bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Multisulphated N‐Linked Glycans in the L2/HNK‐1 Carbohydrate Epitope Expressing Neural Adhesion Molecule P0
Authors: Field M. C. , Wing D. R. , Dwek R. A. , Rademacher T. W. , Schmitz B. , Bollensen E. , Schachner M.
Published: October 2006

Abstract: P0, the most abundant glycoprotein of PNS myelin, is a homophilic and heterophilic adhesion molecule. P0 is known to contain a glycofonn population that expresses the L2/HNK‐1 carbohydrate epitope found on other neural adhesion molecules, and to be functionally implicated centrally in neural cell adhesion and neurite outgrowth. This carbohydrate epitope has been characterized previously from glycolipid structures and contains a sulphated glucuronic acid residue. However, the L2/HNK‐1 carbohydrate epitope has not been characterized in glycoproteins. Because P0 possesses only one glycosylation sequon, the number of P0 glycoforms is equal to the heterogeneity of the glycan species. Here we report that the carbohydrate analysis of L2/HNK‐1‐reactive P0 showed the presence of anionic structures containing sialic acid and sulphate in various combinations. At least one sulphate residue was present in 80% of the monosaccharide sequences, and 20% contained three sulphates. High‐resolution P4 gel chromatography of the desialylated and desulphated oligosaccharides showed substantial heterogeneity of monosaccharide sequences. Sequential exoglycosidase digestions indicated that the majority of the structures were of the hybrid class, although the sulphated structures were found to be en‐doglycosidase H‐resistant.
Brain Serotonin2 and Serotonin1A Receptors Are Altered in the Congenitally Hyperammonemic Sparse Fur Mouse
Authors: Robinson Michael B. , Anegawa Norifusa J. , Gorry Eileen , Qureshi Ijaz A. , Coyle Joseph T. , Lucki Irwin , Batshaw Mark L.
Published: October 2006

Abstract: In previous studies we documented an increase in the levels of the serotonin metabolite, 5‐hydroxyindoleacetic acid, in the congenitally hyperammonemic sparse fur mouse. To extend these findings, brain serotonin receptors were studied in these animals. Radioligand binding assays were performed using [3H]ketanserin to label Serotonin2 sites and 8‐[3H]hydroxy(di‐H‐propylamino)tetralin to label serotonin iA sites in cortical membrane homogenates. The capacity (5max) for [3H]ketanserin binding was significantly lower (−21%; p < 0.05) in sparse fur animals than in control animals; there was no change in affinity (ATD). In contrast, the capacity for 8‐[3H]hydroxy(di‐n‐propylamino)tetralin binding was significantly greater (26%; p < 0.05) in sparse fur compared with control animals. No difference in affinity was observed. Using two behavioral assays, the functional responsiveness of these serotonin receptors was compared in sparse fur and control animals. Head twitch activity elicited by administration of the serotonin agonist quipazine was studied as a behavior mediated by serotonin receptors. Compared with controls, sparse fur mice demonstrated a significantly decreased head twitch response (p < 0.005). Hypothermia elicited by administration of 8‐hydroxy(di‐n‐propylamino)tetralin was studied as a physiologic response mediated by serotoniniA receptors. Although mere were not overall group differences in the dose‐response data, there was a significant increase in the hypothermia induced by 8‐hydroxy(di‐n‐propylami‐no)tetralin in sparse fur compared with control mice (p < 0.02) at the highest dose. These data provide further support for a link between hyperammonemia and alterations in the serotonin system.
Agonist‐Stimulated Inositol Polyphosphate Formation in Cerebellum
Authors: Sastry Pidiparty S. , Dixon John F. , Hokin Lowell E.
Published: October 2006

Abstract: The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5‐trisphosphate [Ins(l,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second‐messenger inositol 1,3,4,5‐tetra‐kisphosphate [Ins(l,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4‐trisphosphate [Ins(l,3,4)P3], eightfold; Ins(l,3,4,5)P4, 6.4‐fold; Ins(l,4,5)P3, 75%; inositol 1,4‐bisphosphate [Ins(1,4)P2, 0%; inositol 3,4‐bisphosphate, 100%; inositol 1‐phosphate/inositol 3‐phosphate, 30%; and inositol 4‐phosphate, 40%. [3H]Inositol 1,3‐bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin‐stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(l,4,5)P3 is metabolized primarily by Ins(1,4,5)P3‐3’ kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 nM), [3H]Ins(l,3,4,5)P4 and [3H]Ins(l,3,4)P3 levels were increased, even at 1 μM serotonin. Ketanserin (7 μM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 μM) also increased the levels of [3H]Ins(l,4,5)P3, [3H]Ins(l,3,4,5)P4, and [3H]Ins(l,3,4)P3, but the profile of these increases was different. The quisqualic acid‐stimulated formation of inositol phosphate isomers was not affected by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, indicating that it was not due to the ionotropic properties of the quisqualate receptor. Similar results were obtained on stimulation of the labeled slices with glutamate, but the magnitudes were less. The data show that in the guinea pig cerebellum, stimulation of the serotonin2 and metabotropic quisqualic acid receptors leads to the initial formation of Ins(l,4,5)P3, but its subsequent metabolism varies, presumably owing to two kinds of receptors, localized on different cell types in the cerebellum with varying levels of inositol phosphate‐metabolizing enzymes.
Bicuculline‐ and Baclofen‐Insensitive γ‐Aminobutyric Acid Binding to Rat Cerebellar Membranes
Authors: Drew Colleen A. , Johnston Graham A. R.
Published: October 2006

Abstract: Up to 60% of γ‐[3H]aminobutyric acid ([3H]GABA) bound specifically to rat cerebellar membranes in the absence of Ca2+ was insensitive to the GABAA antagonist bicuculline and to the GABAB agonist baclofen. This indicates that a significant component of specifically bound [3H]GABA is associated with non‐GABAA, non‐GABAB binding sites. The presence of this binding component appeared seasonal, peaking in the month of September (early spring) each year over a 4‐year period. The calcium independence and bicuculline and baclofen insensitivity of the binding indicate that this binding is not to the classical GABAA and GABAB binding sites. High concentrations of muscimol and isoguvacine inhibited non‐GABAA, non‐GABAB binding. Scatchard analysis of the non‐GABAA, non‐GABAB binding sites indicated two kinetic components: KDI= 42 nM and KD2= 9.2 μM; Bmaxi= 1.6 pmol/mg of protein and Bmax2= 28 pmol/mg of protein.
Expression of Size‐Selected RNA Encoding Brain Serotonin Transporter in Xenopus laevis Oocytes
Authors: Haber Roberta , Goldman David
Published: October 2006

Abstract: The mRNA that encodes a serotonin transporter was expressed using the Xenopus laevis oocyte expression system. Poly(A)+ RNA isolated from mouse brainstem was injected into Xenopus laevis oocytes, and the ability of oocytes to take up serotonin was measured 3 days postinjection. RNA‐dependent serotonin uptake was sensitive to citalopram, a specific inhibitor of serotonin uptake, whereas background levels of serotonin uptake were not citalopram sensitive. Two RNA size fractions, 4.0 and 4.5 kb, were most efficient in stimulating uptake. Injection into Xenopus laevis oocytes of the 4.5‐kb size fraction of mouse brainstem RNA resulted in threefold more serotonin uptake than did injection of unfractionated poly(A)+ RNA.
Highly Ouabain‐Sensitive α3 Isoform of Na+,K+‐ATPase in Human Brain
Authors: Peng J. H. Frank , Zeng Yucheng , Parker Joseph C.
Published: October 2006

Abstract: The Na+,K+‐ATPase α3 isoform has recently been demonstrated immunochemically in human brain. Conclusive biochemical evidence, however, is still lacking. In this study, a unique 50‐kDa polypeptide, which is known to be specific to the rat α3 isoform, has been found in human brainstem Na+,K+‐ATPase following formic acid treatment of the purified a isoform proteins. Human α3 Na+,K+‐ATPase is also highly sensitive to ouabain inhibition, with a 50% ouabain inhibition value of 1.0 X 10−7 M. These results provide clear and direct evidence for the existence of the α3 isoform in human brain. Na+,K+‐ATPase‐α1, α2, and α3 isoforms—Human and rat brainstem axolemma—Ouabain sensitivity—Formic acid treatment. Peng J. H. F. et al. Highly ouabain‐sensitive α3 isoform of Na+,K+‐ATPase in human brain. J. Neurochem.58, 1180–1183(1992).
Brain‐Derived Neurotrophic Factor Is More Highly Conserved in Structure and Function than Nerve Growth Factor During Vertebrate Evolution
Authors: Götz Rudolf , Raulf Friedrich , Schartl Manfred
Published: October 2006

Abstract: Mammalian nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three‐dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fish Xiphophorus, with the mammalian homlogues. Genomic clones encoding fish NGF and BDNF were isolated by cross‐hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half‐maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purified recombinant BDNF. However, the activity of recombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng/ml, 75‐fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA‐deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF had reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates from a common ancestral gene about 600 million years ago. By RNA gel blot analysis NGF mRNA was detected during late embryonic development; BDNF was present in adult brain.
Effects of Muscarinic Agonists and Depolarizing Agents on Inositol Monophosphate Accumulation in the Rabbit Vagus Nerve
Authors: Sierro Christian D. , Vitus Josée , Dunant Yves
Published: October 2006

Abstract: The effects of muscarinic agonists and depolarizing agents on inositol phospholipid hydrolysis in the rabbit vagus nerve were assessed by the measurement of [3H]inositol monophosphate production in nerves that had been preincubated with [3H]inositol. After 1 h of drug action, carbachol, oxotremorine, and arecoline increased the inositol monophosphate accumulation, though the maximal increase induced by these agonists differed. Addition of the muscarinic antagonists atropine or pirenzepine shifted the carbachol dose‐response curves to the right, without decreasing the carbachol maximal stimulatory effects. The KB for pirenzepine was 35 nM, which is characteristic of muscarinic high‐affinity binding sites coupled to phosphoinositide turnover and often associated with the M1 receptor subtype. On the other hand, agents known to depolarize or to increase the intracellular Ca2+ concentration, e.g., elevated extracellular K+, ouabain, Ca2+, and the Ca2+ ionophore A23187, also increased inositol monophosphate accumulation. These effects were not mediated by the release of acetylcholine, as suggested by the fact that they could not be potentiated by the addition of physostigmine nor inhibited by the addition of atropine. The Ca2+‐channel antagonist Cd2+, also known to inhibit the Na+/Ca2+ exchanger, was able to block the effects of K+ and ouabain, but did not alter those of carbachol. These results suggest that depolarizing agents increase inositol monophosphate accumulation in part through elevation of the intracellular Ca2+ concentration and that muscarinic receptors coupled to phosphoinositide turnover are present along the trunk of the rabbit vagus nerve.
G Protein Modulation of ω‐Conotoxin Binding Sites in Neuroblastoma X Glioma NG 108‐15 Hybrid Cells
Authors: Bergamaschi S. , Govoni S. , Battaini F. , Trabucchi M. , Monaco S. Del , Parenti M.
Published: October 2006

Abstract: Electrophysiological evidence shows that voltage‐dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)‐mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for ω‐conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high‐threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108‐15 cells were used. In both crude cellular extracts specific binding of 125I‐CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP‐PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I‐CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5′‐O‐(2‐thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP‐PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the a subunits of Gi1‐2 and Go suggest that Go is the G protein responsible for the observed effect.
Reconstitution of the Brain Mixed Function Oxidase System: Purification of NADPH‐Cytochrome P450 Reductase and Partial Purification of Cytochrome P450 from Whole Rat Brain
Authors: Bergh Arthur F. , Strobel Henry W.
Published: October 2006

Abstract: NADPH‐cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blot techniques. Kinetic studies using cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P450c (P4501A1) as measured by 7‐ethoxycoumarin and γ‐ethoxyresorufin O‐deethylation. Our results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.
Novel Procedure for Measuring Psychosine Derivatives by an HPLC Method
Authors: Nozawa Masayo , Iwamoto Takeo , Tokoro Toshiharu , Eto Yoshikatsu
Published: October 2006

Abstract: We developed a sensitive and simple method to determine galactosylsphingosine and glucosylsphingosine as a 4‐fluoro‐7‐nitrobenzofurazan autofluorescent compound, using HPLC equipped with a Showdex sugar column. Amounts of galactosylsphingosine were successfully measured in the picomole range. This novel procedure is more stable and simpler than the previous method using o‐phthalaldehyde. It was applied to tissues from the twitcher mouse, an animal model of human globoid cell leukodystrophy. The amount of galactosylsphingosine was 34‐102 μ/kg of wet tissues in control cerebrum and cerebellum, whereas in twitcher mice the range was 2,251‐4,228 μ/kg of wet tissues. The psychosine concentration was also increased in the liver and kidney of twitcher mice, respectively, 1,513 μg; and 1,106 μ/kg of wet tissue (normal liver, 125 μg; normal kidney, 74 μ/kg of wet tissue). This novel procedure is useful for the pathochemical evaluation of lysosphingolipids in various sphingolipidoses as well as in other neuropathological and cellular conditions.
Characterization of D‐Aspartic Acid Uptake by Rat Hippocampal Slices and Effect of Ischemic Conditions
Authors: Kuwahara Osamu , Mitsumoto Yasuhide , Chiba Kenzo , Mohri Tetsuro
Published: October 2006

Abstract: The cellular uptake of D‐aspartic acid (D‐Asp) as a model compound for glutamic acid transport was studied in rat hippocampal slices. 1‐Asp is accumulated by both Na+‐dependent and Na+‐independent processes in hippocampal slices, and both processes are dependent on temperature. The Na+‐dependent uptake is assumed to be high in affinity (apparent Km = 0.17 mM), but low in capacity, whereas the Na+‐independent uptake is much lower in affinity (Km = 2.86 mM), but higher in capacity. l‐Aspartic acid, l‐glutamic acid, dihydrokainic acid, and threo‐3‐hydroxy‐DL‐aspartic acid markedly inhibited the uptake of D‐Asp with Na+ in the medium, whereas D‐glutamic acid, glycine, and l‐lysine had no significant effect. The Na+‐dependent uptake of D‐Asp was significantly reduced under “hypoglycemic,”“anoxic,” and “ischemic” conditions, whereas the Na+‐independent uptake was unaffected. Metabolic inhibitors such as NaCN and ICH2COOH significantly inhibited the Na+‐dependent uptake, but not the Na+‐independent uptake. These results suggest that the Na+‐dependent component of D‐Asp transport in rat hippocampal cells is inactivated under ischemic conditions, whereas the Na+‐indepen‐dent component is unaffected.
Neuronal Ganglioside Increases Dependent on the Neuron‐Glia Interaction in Primary Culture
Authors: Ogiso Manabu , Ohta Masako , Harada Yasushi , Hirano Shusuke
Published: October 2006

Abstract: Dissociated neuronal cells from rat embryonic hemispheres were cultivated on astroglial layers. The increase in ganglioside content of the cocultures was more rapid than that of neuronal cultures seeded on polylysine surfaces for the first 24 h, and the extent of the increase was greater 7 days after inoculation, probably because of interaction between the preformed astroglial layers and the neuronal cells in vitro. The promoted expression of the a‐pathway gangliosides, GM1 and GD la, was recognized by TLC and the increase in GM 1 was immunologically ascertained. The incorporation of 3H‐labeled N‐acetyl‐D‐mannosamine into GD3 and b‐series gangliosides was elevated for the first 24 h. However, cocultures in which there was no contact between neuronal cells and the astroglial sheet showed no appreciable increase in incorporation. Thus, cell surface changes were induced at the membrane glycolipid level in the neuronal cells by contact with astroglial layers. The synthesis and expression of neuronal gangliosides are discussed in relation to the onset of neuron‐glia interaction.
Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH‐SY5Y Cells
Authors: Larsson Christer , Saermark Torben , Mau Søren , Simonsson Per
Published: October 2006

Abstract: The activation of protein kinase C was investigated in digitonin‐permeabilized human neuroblastoma SH‐SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4‐14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19‐36 and was associated to a translocation of the enzyme to the membrane fractions of the SHSY5Y cells. 1,2‐Dioctanoyl‐sn‐glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12‐myristate 13‐acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 pM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p‐bromophenacyl bromide did not inhibit calcium‐ and dioctanoylglycerol‐induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.
Ecto‐5′‐Nucleotidase Is Associated with Cholinergic Nerve Terminals in the Hippocampus but Not in the Cerebral Cortex of the Rat
Authors: Cunha Rodrigo A. , Sebastião A. M. , Ribeiro J. A.
Published: October 2006

Abstract: The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 μM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. α,β‐Methylene ADP (100 μM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50‐fold enrichment in the specific activity of ecto‐5′‐nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto‐5′‐nucleotidase activity in the rat brain, the ecto‐5′‐nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto‐5′‐nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.
Identification and Purification of Calcium‐Independent Phospholipase A2 from Bovine Brain Cytosol
Authors: Hirashima Yutaka , Farooqui Akhlaq A. , Mills John S. , Horrocks Lloyd A.
Published: October 2006

Abstract: Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KC1. However, when buffer with 1 M KC1 was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero‐3‐phosphoethanolamine, whereas the latter was active with both diacyl‐glycero‐3‐phosphoethanolamine and 1‐alk‐1′‐enyl‐2‐acylglycero‐3‐phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel nitration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high‐resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40‐kDa molecular mass and two minor bands with 14‐ and 25‐kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high‐resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The optimal pH values of phospholipase A2 obtained from peaks I and II were 7.4 and 8.0, respectively. Neither phospholipase required Ca2+ for activity. The optimal concentrations of Triton X‐100 for peak I and peak II enzymes were 0.05 and 0.1%, respectively.
Identification of a Novel N‐Methyl‐D‐Aspartate Receptor Population in the Rat Medial Thalamus
Authors: Beaton J. A. , Stemsrud K. , Monaghan D. T.
Published: October 2006

Abstract: To evaluate the possibility of pharmacologically distinct N‐methyl‐D‐aspartate (NMDA) receptor subtypes, quantitative autoradiography was used to determine the potency of several compounds as inhibitors of l‐[3H]glutamate or [3H]MK‐801 binding to rat brain NMDA receptors in 10 brain regions. Competitive NMDA receptor antagonists displayed differing pharmacological profiles in the forebrain, cerebellum, and medial regions of the thalamus (midline nuclei). For example, compared with other competitive antagonists, 3‐[(±)‐2‐carboxypiperazin‐4‐yl]propyl‐1‐phosphonate (CPP) and LY‐233536 were especially weak displacers of l‐[3H]glutamate binding in the cerebellum. In the medial thalamus, CPP and D‐2‐amino‐5‐phosphonopentanoate displayed relatively low affinities, whereas LY‐233536 was relatively potent. The noncompetitive NMDA receptor antagonists also displayed regional variations in their pharmacological profiles. Relative to other regions, [3H]MK‐801 binding in the cerebellum was weakly displaced by MK‐801 and potently displaced by dextromethorphan and SKF‐10047. In the medial thalamus, 1‐[1‐(2‐thienyl)‐cyclohexyl]piperidine was relatively potent and SKF‐10047 was relatively weak. These results confirm previous suggestions that the cerebellum contains a distinct NMDA receptor subtype and indicate that nuclei of the medial thalamus contain a novel NMDA receptor subtype that is distinct from both those found in the cerebellum and in the forebrain.
Rat Brain Adenosine Deaminase After 2′‐Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2′‐[3H]Deoxycoformycin Ligand Binding
Authors: Padua R. A. , Geiger J. D. , Delaney S. M. , Nagy J. I.
Published: October 2006

Abstract: Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2′‐deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF‐treated compared with drug‐naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF‐labeled ADA in DCF‐treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200‐fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r= 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively. The results suggest that (1) although the biochemical properties of ADA are largely unchanged after treatment of rats with DCF, the enzyme in some brain regions has become less sensitive to this drug and (2) the prolonged reduction in ADA activity after DCF treatment is due to decreased levels of ADA protein possibly involving altered rates of degradation or production.
Expression of the Neuronal Surface Glycoprotein Thy‐1 Does Not Follow Appearance of Its mRNA in Developing Mouse Purkinje Cells
Authors: Xue Gang‐Ping , Morris Roger
Published: October 2006

Abstract: In developing rodent nervous system, although the appearance of Thy‐1 mRNA, as seen by in situ hybridisation, is in general quickly followed by the appearance of immunohistochemically detectable protein, there are certain sites where a delay of several days occurs between expression of detectable message and protein. Mouse Purkinje cells exemplify this behaviour and are the dominant Thy‐1‐expressing cell in early postnatal cerebellum, so allowing quantitative, homogenate‐based methods to be used to test whether such a lag in protein expression does occur. Measurement of Thy‐1 mRNA (by slot blot) and protein (by radioimmunoassay) shows a substantial excess of Thy‐1 message, compared to protein accumulating in the tissue, during the first postnatal week, which is not found in tissues (rat cerebellum, and rat or mouse cerebrum) where no lag is apparent in appearance of Thy‐1 protein from the section‐based methods. The species of Thy‐1 mRNA produced by Purkinje cells does not appear to change during development, as assessed either in terms of its size (by northern blotting) or in the heterogeneous pattern of transcription initiation sites used (assessed by S1 nuclease protection analysis). Appearance of Thy‐1 protein in these cells, therefore, seems to be regulated post‐transcriptionally.
Synapsin II Phosphorylation and Catecholamine Release in Bovine Adrenal Chromaffin Cells: Additive Effects of Histamine and Nicotine
Authors: Firestone Jordan A. , Browning Michael D.
Published: October 2006

Abstract: Primary cultures of bovine adrenal medullary chromaffin cells can be stimulated with nicotine, which mimics the cholinergic stimulus from the splanchnic nerve. Histamine also stimulates catecholamine release in a time‐and dose‐dependent manner. We have previously shown that nicotine stimulates incorporation of 32Pi into the vesicle‐associated phosphoprotein synapsin II. We report here that histamine, too, stimulates an increase in 32Pi incorporation into synapsin II, which is blocked by the H1‐histamine receptor‐specific antagonist pyrilamine. The time course of histamine‐stimulated synapsin II phosphorylation closely paralleled that of histamine‐stimulated catecholamine release. Interestingly, histamine and nicotine produced an additive increase in both catecholamine release and synapsin II phosphorylation, suggesting that these two secretogogues stimulate the phenomena via independent mechanisms. When we investigated the dependence of these two agonists on extracellular calcium, we found that nicotine‐stimulated release and synapsin II phosphorylation were reduced to basal levels at low calcium concentrations. However, the histamine‐stimulated effects remained significantly elevated. This suggests that calcium arising from two separate pools can stimulate catecholamine release and synapsin II phosphorylation in bovine chromaffin cells. Taken together, these data support the hypothesis that synapsin II phosphorylation is a component of the secretory response from these cells.
Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes
Authors: Orr Nadav , Yavin Ephraim , Lester David S.
Published: October 2006

Abstract: The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane‐associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein–lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein–lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.
Protein Phosphotyrosine in Mouse Brain: Developmental Changes and Regulation by Epidermal Growth Factor, Type I Insulin‐Like Growth Factor, and Insulin
Authors: Girault Jean‐Antoine , Chamak Brigitte , Bertuzzi Gloria , Tixier Hervé , Wang James K. T. , Pang Dennis T. , Greengard Paul
Published: October 2006

Abstract: Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of Mr 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing thereafter. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin‐like growth factor (IGF‐I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF‐I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF‐I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.
Protein Tyrosine Kinases in Human Brain and Gliomas
Authors: Erp Helmy E. van , Rijksen Gert , van Veelen Cees W. M. , van der Heijden Jessica H. W. , Maarschalkerweerd Benedikte N. B. , Staal Gerard E. J.
Published: October 2006

Abstract: Tyrosine kinase activity was determined in neonatal and adult human brain, oligodendrogliomas, and astrocytomas. The astrocytomas were divided into low‐ (grade I and grade II) and high‐grade (grade III and grade IV) tumors. We measured the tyrosine kinase activity in the cytosolic and membrane fraction using poly(glutamic acid:tyrosine, 4:1) as an artificial substrate. The cytosolic activity in oligodendrogliomas (n = 7), low‐grade astrocytomas (n = 7), and neonatal brain (n = 1) was increased, on average, two‐ to fourfold compared with that in normal adult brain (n = 14). The cytosolic activities of high‐grade astrocytomas (n = 11) were in approximately the same range as found in normal adult brain. The absence of an increase in cytosolic activity in high‐grade astrocytomas compared with adult brain is likely due to the occurrence of necrosis in these tumors. In contrast to the cytosolic activity, no differences were found in the membrane‐bound activity. By fast protein liquid chromatography, at least three forms of cytosolic protein tyrosine kinase could be separated, which eluted at 0, 115, and 210 mM NaCl. In most cases the highest amount of activity eluted at 210 mM NaCl. However, in oligodendrogliomas, highgrade astrocytomas, and neonatal brain, more activity eluted at 115 mM NaCl than in normal adult brain (p= 0.043). Nevertheless, protein tyrosine kinases from all three peaks contributed to the elevated levels of total cytosolic activity of oligodendrogliomas and low‐grade astrocytomas.
Phosphorylation of the MARCKS Protein (P87), a Major Protein Kinase C Substrate, Is Not an Obligatory Step in the Mitogenic Signaling Pathway of Basic Fibroblast Growth Factor in Rat Oligodendrocytes
Authors: Deloulme Jean Christophe , Janet Thierry , Pettmann Brigitte , Laeng Pascal , Knoetgen Marie‐France , Sensenbrenner Monique , Baudier Jacques
Published: October 2006

Abstract: Basic fibroblast growth factor (bFGF) is a well‐characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain‐derived oligo‐dendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c‐fos in OL. The role of protein kinase C (PKC) in mediating bFGF‐stimulated proliferation as well as c‐fos expression in OL was investigated. The PKC activator phorbol 12‐myristate 13‐acetate (PMA) stimulated c‐fos expression but did not trigger cell proliferation. When PKC was down‐regulated by pretreatment of OL with PMA for 20 h, the bFGF‐mediated stimulations of OL proliferation and c‐fos mRNA expression were still observed, whereas the induction of c‐fos mRNA by PMA was totally inhibited. These data demonstrate that the bFGF mitogenic signaling pathway in OLs does not require PKC. On the other hand, bFGF was found to stimulate specifically the phosphorylation of a limited number of PKC substrates in oligodendroglial cells, including the MARCKS protein. The bFGF‐dependent phosphorylation of MARCKS protein was totally inhibited when PKC was first down‐regulated, indicating that the phosphorylation of this protein is PKC dependent. Tryptic digestion of the phosphorylated MARCKS protein revealed that bFGF stimulated specifically the phosphorylation of the MARCKS protein on a single phosphopeptide. We provide evidence that bFGF also stimulated fatty acylation of the MARCKS protein, which might explain the observed specific bFGF‐dependent phosphorylation of this protein in OL. We propose that bFGF‐dependent fatty acylation and phosphorylation of the MARCKS protein are not essential for the transduction of the bFGF mitogenic signal but are probably linked to differentiation processes elicited by bFGF on OL.
Characterization of Solubilized Opioid Receptors: Reconstitution and Uncoupling of Guanine Nucleotide‐Sensitive Agonist Binding
Authors: Ofri Danielle , Ritter Alain M. , Liu Yafang , Gioannini Theresa L. , Hiller Jacob M. , Simon Eric J.
Published: October 2006

Abstract: Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate‐(CHAPS). High concentrations of NaCl (0.5–1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane‐bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High‐affinity binding of μ, δ, and κ agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, μ‐agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][d‐Ala2, N‐methyl‐Phe4,Gly5‐ol]‐enkephalin ([3H]DAGO) binding to guanosine 5′‐O‐(thiotriphosphate) (GTPγS). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTPγS was observed, whereas membrane‐bound receptors were uncoupled only partially. Treatment with GTPγS, at concentrations that uncoupled the μ receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200‐fold. This indicates that the observed decrease in Bmax is due to a reduction in affinity of the uncoupled receptors to a level too low to be measurable, whereas the residual coupled receptors retain high affinity for μ agonists. The methods described should prove useful for opioid receptor purification and reconstitution with G proteins and second messenger systems.
Aging Produces a Specific Pattern of Striatal Dopamine Loss: Implications for the Etiology of Idiopathic Parkinson's Disease
Authors: Kish Stephen J. , Shannak Kathleen , Rajput Ali , Deck John H. N. , Hornykiewicz Oleh
Published: October 2006

Abstract: To examine the possible causal contribution of normal or accelerated aging to the neurodegenerative process of Parkinson's disease, we measured the influence of aging on subregional striatal dopamine and homovanillic acid levels in postmortem brain of 23 neurologically and psychiatrically normal human subjects 14–92 years old. We observed a significant decline in striatal dopamine levels and increase in the homovanillic acid/dopamine molar ratios with increasing age. The dopamine loss, on average, was of the same magnitude in the caudate nucleus and the putamen (‐60% in the 84‐year‐old group as compared with the 22‐year‐old group), with the caudal component of both nuclei being more affected than the rostral subdivisions. The level of subregional dopamine metabolism, as measured by the homovanillic acid/dopamine ratio, in our young individuals (mean age, 22 years) was found to be inversely correlated to the degree of subregional dopamine loss suffered by the individuals in the older age groups. We conclude the following: (a) Striatal subdivisions with physiologically higher dopamine metabolism are not at a greater risk of suffering dopamine neuronal damage with advancing age, as would seem to be implied by the oxidative stress hypothesis; thus, formation of dopamine‐derived oxy radicals in the human striatum appears unlikely to be a primary factor responsible for the age‐related striatal dopamine loss. (b) The regional and subregional pattern of striatal dopamine loss in normal aging differs substantially from the pattern typically observed in idiopathic Parkinson's disease; therefore, the cause of idiopathic Parkinson's disease cannot be primarily an age‐dependent neurodegenerative process.
Opiate‐Dependent Changes in the Sensitivity of Adenylate Cyclase to Stimulatory Agonists and 5′‐Guanylylimidodiphosphate Are Independent of G Protein Abundance and Eukaryotic ADP‐Ribosyltransferase Activity in NG108‐15 Cells
Authors: Boyd Robert S. , Donnelly Louise E. , MacDermot John
Published: October 2006

Abstract: NG108‐15 cells were exposed in culture to 1 μM [d‐Ala2, d‐Leu5]enkaphalin (DADLE) for 17 h. This treatment increased the maximum iloprost‐ and 5′‐(N‐ethylcarboxamido)adenosine‐dependent activation of adenylate cyclase, as well as basal enzyme activity. In addition, there was an increase in the capacity of 5′‐guanylylimidodiphosphate [Gpp(NH)p] to inhibit adenylate cyclase activity by direct interaction with the α‐subunit of the Gi regulatory protein. A similar effect was observed if the cells were exposed to 10 μM carbachol. These treatments of NG108‐15 cells did not alter the capacity of NaF to activate adenylate cyclase by direct interaction with Gsα. Exposure of NG108‐15 cells to DADLE alone or DADLE plus carbachol had no effect on the capacity of pertussis toxin to ADP‐ribosylate membrane proteins in these cells; neither was there any change in the activity of eukaryotic ADP‐ribosyltransferase expressed in these cells. Under these conditions, the endogenous enzyme did not label any protein with a molecular mass similar to Giα, 41 kDa. Treatment of the cells with DADLE or carbachol had no effect on the abundance of Gsα, Giα, or Gβ. The underlying mechanism for the changes in agonist‐dependent stimulatory responses or Gpp(NH)p‐dependent inhibition of adenylate cyclase remains obscure, but appears not to be mediated by eukaryotic ADP‐ribosyltransferase activity or a change in the abundance of G proteins known to regulate adenylate cyclase.
Role of the Central Adrenergic System in the Regulation of Prostaglandin Biosynthesis in Rat Brain
Authors: Weidenfeld J. , Kahbha K. , Reches A. , Shohami E.
Published: October 2006

Abstract: The role of endogenous catecholamines in the regulation of brain prostaglandin (PG) synthesis was studied in the rat. Male rats were injected in the brain lateral ventricle or in the ventral noradrenergic bundle with either the cate‐cholaminergic neurotoxin 6‐hydroxydopamine or vehicle. Other groups of rats were injected intraperitoneally with the tyrosine hydroxylase inhibitor, α‐methyl‐p‐tyrosine, or with the inhibitor of dopamine‐β‐hydroxylase, FLA‐63. All these drugs produced a significant depletion of norepinephrine (NE) content in the cortex and hypothalamus. The rats that had lower levels of NE exhibited reduced capacity to synthesize PGE2 but not thromboxane B2 and 6‐keto‐PGE1α in the cortex and hypothalamus. However, induced production of PG, stimulated by the bacterial endotoxin lipopolysaccharide (LPS), remained unchanged, namely, a similar (2‐ to 2.5‐fold) increase of PG synthesis was noted in control and in NE‐depleted rats. We suggest that the regulation of PG synthesis under basal condition requires intact adrenergic input, whereas LPS‐induced production of PG is independent of the adrenergic innervation.
Quisqualate‐Stimulated Phosphatidylinositol Breakdown in Rat Cerebellar Membranes
Authors: Llahi Silvia , Claro Enrique , Fain John N.
Published: October 2006

Abstract: The effect of quisqualate, an excitatory amino acid agonist, on the breakdown of exogenously added phosphatidylinositol was investigated in a membrane preparation from the cerebellum of young rats. Quisqualate stimulated phospholipase C activity in a dose‐dependent manner in the presence of guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS). Half‐maximal activation of the quisqualate response required 0.15 γM GTPγS and was optimal at a free Ca2+ concentration of 300 nM. Phosphoinositide breakdown was also stimulated by quisqualate using either exogenous phosphatidylinositol 4,5‐bisphosphate or endogenous labeled phosphoinositides as the substrate for phospholipase C in cerebellar membranes. In the presence of guanine nucleotides, other excitatory amino acid agonists, such as l‐glutamate, trans‐d, l‐1‐aminocyclo‐pentyl‐1,3‐dicarboxylic acid, and ibotenate, but not N‐methyl‐d‐aspartate, stimulated phosphatidylinositol breakdown. However, quisqualate displayed the highest response among these excitatory amino acid agonists. These data indicate that there is a direct activation of phosphoinositide‐specific phospholipase C by excitatory amino acids through a process dependent on the presence of guanine nucleotides.