were obtained with quail myotubes cultured under the same conditions. This study shows that, in avian muscle cultures, the ionic balance across myotube membranes, rather than muscular activity per se, can regulate the level of A forms and the rate of AChE secretion. These results do not exclude the possible involvement of other factors, such as Ca2+ and/or peptidic factors. In addition, taking together our results and data from the literature, we conclude that the expression of AChE molecular forms depends both on the species and on the culture conditions used.
Effect of Aspartame‐Derived Phenylalanine on Neutral Amino Acid Uptake in Human Brain: A Positron Emission Tomography Study
Authors: Koeppe Robert A. , Shulkin Barry L. , Rosenspire Karen C. , Shaw Leslie A. , Betz A. Lords , Mangner Thomas , Price Joy C. , Agranoff Bernard W.
Published: October 2006

Abstract: The possible effects of elevation of the plasma phe‐nylalanine level secondary to the ingestion of aspartame on brain amino acid uptake in human subjects have been investigated by means of positron emission tomography (PET). 1‐[11C]Aminocyclohexanecarboxylate ([11C]ACHC) is a poorly metabolized synthetic amino acid that crosses the blood‐brain barrier by the same carrier that transports naturally occurring large neutral amino acids. Quantitative test‐retest PET studies were performed on 15 individuals. Seven received two identical baseline scans, whereas eight received a baseline scan followed by a scan performed ∼40–45 min following ingestion of an orange‐flavored beverage containing 34 mg/kg of body weight of the low‐calorie sweetener aspartame, a dose equivalent to the amount in 5 L of diet soft drink consumed all at once by the study subjects, weighing an average of 76 kg. The 40–45‐min interval was selected to maximize the detection of possible decreases in ACHC uptake resulting from increased competition for the carrier, because the plasma phenylalanine level is known to peak at this time. We observed an 11.5% decrease in the amino acid transport rate constant Kt and a smaller decrease in the tissue distribution volume of ACHC (6%). Under conditions of normal dietary use, aspartame is thus unlikely to cause changes in brain amino acid uptake that are measurable by PET.
Effects of a New Thyrotropin Releasing Hormone Analogue, YM‐14673, on the In Vivo Release of Acetylcholine as Measured by Intracerebral Dialysis in Rats
Authors: Okada Masamichi
Published: October 2006

Abstract: The effects of a new thyrotropin releasing hormone (TRH) analogue, YM‐14673 (Nα‐[[(S)‐4‐oxo‐2‐azetidi‐nyl]carbonyl]‐l‐histidyl‐l‐prolinamide dihydrate), on the release of acetylcholine (ACh) in free‐moving rats were examined in vivo by intracerebral dialysis. In the frontal cortex, YM‐14673 (0.1–0.3 mg/kg) caused a significant dose‐dependent increase in the extracellular levels of ACh, suggesting that YM‐14673 stimulated the ACh release. These actions of YM‐14673 were about 50 times more potent than those of TRH. On the other hand, extracellular levels of ACh in caudate nucleus were not changed following injection of YM‐14673 even at 3 mg/kg. TRH and methamphetamine also increased the release of ACh in frontal cortex. Haloperidol prevented the increase in the methamphetamine‐induced release of ACh, whereas the increased release of ACh produced by YM‐14673 was partially antagonized by haloperidol. These results suggest that the dopaminergic system affects the fa‐cilitatory effects on the ACh release in the frontal cortex and that the stimulatory effect of YM‐14673 on the frontal cholinergic neurons is partially mediated by dopaminergic neurons.
Effect of Tryptophan on Extracellular Concentrations of Tryptophan and 5‐Hydroxyindoleacetic Acid in the Striatum and Cerebellum
Authors: Sarna G. S. , Hutson P. H. , O'Connell M. T. , Curzon G.
Published: October 2006

Abstract: The effects of l‐tryptophan (50 mg/kg i.p.) on extracellular concentrations of tryptophan and the 5‐hydroxy‐tryptamine (5‐HT) metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) were determined in the rat striatum and cerebellum, regions with rich and poor 5‐HT innervation, respectively. Determinations were on perfusates from dialysis probes in the brains of conscious, freely moving rats. The pharmaco‐kinetic profiles of dialysate tryptophan after tryptophan load (peak concentration, time to peak concentration, area under curve, and half‐life) in the two regions did not differ significantly. The dialysate 5‐HIAA concentration in the striatum rose two‐ to threefold after the administration of tryptophan. Therefore, as 5‐HIAA was undetectable in the cerebellum either before or after the administration of tryptophan, the increase of 5‐HIAA in the striatum is unlikely to depend appreciably on its production within the cerebral vasculature or outside the brain or on its entering the striatum through a blood‐brain barrier damaged by placement of the dialysis probe. Overall, the findings strengthen previous evidence that extracellular 5‐HIAA concentrations determined by cerebral dialysis are a valid measure of the metabolism of 5‐HT of brain neuronal origin.
Characterisation of Distinct Inositol 1,4,5‐Trisphosphate‐Sensitive and Caffeine‐Sensitive Calcium Stores in Digitonin‐Permeabilised Adrenal Chromaffin Cells
Authors: Robinson Iain M. , Burgoyne Robert D.
Published: October 2006

Abstract: The effect of inositol 1,4,5‐trisphosphate [Ins‐(1,4,5)P3] and caffeine on Ca2+ release from digitonin‐per‐meabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura‐2 to monitor [Ca2+]. Permea‐bilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40–50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(l,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(l,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(l,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m‐chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(l,4,5)P3‐ or caffeine‐sensitive stores. Ca2+ release from the caffeine‐sensitive store was selectively blocked by ryano‐dine. The Ins(l,4,5)P3‐sensitive store was emptied by thap‐sigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2+‐ATPases.
The GTP‐Insensitive Component of High‐Affinity [3H]8‐Hydroxy‐2‐(Di‐n‐Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5‐Hydroxytryptamine1A Receptor
Authors: Emerit M. B. , Miquel M. C. , Gozlan H. , Hamon M.
Published: October 2006

Abstract: Previous studies on central 5‐hydroxytryptamine1A (5‐HT1A) receptors have consistently shown the existence of a GTP‐insensitive component of agonist binding, i.e., binding of [3H]8‐hydroxy‐2‐(di‐n‐propylamino)tetralin ([3H]8‐OH‐DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp‐insensitive component of [3H]8‐OH‐DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3‐[3‐(cholamidopropyl)dimethylammonio]‐1‐propane sulfonate‐soluble extracts to air; it was reduced by preincubation of solubilized 5‐HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short‐cross‐linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp‐insensitive over total [3H]8‐OH‐DPAT binding. The pharmacological properties of this GppNHp‐insensitive component of [3H]8‐OH‐DPAT binding were similar to those of 5‐HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5‐HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5‐HT1A receptor binding subunit (R[5‐HT1A]) and a guanine nucleotide‐binding protein (G protein). Moreover, the oxidation of ‐SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8‐OH‐DPAT specific binding by N‐ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same ‐SH groups on the R[5‐HT1A]–G protein complex. These data suggest that the appearance of GTP‐insensitive [3H]8‐OH‐DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential ‐SH groups (different from those preferentially inactivated by N‐ethylmaleimide) on the R[5‐HT1A]–G protein complex.
Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ‐Aminobutyric AcidA Receptors
Authors: Khrestchatisky Michel , MacLennan A. John , Tillakaratne Niranjala J. K. , Chiang Ming‐Yi , Tobin Allan J.
Published: October 2006

Abstract: A cDNA from a rat hippocampal cDNA library encodes an isoform of the α polypeptide of the γ‐aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the α2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor α polypeptides and shares 71–76% identity with previously described rat α polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the α2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined “BZ type II” receptors. Other workers have previously shown that the α polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of α2 mRNAs in rat brain suggests that the α2 subunit may indeed be involved in the BZ type II receptors.
Levels of Proteolipid Protein mRNAs in Peripheral Nerve Are Not Under Stringent Axonal Control
Authors: Gupta Sanjoy K. , Pringle Joan , Poduslo Joseph F. , Mezei Catherine
Published: October 2006

Abstract: The proteolipid protein (PLP) is the major protein in the myelin sheath of the CNS. It was recently reported that PLP coding transcripts are also found in the PNS, although the protein was not detectable in peripheral nerve myelin. In the present investigation, levels of mRNA for PLP in sciatic nerve were studied during development and following transection and crush injury. Results were compared to those for P0, the major PNS myelin protein, and the myelin‐associated glycoprotein (MAG). PLP transcript levels were very low at 21 days in sciatic nerve and remained unchanged in the adult sciatic nerve. This contrasts markedly with P0 and MAG mRNAs, which are expressed at high levels during development and decrease in content significantly by adulthood. The level of PLP messages was reduced ∼40% in the quiescent Schwann cells in the distal segment of the sciatic nerve at 21 days after permanent transection, yet P0 mRNA levels were very low, and MAG mRNAs were undetectable in this tissue. The distal segment of the crush‐injured sciatic nerve is characterized by transient demyelination followed by rapid myelination. PLP mRNA levels remained comparatively unaffected in the 3‐week period following crush injury. RNase protection experiments using two antisense riboprobes confirmed that levels of PLP‐derived protected fragments, corresponding to PLP and DM‐20 messages, remained unchanged in the developing and adult sciatic nerve. These results indicate that myelin‐specific P0 and MAG genes are tightly controlled at the level of transcription through Schwann cell‐axonal interactions, whereas PLP transcription in the peripheral nerve remains nearly dissociated from axonal influences.
In Vitro and In Vivo Pharmacology of trans‐ and cis‐(±)‐1‐Amino‐1,3‐Cyclopentanedicarboxylic Acid: Dissociation of Metabotropic and Ionotropic Excitatory Amino Acid Receptor Effects
Authors: Schoepp Darryle D. , Johnson Bryan G. , Salhoff Craig R. , McDonald John W. , Johnston Michael V.
Published: October 2006

Abstract: This study explored further the function of the metabotropic excitatory amino acid receptor in the rat brain. The trans and cis isomers of (±)‐1‐amino‐1,3‐cyclopentane‐dicarboxylic acid (ACPD) were characterized for relative affinities at ionotropic and metabotropic excitatory amino acid receptors in vitro, as well as ability to produce in vivo excitatory or excitotoxic effects in rats. trans‐ACPD was about 12 times more potent in vitro as an agonist for metabotropic excitatory amino acid receptors when compared to its ability to displace N‐methyl‐D‐aspartate (NMDA) ([3H]CGS‐19755) receptor binding. cis‐ACPD was about 30 times more potent as a displacer of [3H]CGS‐19755 binding than as a stimulant of phosphoinositide hydrolysis. When administered intra‐peritoneally to neonatal rats, both cis‐ and trans‐ACPD produced convulsions that were prevented by the competitive NMDA receptor antagonists, LY233053 and LY274614. cis‐ACPD was six times more potent as a convulsant when compared to trans‐ACPD. Both compounds were examined for excitotoxic effects in vivo following stereotaxic injection into the mature or neonatal rat striatum. Doses of trans‐ACPD of up to 5,000 or 1,200 nmol produced few signs of striatal neuronal degeneration in the mature or neonatal brain, respectively. However, cis‐ACPD produced extensive dose‐related neuronal degeneration at doses of 100–1,000 nmol in the mature brain and 50–200 nmol in the neonatal brain. These studies suggest that, unlike the ionotropic excitatory amino acid receptors, activation of the metabotropic excitatory amino acid receptor does not result directly in excitatory effects, such as excitotoxicity.
Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes
Authors: Mannon Peter J. , Mervin Sabrena J. , Taylor Ian L.
Published: October 2006

Abstract: The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS). The binding of 125I‐NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I‐NPY binding to solubilized receptors with relative potencies similar to those seen with membrane‐bound receptors: NPY > PYY ± pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 ± 0.4 nM (mean ± SEM) and a Bmax of 5.0 ± 0.2 pmol/mg of protein. In addition the 125I‐NPY binding to the soluble receptor was not inhibited by guanosine‐5′‐O‐(3‐thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane‐bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of ∼67,000, and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis after chemical cross‐linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50‐ to 100‐fold purification of the NPY receptor was obtained.
Removal of C6 Astrocytoma Cell Surface Molecules with Phosphatidylinositol Phospholipase C: Effect on Regulation of Neurol Cell Adhesion Molecule Isoforms
Authors: Theveniau Magali , Guo Xiao‐Jun , Rage* Pierre , Rougon GeneViève
Published: October 2006

Abstract: Different membrane‐associated isoforms of the Neurol cell adhesion molecules have been described. One of them, N‐CAM120, has been shown to be anchored to the membranes by a complex glycan‐phosphatidylinositol group and to be releasabie, under soluble form, by the bacterial enzyme phosphatidylinositol‐phospholipase C. We used the C6 rat astrocytoma cell line expressing both N‐CAM120 and the transmembrane isoform N‐CAM 140 as a model system. We investigated whether artificial depletion of cell membrane N‐CAM 120 influences the synthesis and the messenger RNA transcript levels of the isoforms of the Neurol cell adhesion molecules. Our results showed an increase in the rate of N‐CAM120 protein synthesis, whereas the expression of N‐CAM140 decreased. Additionally, perturbations in the levels of the 6.7‐kb messenger RNA encoding for N‐CAM 140 were observed, whereas the 2.7‐kb transcript encoding for N‐CAM120 remained stable. Examination of the time course for the reexpression of N‐CAM 120 showed that control levels were recovered after 24 h. We provide evidence that: N‐CAM 120 spontaneously released in the culture medium is not incorporated into the extracellular matrix; however, its concentration is important because, if the medium was changed, cells rapidly released a new pool.
A Pi Form of Glutathione‐S‐Transferase Is a Myelin‐and Oligodendrocyte‐Associated Enzyme in Mouse Brain
Authors: Tansey Francine A. , Cammer Wendy
Published: October 2006

Abstract: The pi form of glutathione‐S‐transferase (GST), previously found to be oligodendrocyte associated, has also been found in myelin. In the brains of adult mice, immunocytochemical staining for a pi form of GST was observed in myelinated fibers, as well as oligodendrocytes. In contrast, and as previously found in rats, positive immunostaining for mu forms occurred in astrocytes and not in oligodendrocytes or myelinated fibers. Double immunofluorescence staining strengthened the conclusion that pi was in oligodendrocytes and myelin in mouse brains. The results of enzyme assays performed on brain homogenates and purified myelin indicated that GST specific activities in mouse brain myelin were almost as high (0.8‐fold) as those in mouse brain homogenates. Low, but reproducible, GST activities were also observed in myelin purified from rat brains, in which pi had been demonstrated in oligodendrocytes and mu in astrocytes. The pi form was also stained by the immunoblot technique in myelin purified from mouse brain. It was concluded that pi is a myelin associated, as well as oligodendrocyte associated, enzyme in mouse brain, and possibly also in rat brain. This is the first report of localization of GSTs in mouse brain and of GST in myelin.
Quantitative Microdialysis: Analysis of Transients and Application to Pharmacokinetics in Brain
Authors: Morrison Paul F. , Bungay Peter M. , Hsiao John K. , Ball Beth A. , Mefford Ivan N. , Dedrick Robert L.
Published: October 2006

Abstract: The behavior of a microdialysis probe in vivo is mathematically described. A diffusion‐reaction model is developed that not only accounts for transport of substances through tissues and probe membranes but also accounts for transport across the microvasculature and metabolism. Time‐dependent equations are presented both for the effluent microdialysate concentration and for concentration profiles about the probe. The analysis applies either to measuring the tissue pharmacokinetics of drugs administered systemically, or for sampling of endogenously produced substances from tissue. In addition, an expression is developed for the transient concentration about the probe when it is used as an infusion device. All mathematical expressions are found to be a sum of an algebraic and an integral term. Theoretical prediction of time‐dependent probe behavior in brain has been compared with experimental data for acetaminophen administered at 15 mg/kg to rats by intravenous bolus. Plasma and whole striatal tissue samples were used to describe plasma kinetics and to estimate a capillary permeability‐area product of 0.07 min‐1. Theoretical prediction of transient effluent dialysate concentrations exhibited close agreement with experimental data over 60 min. Terminal decline of the dialysate effluent concentration was slightly overestimated but theoretical concentrations still lay within the 95% confidence interval of the experimental data at 112 min. Microvasculature transport and metabolism play major roles in determining microdialysate transient responses. Extraction fraction (recovery) has been shown to be a declining function in time for five probe operating conditions. High rates of metabolism and/or capillary transport affect the time required to approach steady‐state extraction, shortening the time as the rates increase. Conversely, for substances characterized by low permeabilities and negligible metabolism, experimental situations exist that are predicted to have very slow approaches to microdialysis steady state.
Rapid Degradation of Newly Synthesized Tubulin in Lithium‐Treated Sensory Neurons
Authors: Bennett Gudrun S. , Hollander Brian A. , Laskowska Danuta , DiLullo Camille
Published: October 2006

Abstract: When cultured chick sensory neurons were labeled with [35S]methionine for 1 h or longer in the presence of 5–25 mM LiCl, we found a dose‐dependent reduction in the level of radiolabeled tubulin, to one third of control levels, with no noticeable effect on other proteins. The magnitude of this response was identical after a 1‐h or 72‐h preincubation in 25 mM LiCl and returned to control values within 1 h after removal of LiCl. Short (5‐min) pulse‐chase experiments revealed that tubulin synthesis was not affected by Li+, but that newly synthesized tubulin was rapidly degraded, such that 50% of the labeled β‐tubulin was lost within 5 min. There was no enhanced degradation of tubulin present before exposure to Li+. Addition of LiCl at various times before and after a 10‐min pulse suggested that tubulin becomes completely refractory to Li+‐induced degradation within 10 min after translation. Although Li+ treatment resulted in a decrease in the fraction of extant tubulin present in the unassembled form, the Li+‐induced degradation of nascent tubulin is not a consequence of shifts in assembly state, because colcemid or taxol treatment did not lead to rapid degradation of newly synthesized tubulin, and neither drug altered the response to Li+. We suggest that Li+ interferes with the corre ct folding of tubulin polypeptides, exposing sites, normally hidden, to the action of a protease(s).
Decreased Long‐Chain Fatty Acyl CoA Elongation Activity in Quaking and Jimpy Mouse Brain: Deficiency in One Enzyme or Multiple Enzyme Activities?
Authors: Suneja S. K. , Nagi M. N. , Cook L. , Cinti D. L.
Published: October 2006

Abstract: Using long‐chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a decrease in overall fatty acid chain elongation activity was observed in the quaking and jimpy mouse brain microsomes relative to controls. Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities were depressed to about 50% and 80% of control values in quaking and jimpy mice, respectively. Measurement of the individual enzymatic activities of the elongation system revealed a single deficiency in enzyme activity; only the condensation activity was reduced to the same extent as total elongation in both quaking and jimpy mice. The activities of the other three enzymes, β‐ketoacyl CoA reductase, β‐hydroxyacyl CoA dehydrase, and trans‐2‐enoyl CoA reductase, in both mutants were similar to the activities present in the control mouse. In addition, the activities of these three enzymes were more than two to three orders of magnitude greater than the condensing enzyme activity in all three groups, establishing that the condensing enzyme catalyzes the rate‐limiting reaction step of total elongation. When the elongation of palmitoyl CoA was measured, only a 25% decrease in total elongation occurred in both mutants; a similar percent decrease in the condensation of palmitoyl CoA also was observed. The activities of the other three enzymes were unaffected. These results support the concept of either multiple elongation pathways or multiple condensing enzymes.
Biosynthesis of Prostaglandins D2 and E2 in Chick Dorsal Root Ganglion During Development
Authors: Vesin Marie‐Françoise , Droz Bernard
Published: October 2006

Abstract: Newly formed prostaglandins (PGs), which are assumed to act as modulators of afferent sensory messages, were studied in chick dorsal root ganglia (DRG) during development, [1‐14C]Arachidonic acid was converted by DRG homogenates from 1‐week‐old chickens into two major 14C‐PGs: PGE2 and PGD2. The enzymatic conversion of arachidonic acid was characterized as follows: (a) Boiled preparations were inactivated; (b) synthesis of PGs was inhibited by pretreatment with aspirin or indomethacin and enhanced by esculetin, a protector of cyclooxygenase; and (c) [14C]PGE2and [14C]PGD2 accumulation was a protein dose‐dependent process. Further fractionation of crude homogenates indicated that PG endoperoxide synthetase (EC 1.14.99.1) and PGE2synthetase (EC 5.3.99.3) were membrane‐bound enzymes, whereas PGD2 synthetase (EC 5.3.99.2) was recovered in the cytosol. During development, from embryonic day 10 to day 14 after hatching, PGD2 synthetase activity remained constant; in contrast, a sharp rise in [14C]PGE2 synthesis was observed from embryonic day 14 to 18. The time curves of PGD2 and PGE2 synthetase specific activity may be related to changes taking place in the cell population of developing DRG. It is therefore suggested that arachidonic acid would be enzymatically converted early into PGD2 by maturing ganglion cells and then later into PGE2 by proliferating fibroblasts.
Down‐Regulation of Pinealocyte Protein Kinase C: Effect on α1‐Adrenergic Potentiation of β‐Adrenoceptor Stimulation of Cyclic AMP Accumulation and Induction of Serotonin N‐Acetyltransferase Activity
Authors: Sugden David
Published: October 2006

Abstract: Treatment of rat pinealocytes with 4β‐phorbol 12, 13‐dibutyrate down‐regulated protein kinase C (PKC) activity. Loss of activity was concentration‐dependent (50% loss at 8 × 10‐7M after 18 h of treatment) and time‐dependent (50% loss after 2 h with 3 × 10‐6M). Phenylephrine, an α1‐adrenergic agonist, and phorbol esters unable to activate PKC did not down‐regulate the enzyme. α1‐Adrenergic amplification of β‐adrenergic stimulation of cyclic AMP accumulation, a response previously shown to be mediated by PKC activation, was reduced by only 50% in cells in which PKC activity was down‐regulated by approximately 95%. These data suggest that there is not a simple proportional relationship between the degree of activation of pinealocyte PKC and the α1‐adrenergic amplification of β‐adrenergic cyclic AMP synthesis. In down‐regulated cells, α1‐adrenergic amplification of β‐adrenergic induction of serotonin N‐acetyltransferase activity, a key cyclic AMP‐responsive enzyme involved in the nocturnal synthesis of the pineal hormone melatonin, was unchanged. Thus, even though α1‐adrenergic amplification of cyclic AMP synthesis is impaired, sufficient cyclic AMP is generated to allow a full induction of serotonin N‐acetyltransferase activity. This finding raises the important question of whether the α1‐adrenergic amplification mechanism has a physiological role in regulating melatonin synthesis in vivo.
Changes in Extracellular Amino Acids and Spontaneous Neuronal Activity During Ischemia and Extended Reflow in the CA1 of the Rat Hippocampus
Authors: Andiné Peter , Orwar Owe , Jacobson Ingemar , Sandberg Mats , Hagberg Henrik
Published: October 2006

Abstract: This study addresses the possible involvement of an agonist‐induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four‐vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple‐unit activity. Multiple‐unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and ‐γ‐aminobutyric acid increased in both acute and chronic experiments (seven‐to 26‐fold). Multiple‐unit activity recovered to preischemic levels following 4–6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple‐unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple‐unit activity recovered faster and reached 140% of control at 8h, paralleled by an increase in extracellular aspartate (5.5‐fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.
Extracellular Acidic Sulfur‐Containing Amino Acids and γ‐Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin‐Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus
Authors: Andiné Peter , Orwar Owe , Jacobson Ingemar , Sandberg Mats , Hagberg Henrik
Published: October 2006

Abstract: An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia‐induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple‐unit activity and extracellular changes in acidic sulfur‐containing amino acids and‐γ‐glutamyl peptides during ischemia (20‐min, four‐vessel occlusion) and during 8 h of reflow. Multiple‐unit activity was abolished during ischemia and for the following 1h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and γ‐glutamyltaurine increased (1.5‐to threefold) during ischemia, and extracellular glutathione and γ‐glutamylaspartate plus γ‐glutamylglutamine increased during early reflow (two‐to threefold). The recovery of neuronal activity at 4–8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5‐fold at 8 h of reflow). Perfusion with 10 μM tetrodotoxin at 8 h of reflow abolished the multiple‐unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate‐like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.