bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Immunohistochemical Localization of a Chondroitin Sulfate Proteoglycan from Adult Rat Brain
Authors: Normand Guy , Kuchler Sabine , Meyer Alphonse , Vincendon Guy , Zanetta Jean‐Pierre
Published: October 2006

Abstract: A chondroitin sulfate proteoglycan called PGM 1 has been isolated from the particulate fraction of adult rat forebrain. Delipidation of the material, solubilization of proteoglycans in guanidinium chloride, precipitation at low ionic strength, and final extraction at pH 5.0 were used for its isolation. Proteoglycans were subjected to further purification by diethylaminoethyl‐cellulose chromatography. Individual components were separated by gel filtration. PGM 1 appeared to be a high‐molecular‐weight chondroitin sulfate proteoglycan, capable of strong interaction with hy‐aluronic acid. It was finally isolated by gel filtration on Ultrogel AcA 22 in the presence of 4 M guanidinium chloride. Monospecific antibodies obtained in rabbits against the purified molecule did not cross‐react with other brain proteoglycans. Immunocytochemical techniques revealed an almost unique association of this compound with axons, particularly those known to contain neurofilaments. However, not all these axons and all parts of these axons contained PGM 1. This component was not detectable in liver, intestine, spleen, kidney, lung, heart, skin, hair, lens, and muscle, a finding suggesting a specificity for the nervous tissue. This component is expressed in neural cell cultures. Despite the preservation of the neuronal specificity, it seems to lose its specific axonal localization in vitro.
Distribution of Thiamine, Thiamine Phosphates, and Thiamine Metabolizing Enzymes in Neuronal and Glial Cell Enriched Fractions of Rat Brain
Authors: Laforenza Umberto , Patrini Cesare , Rindi Gianguido
Published: October 2006

Abstract: The distribution of thiamine, thiamine phos‐phoesters, and the thiamine pyrophosphate synthetizing [thiamine‐pyrophosphokinase (TPKase)] as well as hydro‐lyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono‐ and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0‐, 2.2‐, 1.3‐, 2.8‐, 3.7‐, and 20.8‐fold higher in neuronal than in glial fractions.
Neurite Extension and Increased Expression of R1 Cyclic AMP‐Binding Protein in Ouabain‐Resistant Neuroblastoma Mutants
Authors: Meyer Sharon A. , Lin Zheng , Liu Alice Y.‐C.
Published: October 2006

Abstract: Morphological and biochemical parameters of neuroblastoma differentiation were assessed in 12 clonal derivatives of the N‐18 mouse neuroblastoma cell line selected for their ouabain‐resistant (ouar) property. When cultured in a normal growth medium, nine of the 12 ouar cell lines exhibited a more complex pattern of neurite outgrowth than the parental N‐18 cells. The morphological pattern most frequently observed with the ouar cells was the extension of several branched processes per cell. This pattern of spontaneous neurite outgrowth in the ouar cell lines can be correlated with an increase in expression of the 47,000‐dalton R1 cyclic AMP (cAMP)‐binding protein. The growth rate, intracellular level of cAMP, and acetylcholin‐esterase activity of the ouar cell lines were not significantly different from those of the parental N‐18 neuroblastoma cells. Treatment of the parental and ouar neuroblastoma cell lines with 1 mM N6,O2‐dibutyryl cAMP promoted an elaborate pattern of neurite outgrowth and marked increases in acetylcholinesterase and R1 cAMP‐binding activities. The distinctive pattern of differentiation phenotype exhibited by the ouar cells and the dibutyryl cAMP‐induced differentiated neuroblastoma cells suggests that these two protocols yielded different degrees of differentiation. Furthermore, our results suggest a linkage of the biochemical events underlying ouabain resistance and expression of differentiation phenotypes in the mouse neuroblastoma cells.
An Inhibitor of the UDP‐N‐Acetylgalactosamine:GM3, N‐Acetylgalactosaminyltransferase: Purification and Properties, and Preparation of an Antibody to This Inhibitor
Authors: Quiroga Santiago , Caputto Ranwel
Published: October 2006

Abstract: An inhibitor of the UDP‐N‐acetylgalactosamine: GM3, N‐acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100‐fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE‐Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. α‐Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment.
Intracellular Calcium Homeostasis in a Human Neuroblastoma Cell Line: Modulation by Depolarization, Cholinergic Receptors, and α‐Latrotoxin
Authors: Sher E. , Gotti C. , Pandiella A. , Madeddu L. , Clementi F.
Published: October 2006

Abstract: Intracellular calcium homeostasis and its modulation by different agents was studied in cells by using the by using the Ca2+‐sensitive fluorescent dye quin2. The results obtained demonstrate the existence in IMR32 cells of (a) voltage‐dependent, verapamil sensitive, Ca2+ channels, which are expressed before differentiation; (b) muscarinic receptors whose activation triggers both Ca2+ influx and Ca2+ redistribution from intracellular stores, whereas nicotinc receptors and α‐bungarotoxin binding sites do not; and (c) receptors for α‐latrotoxin (the major toxin of the black widow spider venom), which are well‐known markers of the neuronal presynaptic membrane. Up to now, no cell lines of human origin sensitive to this toxin have been identified. These results confirm that IMR32 cells are very convenient model cells for studying specific aspects of the neurochemistry and neurobiology of the human neuron at the molecular and cellular levels.
Alterations in Retinal Tyrosine and Dopamine Levels in Rats Consuming Protein or Tyrosine‐Supplemented Diets
Authors: Gibson Candace J.
Published: October 2006

Abstract: The ad libitum ingestion of casein diets varying in protein content altered serum and retinal levels of tyrosine. The serum tyrosine level rose when protein ingestion was increased from 6 to 24% casein. In rats consuming highprotein diets (40% casein), no further increase in serum tyrosine level occurred, although the levels of other large neutral amino acids, which compete with tyrosine for retinal uptake, continued to rise. The activity of the liver enzyme tyrosine aminotransferase varied directly with the percentage of protein in the diet and may partially explain the failure of chronic high‐protein feeding to increase serum tyrosine levels. The retinal tyrosine concentration was significantly correlated with the serum tyrosine level and with the serum tyrosine ratio at all levels of protein intake. Retinal 3,4‐dihydroxyphenylalanine synthesis and dopamine (DA) level varied in parallel with the level of the precursor, tyrosine. Addition of pure l‐tyrosine (1, 2, or 4%) to normal protein diets resulted in a stepwise increase in serum and retinal tyrosine levels and retinal DA turnover. Alterations of retinal tyrosine level as a result of change in amount of dietary protein or by its addition to the normal diet can influence retinal DA synthesis and release.
Developmental Changes in the Molecular Weights of Polypeptides in the Human CNS That Carry the HNK‐1 Epitope and Bind Phaseolus vulgaris Lectins
Authors: Mikol Daniel D. , Wrabetz Larry , Marton Linda S. , Stefansson Kari
Published: October 2006

Abstract: The binding patterns of electrophoresed polypeptides from homogenates of human frontal lobe, cerebellum, and spinal cord obtained at various stages of development were determined for several lectins with specificities for a wide range of oligosaccharides. A discrete developmental change in the molecular‐weight pattern was seen only among polypeptides binding the two Phaseolus vulgaris agglutinins, E‐phytohemagglutinin (E‐PHA) and L‐PHA. With increasing maturity, the apparent molecular weights of the major polypeptides binding these two lectins progressively decreased. Furthermore, at all stages of development, E‐PHA and L‐PHA bound to the same polypeptides as the monoclonal antibody HNK‐1, which recognizes a carbohydrate epitope on polypeptides that may play roles in cell adhesion. Based on the carbohydrate specificities of the two PHAs, we conclude that it is likely that the HNK‐1 epitope resides on a triantennary N‐linked oligosaccharide bisected by N‐acetylglucosamine.
Subcellular Distribution of Mammalian Tachykinins in Rat Basal Ganglia
Authors: Diez‐Guerra F. Javier , Richardson Peter J. , Emson Piers C.
Published: October 2006

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3_10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.
Desensitization of Muscarinic Receptor‐Coupled Phosphoinositide Hydrolysis in Rat Hippocampus: Comparisons with the α1‐Adrenergic Response
Authors: Lenox Robert H. , Hendley Daniel , Ellis John
Published: October 2006

In the presence of lithium, carbamylcholine chloride (carbachol) and epinephrine increase the accumulation of inositol monophosphate severalfold in hippocampal slices from the rat. The stimulation by carbachol (EC50, 31 μM) is mediated by muscarinic receptors, whereas the effects of epinephrine (EC50, 2 μM) are due to activation of α1‐adrenergic receptors. The responses of epinephrine and carbachol are additive, even under conditions that significantly reduce the levels of phosphoinositides and free inositol, suggesting that the muscarinic and adrenergic receptors may be located on separate cells. At concentrations that saturate their respective receptors, epinephrine induces an increase in inositol monophosphate that is linear with time to at least 60 min, whereas the response to carbachol begins to reach a plateau by 20–40 min. When hippocampal slices are preincubated with saturating concentrations of carbachol, the subsequent response to carbachol is reduced by 42%. However, preincubation with carbachol or epinephrine has no effect on the subsequent response to epinephrine. Despite the lack of adrenergic desensitization by this paradigm, preexposure of hippocampal slices to the tumor‐promoting phorbol ester, phorbol 12,13‐dibutyrate, reduces the response to epinephrine to a significantly greater degree (57%) than it reduces the muscarinic response (25%). These studies indicate that, although they utilize the same second messenger, the muscarinic and α1‐adrenergic receptors of hippocampal slices have different characteristics and regulatory mechanisms.
N6‐Cyclohexyladenosine Inhibits Veratridine‐Stimulated Na Uptake by Rat Brain Synaptosomes
Authors: Simoes A. P. , Oliveira P. C. , Sebastião A. M. , Ribeiro J. A.
Published: October 2006

The effect of the stable adenosine analogue, N6‐cyclohexyladenosine, on 22Na uptake by rat brain synaptosomes stimulated by veratridine was investigated. In the presence of N6‐cyclohexyladenosine, both the initial rate and the maximum sodium uptake were decreased. The inhibitory effect of N6‐cyclohexyladenosine on sodium uptake by synaptosomes after 5 s of incubation with 22Na was concentration‐dependent, antagonized by 1,3‐dipropyl‐8‐p‐sulfophenylxanthine, and attenuated by increasing the concentration of veratridine. The possibility that the adenosine analogue, by activating a xanthine‐sensitive adenosine receptor, can operate inhibition of the voltage‐dependent sodium channels is discussed.
Sialyllactotetraosylceramide, a Ganglioside Marker for Human Malignant Gliomas
Authors: Fredman Pam , Hoist Hans von , Collins V. Peter , Granholm Lars , Svennerholm Lars
Published: October 2006

The ganglioside composition of surgically removed human glioma tissue was shown to differ from that of normal adult brain tissue. First, it contained reduced amounts of the major normal brain gangliosides of the gangliotetraose series. Second, it contained increased proportions of gangliosides not detected in normal brain tissue. One of these was isolated and established as being sialyllactotetraosylceramide 3′‐isoLM1. Radioimmunoassay for this ganglioside antigen in human glioma tissue revealed that 14/14 specimens of grades III and IV were positive but only 1/4 of grade II. Normal brain tissue was negative. These results suggest that sialyllactotetraosylceramide is a marker for human malignant gliomas.
Effects of Polyhydric and Monohydric Compounds on the Stability of Type I Receptors for Adrenal Steroids in Brain Cytosol
Authors: Emadian Seyed M. , Luttge William G.
Published: October 2006

Abstract: We have shown previously that unoccupied type I receptors for adrenal steroids in brain cytosol lose their capacity to bind [3H]aIdosterone ([3H]ALDO) in a time‐and temperature‐dependent manner. Based on reports that sugars and polyvalent alcohols are capable of stabilizing a variety of globular proteins, we attempted in the present study to stabilize type I receptors by including polyhydric compounds in our brain cytosol preparations. However, contrary to expectations, adjusting cytosol to a 10% (g/dl) concentration of ethylene glycol, glycerol, erythritol, xylitol, ribitol, or sorbitol failed to stabilize these receptors at 0°C and in fact produced a slight reduction in [3H]ALDO binding capacity. The magnitude of this reduction was greater when cytosol was incubated for 2 h at 22°C prior to incubation with [3H]ALDO. In contrast to these results, when brain cytosol was adjusted to a 10% (g/dl) concentration of the monohydric compound, ethanol, a significant increase in [3H]ALDO binding to type I receptors was found. Under identical conditions, methanol and propanol failed to have a significant effect on the binding capacity of these receptors. When cytosol was aged for 2 h at 22 °C, all three of these monohydric compounds produced a marked loss in the [3H]ALDO binding capacity of type I receptors. An investigation of various doses of ethanol at 0°C on the subsequent binding of [3H]ALDO yielded an inverse U‐shaped curve with 10% ethanol producing the highest level of specific binding, as reflected by an increase in maximal binding in Scatchard plots, and 40% ethanol producing a complete loss in type I receptor binding capacity. In view of these findings, and proposed mechanisms of action of polyand monohydric compounds, we suggest that isolation of type I receptors into aqueous brain cytosol leads to a spontaneous infolding of hydrophobic domains on the receptor surface and a concomitant reduction in ligand binding capacity.
An 82‐Kilodalton Membrane Protein that Inhibits the Activity of Neurite Outgrowth Factor
Authors: Taniura Hideo , Hayashi Yokichi , Miki Naomasa
Published: October 2006

Abstract: Neurite outgrowth factor (NOF), an extracellular matrix glycoprotein of 700 kilodaltons (kDa), promoted neurite outgrowth from cultured ciliary ganglion (CG) neurons of chicken embryo. A fraction solubilized with Nonidet P‐40 of chicken gizzard muscle membranes inhibited the neurite‐promoting activity of NOF in a dose‐dependent manner, but not that of laminin. Binding of CG neurons to the substratum and their survival were not affected by the extract. The inhibitory activity of the extract was abolished by treatment with trypsin or heat. The molecular size was determined to be about 82 kDa by ligand blotting. The active component was partially purified by column chromatography. It is suggested that this molecule interacts with the domain of NOF responsible for its neurite‐promoting activity and may modulate NOF activity during development in vivo.
Enhancement of t‐[35S]Butylbicyclophosphorothionate and [3H]Strychnine Binding by Monovalent Anions Reveals Similarities Between γ‐Aminobutyric Acid‐ and Glycine‐Gated Chloride Channels
Authors: Marvizón Juan Carlos G. , Skolnick Phil
Published: October 2006

Abstract: The characteristics of [3H]strychnine and t‐[35S]‐butylbicyclophosphorothionate ([35S]TBPS) binding to sites associated with glycine‐ and γ‐aminobutyric acid (GABA)‐gated chloride channels were compared in the presence of a series of anions with known permeabilities through these channels. Good correlations were found between (a) the potencies (EC50) of these anions to stimulate radioligand binding and their permeabilities relative to chloride; (b) the affinities (KD) of these radioligands in the presence of fixed concentrations of these anions and their relative permeabilities; (c) the potencies (EC50) of these anions to stimulate [35S]TBPS and [3H]strychnine binding; and (d) the affinities (KD) of [3H]strychnine and [35S]TBPS measured at a fixed concentration of these anions. These studies support electrophysiological and biochemical observations demonstrating similarities between glycine‐ and GABA‐gated chloride channels, and suggest that anions enhance [3H]strychnine and [35S]TBPS binding through specific anion binding sites located at the channels.
Microtubule‐Associated Cyclic AMP‐Dependent Protein Kinase in Drosophila melanogaster
Authors: Ádám Géza , Friedrich Peter
Published: October 2006

Abstract: Microtubules were prepared from head extracts of the adult fruit fly, Drosophila melanogaster, by one‐step, taxol‐assisted polymerization. The microtubular fraction displayed cyclic AMP‐dependent protein kinase (protein kinase A) activity, as witnessed by endogenous protein phosphorylation and by protein kinase assay. Microtubule‐bound protein kinase A amounts to 4–5% of total soluble kinase activity, which is almost an order of magnitude less than in mammals. The high‐molecular‐weight microtu‐bule‐associated protein‐2 (MAP‐2), the main binding species for protein kinase A in mammalian brain microtubules, is not detectable in the fly system by protein staining and immunoblotting with anti‐pig MAP‐2 serum, as well as by hybridization of fly DNA with a cDNA probe for human MAP‐2. Cyclic AMP removes a major part of the regulatory (R) subunit of the enzyme from Drosophila microtubules, as demonstrated by enzyme assay, autophosphoryla‐tion of R subunit, and quantitating cyclic AMP binding sites. It is proposed that permanently elevated cyclic AMP levels may elute protein kinase A from crucial intracellular binding sites, thereby interfering with signal transduction.
Inhibition by Forskolin of Excitatory Amino Acid‐Induced Accumulation of Cyclic AMP in Guinea Pig Hippocampal Slices
Authors: Baba Akemichi , Nishiuchi Yukiko , Uemura Atsuhiro , Tatsuno Tohru , Iwata Heitaroh
Published: October 2006

The effect of forskolin on the excitatory amino acid‐induced accumulation of cyclic AMP was examined in hippocampal preparations of the guinea pig. Forskolin at concentrations of 0.1–10 μM remarkably enhanced the stimulatory effects of histamine and adenosine, whereas it markedly attenuated the stimulation induced by cysteine sulfinate, an excitatory amino acid. Forskolin reduced the maximal response to cysteine sulfinate without affecting the apparent ED50. At a concentration of 1 μM, forskolin also inhibited the stimulatory effects of glutamate, N‐methyl‐dl‐aspartate, and veratridine without affecting those of kainate and quisqualate. Pretreatment of the slices with 0.1 mM N‐ethylmaleimide partially prevented the attenuation by forskolin of cysteine sulfinate‐induced accumulation of cyclic AMP without affecting the stimulation induced by cysteine sulfinate. Forskolin at concentrations of < 1 μM did not affect GTP‐stimulated activity and Cl−‐dependent activity of adenylate cyclase of the hippocampal membranes.
Reevaluation of Taurine Levels and Distribution of Cysteic Acid Decarboxylase in Developing Human Fetal Brain Regions
Authors: Datta Salil C.
Published: October 2006

Abstract: The possibilities of interference by glycerophosphoryl ethanolamine (GPE) in the estimation of taurine levels in cerebral cortex, midbrain, cerebellum, medullapons, and spinal cord of developing human fetal brain regions were eliminated by hydrolyzing tissue extracts with 6 M HC1. Cysteic acid thus produced was separated from taurine by ion‐exchange chromatography using Biorad‐AG resin. Fluorescamine was used as fluorogen. Data reveal that the estimation of taurine in human fetal brain regions is affected if GPE is present as a contaminant in the assay system. Cysteic acid decarboxylase activity was measured using cysteic acid as the substrate. Higher enzymic activity was recorded with increased fetal body weight, but the reverse was true for taurine level.
Inhibition of Type A Monoamine Oxidase by Methylquinolines and Structurally Related Compounds
Authors: Naoi Makoto , Nagatsu Toshiharu
Published: October 2006

Abstract: A series of methylquinolines (MQ) were found to inhibit markedly type A monoamine oxidase (MAO) in human brain synaptosomal mitochondria. 4‐MQ and 6‐MQ inhibited type A MAO (MAO‐A) competitively and 7‐ and 8‐MQ inhibited MAO‐A noncompetitively. Among these four isomers of MQ, 6‐MQ was the most potent inhibitor; the Ki value toward MAO‐A was 23.4 ± 1.8 μM, which was smaller than the Km value toward kynuramine, ± amine substrate, 46.2 ± 2.8 μM. On the other hand, MQ were very weak inhibitors of type B MAO (MAO‐B) and 8‐MQ did not inhibit MAO‐B in brain synaptosomal mitochondria. The inhibition of MAO‐A proved to be reversible; by dialysis the inhibition of MQ was completely reversible. The affinity of these isomers of MQ toward MAO‐A or ‐B was confirmed further with human liver mitochondria as sources of MAO‐A and ‐B and with human placental mitochondria and rat pheochromocytoma PC12h cell line as sources of MAO‐A. The relationship of the chemical structure of structurally related quinoline and isoquinoline derivatives to inhibition of the activity of type A or B MAO was examined.
Effect of Fatty Acids on [3H]Ouabain Binding to Cerebromicrovascular (Na++ K+)‐ATPase
Authors: Caspers Mary Lou , Grammas Paula
Published: October 2006

Abstract: The effects of short‐ and long‐chain fatty acids on the cerebromicrovascular (Na++ K+)‐ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37–110 μg) and was time‐dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose‐dependent manner, with a 105% increase over basal levels at 100 μM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4‐Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of “high‐affinity” binding sites with an apparent binding affinity (KD) of 64.7 ± 2.0 nM and a binding capacity (Bmax) of 10.1 ± 1.5 pmol/mg protein. In the presence of 100 μM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 ± 2.7 nM (p < 0.01), whereas the Bmax remained virtually unchanged (12.5 ± 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4‐pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long‐chain polyunsaturated fatty acids may be involved in the regulation of blood‐brain barrier (Na++ K+)‐ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.
Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells
Authors: DeLorme Eleanor M. , McGee Richard
Published: October 2006

Abstract: To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol‐stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol‐stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol‐stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down‐regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.
Ribonuclease Activities and Distribution in Alzheimer's and Control Brains
Authors: Maschhoff K. , White C. L. , Jennings L. W. , Morrison‐Bogorad M. R.
Published: October 2006

Abstract Levels of free and total alkaline ribonuclease, and levels of acidic ribonuclease, were measured postmortem in control brains and in the brains of patients with Alzheimer's disease. In each brain region assayed, whether control or Alzheimer's, there was a statistically significant difference between the levels of free and total alkaline ribonuclease. Between 59 and 90% of the enzyme activity was associated with alkaline ribonuclease inhibitor in an inactive complex. Levels of free and total alkaline ribonuclease varied widely among different brains and brain regions, and were always lower in cerebellum than in temporal cortex and occipital pole. There was no significant difference in the levels of total alkaline ribonuclease, free alkaline ribonuclease, or acidic ribonu‐cleases between corresponding regions of Alzheimer's and control brains. There was also no qualitative difference in the subcellular distribution of the alkaline and acidic ribonucleases between Alzheimer's and control brain. No significant relationships were found between ribonuclease levels and age, neuritic plaque density, postmortem interval, or storage time.
Protein Fatty Acid Acylation in Developing Cortical Neurons
Authors: Perrone‐Bizzozero Nora I. , Benowitz Larry I. , Apostolides Paul J. , Franck Elizabeth R. , Finklestein Seth P. , Bizzozero Oscar A.
Published: October 2006

Abstract Neuron‐enriched cultures derived from embryonic day 17 rat cerebral cortex were incubated in the presence of [3H]myristic or [3H]palmitic acid. Analysis of radiolabeled proteins by two‐dimensional gel electrophoresis and fluorography revealed extensive incorporation of fatty acids into a small number of neuronal proteins. The major acylated proteins had apparent molecular weights and isoelectric points as follows: 87,000, 4.3; 63,000, 4.4; 45,000, 4.4; and 20,000, 5.3. After labeling with [3H]myristic acid, the radioactivity associated with these proteins was identified as myristic acid, which was attached via an ester linkage. All four of the major acylated neuronal proteins were found to be membrane‐bound and enriched in growth cones. By virtue of its molecular weight, isoelectric point, subcellular distribution, and peptide map, the 87‐kilodalton polypeptide was shown to be equivalent to pp80, a phosphoprotein that has been described in developing and mature synaptic terminals. The 45‐kilo‐dalton acylated protein also appears to coincide with another growth cone phosphoprotein, pp40. Acylation may serve as a mechanism to regulate the function of these proteins, or may play a role in directing them to the nerve terminal membrane.
Characterization of Cellular Transport, Subcellular Distribution, and Secretion of the Neurotoxicant 1‐Methyl‐4‐Phenylpyridinium in Bovine Adrenomedullary Cell Cultures
Authors: Reinhard John F. , Diliberto Emanuel J. , Daniels Alejandro J.
Published: October 2006

Abstract Cultures of bovine adrenomedullary chromaffin cells accumulated l‐[methyl‐3H]methyl‐4‐phenylpyridinium ([3H]MPP+) in a time‐ and concentration‐dependent manner with an apparent Km of 0.7 μM and a Vmax of 3 pmol/min/106 cells. The uptake was sodium dependent and sensitive to inhibitors of the cell‐surface catecholamine transporter. At low concentrations of MPP+, the subcellular distribution was identical to that of endogenous catecholamines in the catecholamine‐containing chromaffin vesicles. However, at a higher concentration of MPP+, a larger proportion of the toxicant was recovered in the cytosolic fraction, with less in the chromaffin vesicle fractions. When cells were prelabeled with [3H]MPP+, at 1 and 300 μM, and then permeabilized with digitonin in the absence of Ca2+, there was a proportionally greater release of MPP+ from the cells labeled at the higher concentration of the toxicant. In the presence of Ca2+, cell permeabilization induced a time‐dependent secretion of catecholamines and a parallel secretion of MPP+. Under these conditions, the secretion of endogenous catecholamines was unaffected by the presence of MPP+. When the permeabilization studies were carried out in the presence of tetrabenazine, a massive release of MPP+ was observed in the absence of Ca2+ and was not further increased by Ca2+. In intact cells prelabeled with 300 μM [3H]MPP+, the secretagogues nicotine and veratridine elicited a Ca2+‐dependent secretion of catecholamines and MPP+ from the cells in similar proportions to their cellular contents. Barium‐induced release of both species was independent of external Ca2+. Collectively, these data reveal that MPP+ is a novel substrate for both the cellular and vesicular catecholamine transporters and that, in intact cells, vesicular transport may become limiting relative to the cell‐surface transporter, leading to the cytosolic accumulation of the toxicant.
Inositol Phospholipid Hydrolysis and Potentiation of Cyclic AMP Formation by Noradrenaline in Rat Cerebral Cortex Slices Are Not Mediated by the Same α‐Adrenoceptor Subtypes
Authors: Robinson J. P. , Kendall D. A.
Published: October 2006

Abstract: A pharmacological study was undertaken to determine whether the noradrenaline‐stimulated breakdown of inositol phospholipids and the potentiation of isoprenaline‐stimulated cyclic AMP by noradrenaline in rat cerebral cortex slices are mediated by the same α‐receptor subtype. The rank order of potency of a range of α1 and α2 antagonists suggests that both responses may involve an α1 receptor, but there were several differences between the pharmacological profiles for the two systems. Although in both cases, all selective α1 antagonists were more potent than α2 antagonists, the rank orders and the absolute potencies differed for the two responses. The inhibition of the inositol phosphate response was characterised by a high α1/α2 antagonist ratio, and in most cases, Hill slopes of inhibition were consistent with the involvement of a single receptor site. Inhibition of the cyclic AMP response had a much lower α1/α2 antagonist ratio and generally exhibited Hill slopes less than one. Evidence has been provided suggesting that adenosine is involved in the potentiation of cyclic AMP and that other, as yet unidentified, factors may also be involved. Even in the absence of an adenosine component, the results presented support the suggestion that the potentiation due to noradrenaline is mediated by a receptor whose identity does not easily fit with the currently accepted classification of α adrenoceptors.
Phosphatidylinositol Metabolism During In Vitro Hypoxia
Authors: Huang Hsueh‐Meei , Gibson Gary E.
Published: October 2006

Abstract: The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium‐stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2‐3H]inositol, depolarization with 60 mMKCl increased [2‐3H]inositol monophosphate and [2‐3H]inositol bisphosphate accumulation in a Ca2+‐dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium‐stimulated [2‐3H]inositol bisphosphate formation. More prolonged hypoxia (10 and 30 min) reduced potassium‐stimulated [2‐3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2‐3H]inositol phosphates.

These results are consistent with the following hypothesis. The hypoxic‐induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.
Medial Forebrain Bundle Stimulation or D‐2 Dopamine Receptor Activation Increases Preproenkephalin mRNA in Rat Striatum
Authors: Bannon Michael J. , Kelland Mark , Chiodo Louis A.
Published: October 2006

Abstract: Electrical stimulation of the medial forebrain bundle, in a manner that augmented the release of dopamine in the forebrain, rapidly increased the striatal content of preproenkephalin (but not preprotachykinin) mRNA. This effect was mimicked by administration of either the indirect (do‐pamine‐releasing) agonist methamphetamine or by the D‐2 dopamine receptor agonist quinpirole, but not by the D‐1 agonist SKF 38393. These data suggest that D‐2 receptors, which mediate a stimulatory effect on enkephalin gene expression, may be subsaturated under basal conditions and, therefore, responsive to increases in synaptic dopamine.
Synaptosomal “Membrane‐Bound” Choline Acetyltransferase Is Most Sensitive to Inhibition by Choline Mustard
Authors: Rylett R. Jane
Published: October 2006

Abstract: The objectives of the present study were to validate the presence of cytoplasmic and membrane‐associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt‐washed presynaptic membranes represents membrane‐bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az‐treated nerve terminals revealed that the membrane‐bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane‐associated enzyme, with the activity of the water‐solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water‐solubilized, ionically membrane‐associated, and detergent‐solubilized membrane‐bound pools of ChAT were not differentially inhibited by either compound. This study suggests that choline mustard transported on the high‐affinity choline carrier may be directed toward a membrane‐associated form of ChAT.
Acetylcholine Synthesis by a Sympathetic Ganglion in the Presence of 2‐(4‐Phenylpiperidino)cyclohexanol (AH5183) and Picrylsulfonic Acid
Authors: Mykita S. , Collier B.
Published: October 2006

Abstract: The present experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2‐(4‐phenylpiperidino)cyclohexanol (AH5183 or vesamicol) and/or picrylsulfonic acid (TNBS), two compounds known to have the ability to block the uptake of ACh by cholinergic synaptic vesicles in vitro. We confirmed that, in stimulated (5 Hz) perfused (30 min) ganglia, AH5183 depressed ACh release and ACh tissue content increased by 86 ± 6% compared to contralateral ganglia used as controls. Preganglionic activity increased ACh release by a similar amount in the presence (19.9 ± 1.0 pmol/min) or absence (20.5 ± 2.4 pmol/min) of TNBS. The final tissue ACh content was also similar in the presence (1,668 ± 166 pmol) or absence (1,680 ± 56 pmol) of TNBS. However, the AH5183‐induced increase of tissue ACh content (86 ± 6%) was abolished completely when AH5183 was perfused with 1.5 mM/TNBS (‐3.0 ± 1.0%). This inhibition of ACh synthesis, observed in TNBS‐AH5183‐perfused ganglia, was not dependent upon further inhibition of ACh release beyond that caused by AH5183 alone, because 14.0 ± 1.9% of the transmitter store was released by preganglionic nerve stimulation in the presence of TNBS plus AH5183 and this was similar in the presence of AH5183 without TNBS (14.0 ± 0.6%). Moreover, when ganglia were first treated with TNBS and then stimulated in the presence of AH5183, an increase of 64 ± 6% of the ganglionic ACh content occurred, and this increase was not statistically different from the increase measured with AH5183 alone (86 ± 6%). Conversely, when ganglia were perfused first with AH5183 and then with TNBS during preganglionic stimulation, no change in the final tissue ACh content was observed, suggesting that AH5183 must be present inside the cells for the inhibitory action of TNBS to be manifest. Moreover, after perfusion of ganglia with [3H]choline, the specific activity of ACh was greater in ganglia treated with AH5183 (76 ± 3 dpm/pmol) than in ganglia treated with TNBS plus AH5183 (48 ± 4 dpm/pmol), whereas the specific activity of choline was less (23 ± 3 and 47 ± 7 dpm/pmol, respectively), suggesting that the enzyme choline acetyltransferase might be inhibited. Considering that TNBS acts extracellularly on the membrane and AH5183 intracellularly on the vesicles, these results suggest that the interaction between the two compounds might well occur during exocytosis and, if the phenomenon involves choline acetyltransferase inhibition, it is presumably a membrane‐associated pool of enzyme that is affected.
Clathrin‐Coated Vesicle Subtypes in Mammalian Brain Tissue: Detection of Polypeptide Heterogeneity by Immunoprecipitation with Monoclonal Antibodies
Authors: Puszkin Saul , Kohtz Jhumku D. , Schook William J. , Kohtz D. Stave
Published: October 2006

Abstract: A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On im‐munoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radio‐iodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A‐7C11 recognized a 40‐kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one‐quarter of the total brain CVs. The mAb S‐l1 D9 reacted with a 44‐kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S‐11 D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C‐10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa > LCb). Each of the mAbs yielded different im‐munofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor‐differentiated PC 12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.
Bovine γ‐Aminobutyric AcidA Receptor Sequence‐Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ‐Aminobutyric AcidA Receptors
Authors: Duggan Michael J. , Stephenson F. Anne
Published: October 2006

Abstract: Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N‐terminal 15 amino acids and the C‐terminal 17 amino acids of the bovine γ‐aminobutyric acidA (GABAA) receptor α1 subunit. These antibodies were shown to react with the denatured GABAA receptor α subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent‐solubilised GABAA receptor as demonstrated by dose‐dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain‐regional distribution of the expression of the al subunit.
Effects of Aging on p‐ and m‐Octopamine, Catecholamines, and Their Metabolizing Enzymes in the Rat
Authors: David Jean‐Claude , Coulon Jean‐Francois , Cavoy Albert , Delacour Jean
Published: October 2006

Abstract: Functions of octopamine in the mammalian brain are still not well known. An important aspect of this problem is the relationship between octopamines and catecholamines. Previous data have shown that their respective ontogenic evolutions are not parallel. Do the changes in brain related to aging also differentially affect these two groups of molecules? In order to check this point, the brain levels of p‐ and m‐octopamine, p‐tyramine, noradrenaline, and dopamine, as well as the activities of metabolizing enzymes, were determined in young adult and aging rats (20–26 months). Unlike catecholamines, there is a drastic decrease of p‐octopa‐mine after 20 months of age in the hypothalamus and telencephalon. p‐Tyramine levels are also lowered. This change appears to be due to a decrease of the aromatic L‐amino acid decarboxylase activity. These data, as those of ontogenic studies, confirm that p‐octopamine and catecholamine metabolisms may have some independent steps and, moreover, that p‐octopamine may have a role in the normal activity of the brain.
Muscarinic Receptor Stimulation Increases Inositol‐Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells
Authors: Horwitz Joel
Published: October 2006

Abstract: Muscarinic receptor stimulation increased the accumulation of 3H‐inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5′‐N‐ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol‐phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 μM and 0.36 μM, respectively. PC 12 cells contained a single class of N‐[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 μM) and pirenzepine (Ki; 0.12 μM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an Mi receptor. Muscarine inhibited cAMP accumulation in cells made de ficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of3H‐inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotrem‐orine inhibited cAMP accumulation but it did not stimulate inositol‐phospholipid metabolism. These data indicate that increased inositol‐phospholipid metabolism and inhibition of cAMP production are two independent effects of muscarinic receptor stimulation in PC12 cells.
Photoaffinity Labeling and Electrophoretic Identification of the H1‐Receptor: Comparison of Several Brain Regions and Animal Species
Authors: Ruat Martial , Schwartz Jean‐Charles
Published: October 2006

Abstract: The photoaffinity probe [l25I]iodoazidophen‐pyramine was used to label irreversibly the H1‐receptor in membranes of several guinea pig brain regions and of the cerebral cortex of the rat, mouse, and pig. Following sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis, two main bands were specifically labeled in all tissues: a 56‐kilodalton (kDa) peptide and a 41‐47‐kDa peptide whose relative importance diminished in the presence of protease inhibitors. This indicates that, in all tissues examined, in spite of evidence for pharmacological heterogeneity, the ligand recognition domain of the H1‐receptor resides in a 56‐kDa peptide.