bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbachol and Bradykinin Increase the Production of Diacylglycerol from Sources Other than Inositol‐Containing Phospholipids in PC12 Cells
Authors: Horwitz Joel
Published: October 2006

Abstract: Both carbachol and bradykinin increased diacylglycerol formation in PC12 pheochromocytoma cells. The effect of carbachol was apparent only in cells that had been treated with nerve growth factor. Incubation of the cells in Ca2+‐free medium attenuated carbachol‐stimulated diacylglycerol formation but did not reduce the response to bradykinin. Pretreatment of the cells with pertussis toxin did not affect either carbachol‐ or bradykinin‐stimulated diacylglycerol formation; therefore, the inhibitory guanine nucleotide Gi probably does not mediate this response. The time course of carbachol‐stimulated diacylglycerol accumulation did not coincide with the time course of inositol 1,4,5‐trisphosphate (IP3) production. IP3 was elevated at the earliest time measured, 15 s, and then slowly declined so that by 5 min IP3 levels were only 50% of maximal. Diacylglycerol levels, in contrast, were not elevated for the first 2 min and then peaked at 5 min. These data indicate that hydrolysis of phosphatidylinositol 4,5‐bisphosphate was not the major source of the diacylglycerol peak at 5 min. To investigate the source of diacylglycerol, I examined the fatty acid composition of the diacylglycerol by prelabeling the cells with [3H]palmitic acid and [14C]stearic acid. The 14C/3H ratio in diacylglycerol should reflect the phospholipid(s) from which it is derived. The 14C/3H ratio of the increment in diacylglycerol produced by carbachol and bradykinin was intermediate between the 14C/3H ratios of phosphatidylcholine and phosphatidylinositol. The 14C/3H ratio in triacylglycerol was similar to that of phosphatidylcholine. These data indicate that carbachol and bradykinin stimulate the formation of diacylglycerol from sources other than inositol‐containing phospholipids; phosphatidylcholine and triacylglycerol are two possible sources of this diacylglycerol.
Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents
Authors: Faissner Andreas , Kruse Jan , Kühn, Klaus , Schachner Melitta
Published: October 2006

Abstract: The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1‐160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1‐220) from developing brain. J1‐160 and J1‐220 share common epitopes but are considered as separate entities, with J1‐220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1‐160 and J1‐220 bound to collagens type I–VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1‐220 bound to all collagen types, whereas J1‐160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1‐220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10‐9 and 1.8 and 5.5 X 10‐8M, respectively, under hypotonic buffer conditions and a single KD of 2.1–8.0 X 10‐8M under isotonic buffer conditions. Binding of J1‐160 to collagens had an apparent KD of 1.9–8.0 X 10‐9M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1‐160 to collagen types V and VI were ˜ 2 × 10–8M. Binding of J1‐220 to collagen type I could be inhibited by J1‐220, J1‐160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1‐160 was inhibited by J1‐220, J1‐160, and collagen type VI (in order of decreasing efficacy of competition). J1‐160 and J1‐220 were retained on a heparin–agarose column and eluted in a salt gradient at ˜ 0.5 M NaCl. The formation of the J1–heparin complexes was inhibited 100‐fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.
Fibroblast‐Derived J 1 Adhesion Glycoproteins Show Binding Properties to Extracellular Matrix Constituents Different from Those of Central Nervous System Origin
Authors: Probstmeier Rainer , Kühn Klaus , Schachner Melitta
Published: October 2006

Abstract: The J 1 extracellular adhesion molecule from mouse brain consists of several immunochemically related glycoproteins of different molecular weights and distinct functional properties. Like the brain J 1 glycoproteins, the fibroblastderived J 1 glycoproteins interact with all collagen types tested (collagen G and type I–VI and IX), as measured by binding of 125I‐labeled J 1 glycoproteins to immobilized collagens. As tested for collagen type I, this binding can be inhibited more effectively by chondroitin sulfate than by heparin. After electrophoretic separation and transfer to nitrocellulose, fibroblast‐derived J 1 only binds to a limited number of collagen types (collagen types I, VI, and IX and G), whereas brainderived J 1 glycoproteins bind to all collagen types tested (collagen types I–VI and IX and G). These results show that fibroblast‐derived J 1 glycoproteins, although immunochemically related to J 1 glycoproteins from brain, differ from these in their binding specificities to extracellular matrix constituents.
Effect of 3,4‐Methylenedioxymethamphetamine on 3,4‐Dihydroxyphenylalanine Accumulation in the Striatum and Nucleus Accumbens
Authors: Nash J. Frank , Meltzer Herbert Y. , Gudelsky Gary A.
Published: October 2006

Abstract: The effect of the racemic mixture of 3,4‐methy‐lenedioxymethamphetamine (MDMA) on the synthesis of dopamine in the terminals of nigrostriatal and mesolimbic neurons was estimated by measuring the accumulation of 3,4‐dihydroxyphenylalanine (DOPA) in the striatum and nucleus accumbens 30 min following the administration of the l‐aromatic amino acid decarboxylase inhibitor, 3‐hydroxybenzylhydrazine. MDMA produced an increase in DOPA accumulation in the striatum which was greater in magnitude and longer in duration than that in the nucleus accumbens. Although the concentrations of serotonin (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) in both the striatum and nucleus accumbens were reduced 3 h following an injection of MDMA (20 mg/kg), 5‐HT and 5‐HIAA concentrations were significantly reduced only in the striatum 7 days after the administration of MDMA. Pretreatment with a 5‐HT2 antagonist, ketanserin, significantly attenuated the reduction in 5‐HT concentration in the striatum 3 h following MDMA administration and completely blocked 5‐HT depletion at 7 days post administration. Moreover, ketanserin completely blocked MDMA‐induced DOPA accumulation in the striatum. The results obtained in these studies suggest that MDMA activates nigrostriatal dopaminergic pathways via 5‐HT2 receptors. In addition, these data are supportive of the hypothesis that dopamine plays a role in MDMA‐induced 5‐HT depletion.
Drosophila GABAergic Systems: Sequence and Expression of Glutamic Acid Decarboxylase
Authors: Jackson F. Rob , Newby Laurel M. , Kulkarni Shankar J.
Published: October 2006

Abstract: A mammalian glutamic acid decarboxylase (GAD) cDNA probe has been utilized to isolate Drosophila cDNA clones that represent a genomic locus in chromosome region 64A. Deletion analysis indicates that this chromosomal locus encodes an enzymatically active GAD protein. The in vitro translation of cRNA representing a Drosophila cDNA clone yields a 57‐kDa protein that can be immunoprecipitated by an anti‐GAD antiserum. A GAD‐immunoreactive protein of the same size can also be detected in Drosophila head extracts. The nucleotide sequence derived from two overlapping Drosophila cDNA clones predicts a 57,759‐dalton protein composed of 510 residues that is 53% identical to mammalian GAD. Sequence comparisons of mammalian and Drosophila GAD identify two highly conserved regions (≥70% identity), one of which encompasses a putative cofactor‐binding domain. Transcriptional analyses show that expression of the Drosophila Gad gene commences early in embryonic development (4–8 h) and continues in all later developmental stages. A 3.1‐kb class of mRNA is detected throughout embryogenesis, in all three larval stages, in pupae, and in adults. This transcript class has a widespread distribution in the adult CNS. A smaller 2.6‐kb transcript is expressed in a developmentally regulated manner; it is detected only in embryos and pupae.
Effects of Inhibitors of Protein Synthesis and Intracellular Transport on the γ‐Aminobutyric Acid Agonist‐Induced Functional Differentiation of Cultured Cerebellar Granule Cells
Authors: Belhage Bo , Hansen Gert H. , Meier Eddi , Schousboe Arne
Published: October 2006

Abstract: The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the γ‐aminobutyric acid (GABA) agonist [4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP)]‐induced changes in morphological differentiation and GABA receptor expression was investigated in cultured cerebellar granule cells. After 4 days in culture the neurons were exposed to the inhibitors for 6 h in the simultaneous presence of THIP. Subsequently, cultures were either fixed for electron microscopic examination or used for preparation of membranes for [3H]GABA binding assays. In some experiments the functional activity of the newly induced low‐affinity GABA receptors was assessed by investigation of the ability of GABA to inhibit neurotransmitter release from the neurons. These experiments were performed to differentiate between an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low‐affinity GABA receptors. In contrast, the inhibitors of intracellular transport as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensintreated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low‐affinity GABA receptors in the membrane preparations. This indicates that the low‐affinity receptors were not located in the plasma membrane. This is in good agreement with the corresponding morphological findings, that monensin treatment led to an intense vacuolization of the Golgi apparatus, thereby preventing intracellular transport of the newly synthesized GABA receptors. No qualitative alteration of the general ultrastructure was observed in cells cultured in the simultaneous presence of THIP and actinomycin, cycloheximide. or colchicine. However, these inhibitors reduced the cytoplasmic density of the different organelles involved in the cellular machinery for synthesis and intracellular transport compared to cells grown in the presence of THIP alone. These results suggest that the THIP‐induced alterations in the GABA receptor expression in cerebellar granule cells requires de novo synthesis of low‐affinity GABA receptors, which in turn is dependent on proper function of the organelles involved in synthesis and intracellular transport of proteins destined for insertion into the plasma membrane.
Effect of Noncompetitive Blockade of N‐Methyl‐d‐Aspartate Receptors on the Neurochemical Sequelae of Experimental Brain Injury
Authors: McIntosh Tracy K. , Vink Robert , Soares Holly , Hayes Ronald , Simon Roger
Published: October 2006

Abstract: Pharmacological inhibition of excitatory neurotransmission attenuates cell death in models of global and focal ischemia and hypoglycemia, and improves neurological outcome after experimental spinal cord injury. The present study examined the effects of the noncompetitive N‐methyl‐d‐aspartate receptor blocker MK‐801 on neurochemical sequelae following experimental fluid‐percussion brain injury in the rat. Fifteen minutes after fluid‐percussion brain injury (2.8 atmospheres), animals received either MK‐801 (1 mg/ kg, i.v.) or saline. MK‐801 treatment significantly attenuated the development of focal brain edema at the site of injury 48 h after brain injury, significantly reduced the increase in tissue sodium, and prevented the localized decline in total tissue magnesium that was observed in injured tissue of saline‐treated animals. Using phosphorus nuclear magnetic resonance spectroscopy, we also observed that MK‐801 treatment improved brain metabolic status and promoted a significant recovery of intracellular free magnesium concentrations that fell precipitously after brain injury. These results suggest that excitatory amino acid neurotransmitters may be involved in the pathophysiological sequelae of traumatic brain injury and that noncompetitive N‐methyl‐d‐aspartate receptor antagonists may effectively attenuate some of the potentially deleterious neurochemical sequelae of brain injury.
Pretreatment of Mouse Striatal Neurons in Primary Culture with 17β‐Estradiol Enhances the Pertussis Toxin‐Catalyzed ADP‐Ribosylation of Gαo,i Protein Subunits
Authors: Maus M. , Hdmburger V. , Bockaert J. , Glowinski J. , Premont J.
Published: October 2006

Abstract: Pretreatment of striatal neurons from mouse embryos in primary culture with 17β‐estradiol (10−9M, 24 h) enhanced the ADP‐ribosylation of Gαo,i proteins catalyzed by pertussis toxin (PTX). As estimated by quantitative ADP‐ribosylation of Gαs with cholera toxin and immunoblot experiments using anti‐Gαo and anti‐Gβ sera, 17β‐estradiol pretreatment did not modify the levels of the major GTP‐binding protein (G protein) constituent subunits Gαs, Gαo, and Gβ. Thus, 17β‐estradiol should induce a qualitative modification of these G proteins, perhaps by stabilizing the association of the heterotrimers Gαo,iβγ, which are the targets of PTX. Such a hypothesis is in agreement with observations indicating that 17β‐estradiol both suppressed the D2 dopamine‐ and opiate receptor‐induced inhibitions of adenylate cyclase activity and enhanced the positive coupling between biogenic amine receptors (D1 dopamine, β‐adrenergic, and A2 adenosine) and adenylate cyclase. In addition, PTX pretreatment, which is known to uncouple receptors associated with Go,i proteins and thus to impair the dissociation of the heterotrimers Gαo,iβγ, mimicks the effects of the steroid on the responses of adenylate cyclase to inhibitory and stimulatory agonists. Finally, the chemical specificity of the steroids was the same in the ADP‐ribosylation as in the adenylate cyclase experiments: Testosterone (10−9M) mimicked the effects of 17β‐estradiol, whereas 17α‐estradiol, progesterone, and dexamethasone did not. Because 17β‐estradiol enhanced uniformly the PTX‐catalyzed ADP‐ribosylation of Gαo and Gαi proteins, it can be expected that transducing systems other than adenylate cyclase involving these G proteins, such as ionic channels or phospholipases, are also affected by the steroid pretreatment of striatal neurons.
Oligosaccharide‐Related Epitope Specific for a Brain‐Specific Glycoprotein, 1D4 Antigen
Authors: Sano Shin‐ichiro , Ito Shunji , Nakamura Masahiko , Nakagawa Hachiro
Published: October 2006

Abstract: The characteristics of glycosylation of a brain‐specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high‐mannose and hybrid types of N‐linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N‐linked oligosaccharides by treatment with glycopeptidase F or removal of both N‐ and O‐linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex‐type oligosaccharide‐related epitope specific for the 1D4 antigen. Results also showed that N‐linked giycosy lation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain‐related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide‐related epitope is species specific. Furthermore, results with neuraminidase‐treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide‐related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.
Recycled Synaptic Vesicles Contain Vesicle but Not Plasma Membrane Marker, Newly Synthesized Acetylcholine, and a Sample of Extracellular Medium
Authors: Bonzelius F. , Zimmermann H.
Published: October 2006

Abstract: To monitor the fate of the synaptic vesicle membrane compartment, synaptic vesicles were isolated under varying experimental conditions from blocks of perfused Torpedo electric organ. In accordance with previous results, after low‐frequency stimulation (0.1 Hz, 1,800 pulses) of perfused blocks of electric organ, a population of vesicles (VP2 type) can be separated by density gradient centrifugation and chromatography on porous glass beads that is denser and smaller than resting vesicles (VP1 type). By simultaneous application of fluorescein isothiocyanate‐dextran as extracellular volume marker and [3H]acetate as precursor of vesicular acetylcholine, and by identifying the vesicular membrane compartment with an antibody against the synaptic vesicle transmembrane glycoprotein SV2, we can show that the membrane compartment of part of the synaptic vesicles becomes recycled during the stimulation period. It then contains both newly synthesized acetylcholine and a sample of extra cellular medium. Recycled vesicles have not incorporated the presynaptic plasma membrane marker acetylcholinesterase. Cisternae or vacuoles are presumably not involved in vesicle recycling. After a subsequent period of recovery (18 h), all vesicular membrane compartments behave like VP1 vesicles on subcellular fractionation and still retain both volume markers. Our results imply that on low‐frequency stimulation, synaptic vesicles are directly recycled, equilibrating their luminal contents with the extracellular medium and retaining their membrane identity and capability to accumulate acetylcholine.
Effects of Different Types of Stimulation on Cyclic AMP Content in the Rabbit Carotid Body: Functional Significance
Authors: Pérez‐García M. T. , Almaraz L. , González C.
Published: October 2006

Abstract: Cyclic AMP levels in rabbit carotid bodies incubated under control conditions, 100% O2‐ or 95% O2/5% CO2‐equilibrated medium, are close to 1 pmol/mg wet tissue (range 0.4–2.43 pmol/mg). Isobutylmethylxanthine (0.5 mM) increases cyclic AMP levels by a factor of 14 and 8 in HEPES‐and CO2/CH3O−‐buffered medium, respectively. Forskolin (0.5–10 μM) applied during 30 min increases cyclic AMP levels in a dose‐dependent manner. Incubation of carotid bodies at low O2 tensions resulted in an elevation of cyclic AMP levels both in the absence and in the presence of isobutylmethylxanthine. In the latter conditions cyclic AMP increase was maximum at an O2 tension of 46 mm Hg and tended to decrease at extremely low Po2. In isobutylmethylxanthine‐containing Ca2+‐free medium, cyclic AMP increased linearly with decreasing Po2 from 66 to 13 mm Hg; the absolute cyclic AMP levels attained in Ca2+‐free medium were smaller than those observed in Ca2+‐containing medium at any Po2. The differences between Ca2+‐free and Ca2+‐containing media appear to be due to the action of released neurotransmitters in the latter conditions, because dopamine and norepinephrine, which are known to be released by hypoxia in a Ca2+‐dependent manner, increase cyclic AMP in the carotid body. Low pH/high Pco2 and high [K+]e increase cyclic AMP levels only in Ca2+‐containing medium. Forskolin potentiates the release of catecholamines induced by low Po2. These results suggest that cyclic AMP plays an important role in the modulation of the chemoreception process.
Stereoselectivity for the (R)‐Enantiomer of HA‐966 (l‐Hydroxy‐3‐Aminopyrrolidone‐2) at the Glycine Site of the N‐Methyl‐d‐Aspartate Receptor Complex
Authors: Pullan L. M. , Britt M. , Chapdelaine M. J. , Keith R. A. , LaMonte D. , Mangano T. J. , Patel J. , Powel R. J. , Stumpo R. J. , Warwick P. J. , Zinkand W. C. , Salama A. I.
Published: October 2006

Abstract: HA‐966 (1‐hydroxy‐3‐aminopyrrolidone‐2) is an antagonist at the glycine allosteric site of the N‐methyl‐d‐aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA‐966 is chiral. We report stereoselectivity for the (R)‐enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)‐enantiomer has an IC50 of 4.1 ± 0.6 μM. The racemic mixture has an IC50 of 11.2 ± 0.5 μM, whereas (S)‐HA‐966 has an IC50 greater than 900 μM. In glycine‐stimulated [3H]l‐[1‐(2‐thienyl)cyclohexyl] piperidine binding, the (R)‐enantiomer inhibits with an IC50 of 121 ± 61μM, whereas the racemic mixture has an IC50 of 216 ± 113 μM and (S)‐HA‐966 is inactive. The inhibition by (R)‐HA‐966 can be prevented by the addition of glycine. (R)‐HA‐966 and racemic HA‐966, but not (S) HA‐966, also prevent N‐methyl‐d‐aspartate cytotoxicity in cortical cultures. The (R)‐enantiomer and, less potently, the (S)‐enantiomer inhibit N‐methyl‐d‐aspartate‐evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)‐HA‐966 is reversed by added glycine. In glutamate‐evoked contractions of the guinea pig ileum, (R)‐HA‐966 causes a glycine‐reversible inhibition (IC50 of about 150 μM), whereas (S)‐HA‐966 is much less potent (IC50 of greater than 1mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N‐methyl‐d‐aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N‐methyl‐d‐aspartate receptors in glutamate‐evoked contractions of the guinea pig ileum, and supports their similarity to central N‐methyl‐d‐aspartate receptors.
Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations
Authors: Yorek Mark A. , Dunlap Joyce A. , Leeney Ellen M. , Stefani Mark R.
Published: October 2006

Abstract: Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels, myo‐lnositol accumulation and incorporation into inositol phospholipids and intracellular myo‐inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/ 30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo‐inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo‐inositol accumulation and incorporation into phospholipids at near normal levels, myo‐Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil‐resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.
Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Cerebral Cortex: Effect of Quinolinic Acid Lesions of the Basal Forebrain
Authors: Lapchak Paul A. , Araujo D. M. , Quirion R. , Beaudet A.
Published: October 2006

Abstract: The effects of neurotensin (NT) on endogenous acetylcholine (ACh) release from basal forebrain, frontal cortex, and parietal cortex slices were tested. The results show that NT differentially regulates evoked ACh release from frontal and parietal cortex slices without altering either spontaneous or evoked ACh release from basal forebrain slices. In the frontal cortex, NT significantly inhibited evoked ACh release by a tetrodotoxin (TTX)‐insensitive mechanism, suggesting an action directly on cholinergic terminals. In the parietal cortex, NT enhanced evoked ACh release by a TTX‐sensitive mechanism, suggesting an action of NT on the cholinergic neuron or in close proximity to the cholinergic neuron. The effects of NT on ACh release were confined to evoked ACh release; that is, spontaneous ACh release was not affected. NT did not affect spontaneous or potassium‐evoked ACh release from occipital cortex slices. The second set of experiments tested the effects of quinolinic acid (QUIN) lesions of the basal forebrain cell bodies on the NT‐induced regulation of evoked ACh release in the cerebral cortex. QUIN lesions of basal forebrain cell bodies caused decreases in choline acetyltransferase activity (27 and 28%), spontaneous ACh release (14 and 21%), and evoked ACh release (38 and 44%) in frontal and parietal cortex, respectively. In addition, 11 days following QUIN lesions of basal forebrain cell bodies, the action of NT to regulate evoked ACh release in frontal cortex or parietal cortex was no longer observed. The results suggest that in the rat frontal and parietal cortex, NT differentially regulates the activity of cholinergic neurons by decreasing and increasing evoked ACh release, respectively. In addition, the effect of NT is confined to the modulation of ACh release from terminals of cholinergic projection neurons ascending from the basal forebrain to the cerebral cortex.
Specific Binding of [3H]Pyrilamine to Histamine H1 Receptors in Guinea Pig Brain In Vivo: Determination of Binding Parameters by a Kinetic Four‐Compartment Model
Authors: Yanai Kazuhiko , Yagi Naoto , Watanabe Takehiko , Itoh Masatosi , Ishiwata Kiichi , Ido Tatsuo , Matsuzawa Taiju
Published: October 2006

The binding of [3H]pyrilamine, a selective ligand of histamine H1 receptors, to guinea pig brain in vivo was compared with its binding to a brain homogenate. The pharmacological properties (regional distribution, saturability, and stereoselectivity) of the [3H]pyrilamine binding in vivo were similar to those of the in vitro binding to brain homogenate. A dynamic four‐compartment model was proposed for the analysis of the kinetics of [3H]pyrilamine binding in vivo. The receptor constants in vivo were determined by a computer‐fitting method after correcting the radioactivity of arterial plasma and brain for the presence of radioactive metabolites. The in vivo association and dissociation were 213 and 42 times, respectively, slower than those of in vitro binding at 37°C. A possible mechanism for slow association and dissociation in vivo is discussed.
Analysis of Carnosine, Homocarnosine, and Other Histidyl Derivatives in Rat Brain
Authors: O'Dowd John J. , Cairns Maria T. , Trainor Maxine , Robins David J. , Miller David J.
Published: October 2006

Isocratic reverse‐phase analytical HPLC has been used to examine naturally occurring imidazoles of rat brain. Elution of brain extracts with a phosphate buffer mobile phase from columns packed with Hypersil ODS (5 μm) resulted in good separation of the well‐documented brain imidazole‐containing dipeptides carnosine and homocarnosine. Measured concentrations corresponded to published values. Several further peaks observed had properties consistent with those of N‐acetyl derivatives of compounds related to carnosine and homocarnosine. N‐Acetyl forms not commercially available were prepared and their identities verified by nuclear magnetic resonance spectroscopy. A number of these had chromatographic properties identical to those of compounds in brain extracts. Fractions corresponding to some of the peaks were examined using staining systems specific for certain chemical features and compared with results obtained for commercial or synthetic standards. The results of these tests supported the chromatographic data. Thus, chromatographic and microchemical evidence is presented for the existence of N‐acetyl forms of histidine, 1‐methylhistidine, carnosine, anserine, and homocarnosine in rat brain.
Neurofilament Proteins Are Distributed in a Diminishing Proximodistal Gradient Along Rat Sciatic Nerve
Authors: Schlaepfer William W. , Bruce Jennifer
Published: October 2006

Neurofilament (NF) proteins are distributed in a diminishing proximodistal gradient along rat sciatic nerve when compared with total noncollagen or other proteins in nerve. About a twofold decline of NF proteins can be detected by quantitating nerve proteins that have been separated by gel electrophoresis. A similar decrease of immunoreactivity to each NF subunit is seen in distal nerve segments when noncollagen nerve proteins are immunoblot ed. Parallel decreases occur in all three NF proteins, thereby maintaining neurofilament subunit stoichiometry along the neuraxis. The same NF gradient can be detected when the NF contents in nerve branches to the gluteus and gastrocnemius muscles are compared with each other and with those in nerve segments taken from the same proximodistal levels of the parent sciatic nerve. The gradient of NF proteins increases during postnatal development and is readily detected by postnatal day 16. During the same period of development, the heavy NF subunit appears for the first time and is rapidly incorporated throughout the sciatic nerve. Hence, the NF gradient becomes manifest during the development and maturation of the adult form of the axonal cytoskeleton. The basis for the proximodistal gradient of NF proteins in peripheral nerve is presently unknown. The extent of the gradient cannot be accounted for on the basis of diminishing numbers of nerve fibers or increasing amounts of other nerve proteins, e.g., collagen, in distal nerve. An alternative interpretation is that the gradient reflects a low level of NF protein turnover during axonal transport.
Reflex Splanchnic Nerve Stimulation Increases Levels of Carboxypeptidase E mRNA and Enzymatic Activity in the Rat Adrenal Medulla
Authors: Flicker Lloyd D. , Rigual Ricardo J. , Diliberto Emanuel J. , Viveros O. Humberto
Published: October 2006

Carboxypeptidase E (CPE; EC 3.4.17.10) is a carboxypeptidase B‐like enzyme involved with the biosynthesis of numerous peptide hormones and neurotransmitters, including the enkephalins. Reflex splanchnic stimulation of the rat adrenal medulla, which has previously been found to substantially increase enkephalin mRNA and enkephalin peptide levels, was examined for an influence on CPE mRNA and enzymatic activity. Several hours after insulin‐induced reflex splanchnic stimulation, the levels of CPE activity in rat adrenal medulla are reduced to 40–60% of control. CPE activity returns to the control level 2 days after the treatment and then continues to increase, reaching ˜200% of control 1 week after the treatment. The time course of the changes in CPE activity is different from those of the changes in epinephrine levels and the previously reported changes in enkephalin peptide levels. CPE mRNA is also influenced by the insulin shock, with levels increasing to 155% of the control level after 6 h and 170% after 2 days. The time course of the change in CPE mRNA levels is similar to that previously found for proenkephalin mRNA. However, the magnitude of the change is much different: Proenkephalin mRNA has been reported to increase by 1,600%. The changes in CPE mRNA and enzymatic activity are consistent with the proposal that CPE is not a rate‐limiting enzyme in the biosynthesis of enkephalin.
High Level of Expression of the Myelin Protein P0 in Chinese Hamster Ovary Cells
Authors: Filbin Marie T. , Tennekoon Gihan I.
Published: October 2006

The major PNS myelin protein, P0, has been expressed in Chinese hamster ovary cells by transfection with a plasmid containing the P0‐cDNA. The expression of P0 at both the RNA and the protein level was greatly increased, without detriment to the cell, by the dihydrofolate reductase/methotrexate strategy of gene amplification. The P0 expressed by these cells was glycosylated (containing approximately equal amounts of the complex and high‐mannose type glycoproteins) and reached the plasma membrane. This system is suitable not only for addressing the function of P0 directly, but is also applicable to any protein of which an abundance is needed.
Glutamate‐Dependent Active‐Site Labeling of Brain Glutamate Decarboxylase
Authors: Martin David L. , Wu Susan J. , Martin Sandra Bloom
Published: October 2006

A major regulatory feature of brain glutamate decarboxylase (GAD) is a cyclic reaction that controls the relative amounts of holoenzyme and apoenzyme [active and inactive GAD with and without bound pyridoxal 5′‐phosphate (pyridoxal‐P, the cofactor), respectively]. Previous studies have indicated that progression of the enzyme around the cycle should be stimulated strongly by the substrate, glutamate. To test this prediction, the effect of glutamate on the incorporation of pyridoxal‐P into rat‐brain GAD was studied by incubating GAD with [32P]pyridoxal‐P, followed by reduction with NaBH4 to link irreversibly the cofactor to the enzyme. Adding glutamate to the reaction mixture strongly stimulated labeling of GAD, as expected. 4‐Deoxypyridoxine 5′‐phosphate (deoxypyridoxine‐P), a close structural analogue of pyridoxal‐P, was a competitive inhibitor of the activation of glutamate apodecarboxylase by pyridoxal‐P (K1= 0.27 μM) and strongly inhibited glutamate‐dependent labeling of GAD. Analysis of labeled GAD by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis showed two labeled proteins with apparent molecular masses of 59 and 63 kDa. Both proteins could be purified by immunoaffinity chromatography on a column prepared with a monoclonal antibody to GAD, and both were labeled in a glutamate‐dependent, deoxypyridoxine‐P‐sensitive manner, indicating that both were GAD. Three peaks of GAD activity (termed peaks I, II, and III) were separated by chromatography on phenyl‐Sepharose, labeled with [32P]pyridoxal‐P, purified by immunoaffinity chromatography, and analyzed by SDS‐polyacrylamide gel electrophoresis. Peak I contained only the 59‐kDa labeled protein. Peaks II and III contained the both the 59‐ and 63‐kDa proteins, but in differing proportions. The 59‐kDa protein predominated in peak II and the 63‐kDa protein in peak III. A similar pattern of proteins was observed with each of the immunoaffinity‐purified peaks of enzyme activity on silver‐stained SDS‐polyacrylamide gels. 32P‐Labeled peak‐I GAD and the 59‐ and 63‐kDa bands of peak III were each exhaustively digested with chymotrypsin and chromatographed on sulfopropyl and reverse‐phase HPLC columns. The elution profiles of the digests were very similar, and each contained a single major peak of radioactivity, as would be expected if the same active‐site peptide was labeled. The strong stimulation by glutamate of labeling with [32P]pyridoxal‐P is entirely consistent with the mechanism of GAD inferred from previous studies and provides further evidence for the glutamate‐dependent cyclic interconversion of the apo‐ and holoenzymes.
Dephosphorylation of r Factor by Protein Phosphatase 2A in Synaptosomal Cytosol Fractions, and Inhibition by Aluminum
Authors: Yamamoto Hideyuki , Saitoh Yoshiki , Yasugawa Setsuko , Miyamoto Eishichi
Published: October 2006

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE‐cellulose column and assayed for protein phosphatases for τ factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form on Sephacryl S‐300 column chromatography. Both peaks 1 and 2 dephosphorylated τ factor phosphorylated by Ca2+/calmodulin‐dependent protein kinase II and the catalytic subunit of cyclic AMP‐dependent protein kinase. The Km values were in the range of 0.42–0.84 μM for τ factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca2+, Mn2+, and Mg2+. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC50 values of 40–60 μM. These results suggest that dephosphorylation of r factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of τ factor
Rapid Isolation of Metabolically Active Mitochondria from Rat Brain and Subregions Using Percoll Density Gradient Centrifugation
Authors: Sims Neil R.
Published: October 2006

Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing‐out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.
Presence of 3‐Hydroxyanthranilic Acid in Rat Tissues and Evidence for Its Production from Anthranilic Acid in the Brain
Authors: Baran Halina , Schwarcz Robert
Published: October 2006

Abstract: As assessed by HPLC with electrochemical detection, 3‐hydroxyanthranilic acid (3‐HANA) was found to be present in the rat brain and peripheral organs. The highest concentrations were measured in the kidney (86 fmol/mg of tissue) and spleen (56 fmol/mg of tissue), whereas the adrenal gland, liver, heart, and several forebrain areas (hippocampus, striatum, parietal cortex, thalamus, amygdala/pyriform cortex, and frontal cortex) contained less 3‐HANA (between 15 and 22 fmol/mg of tissue). Slightly lower concentrations of 3‐HANA were found in the brainstem and the cerebellum. The metabolic disposition of 3‐HANA was examined in tissue slices which were incubated in Krebs‐Ringer buffer at 37°C in vitro. Incubation for up to 2 h did not affect 3‐HANA concentration in brain tissue. However, inhibition of 3‐HANA degradation by the specific 3‐hydroxyanthranilic acid oxygenase blocker 4‐chloro‐3‐hydroxyanthranilic acid (4‐C1‐3‐HANA; 10 μM) resulted in a rapid (within 2.5 min) doubling of 3‐HANA levels in slices from cerebral cortex. No further increases were observed after incubations of up to 120 min. Exposure of cortical slices to 3‐HANA's putative bioprecursors, 3‐hydroxykynurenine (3‐HK) and anthranilic acid (ANA), in the absence of 4‐C1‐3‐HANA resulted in rapid, transient increases in 3‐HANA production. Maximal 3‐HANA synthesis from ANA exceeded the maximal effect of 3‐HK by approximately 11‐fold. In the presence of 4‐C1‐3‐HANA, 1 mM 3‐HK and 1 mM ANA produced 9.0 ± 0.3 and 89.0 ± 9.3 (5 min) or 51.6 ± 7.9 and 187.5 ±11.2 (120 min) fmol of newly synthesized 3‐HANA/mg of brain tissue, respectively. In the brain, but not in the spleen, ANA proved to be a superior 3‐HANA precursor at lower concentrations as well. Time and dose relationships for the de novo production of 3‐HANA from ANA in brain slices were established in the presence of 10 μM 4‐C1‐3‐HANA. Biosynthesis of 3‐HANA from ANA in the brain may be critically involved in the function or dysfunction of 3‐HANA's principal cata‐bolic product, the endogenous excitotoxin quinolinic acid.
Studies on the Topography of the Catalytic Site of Acetylcholinesterase Using Polyclonal and Monoclonal Antibodies
Authors: Ogert Robert A. , Gentry Mary K. , Richardson Earl C. , De Al Carolyn D. , Abramson Stewart N. , Alving Carl R. , Taylor Palmer , Doctor B. P.
Published: October 2006

Abstract: Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme‐linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross‐reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggests that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket‐like conformation in the native enzyme.
Inhibition of Monoamine Oxidase by 7‐Chloro‐4‐Nitrobenzofurazan
Authors: Fu Richard C. , Liu Xian‐Fang , Chen Shiuan
Published: October 2006

Abstract: 7‐Chloro‐4‐nitrobenzofurazan (NBD‐C1) is a potent inhibitor of both types of monoamine oxidase (MAO). NBD‐Cl competitively inhibited the oxidative deamination of kynuramine catalyzed by human placenta MAO‐A, the oxidative deamination of benzylamine catalyzed by bovine liver MAO‐B, the oxidative deamination of serotonin catalyzed by rat brain MAO‐A, and the oxidative deamination of phenylethylamine catalyzed by rat brain MAO‐B. In addition, a timedependent inactivation of MAOs by NBD‐Cl has been demonstrated upon incubation of the enzyme preparations with NBD‐C1 at pH 9, but not at pH 7.5. The time‐dependent inhibition of MAO by NBD‐C1 could be prevented by the addition of 4‐nitrophenyl azide, an active site‐directed label of MAO, during incubation of the enzyme with NBD‐C1. On the basis of these findings, it is suggested that at pH 9, NBD‐Cl modifies one (or more) essential lysine residue(s) in the active sites of the two types of MAO.
A Retinal Ganglion Cell Neurotrophic Factor Purified from the Superior Colliculus
Authors: Schulz Mark , Raju Trichur , Ralston Gregory , Bennett Max R.
Published: October 2006

Abstract: Dissociated neonatal rat retinal ganglion cells can be maintained by the addition of an extract from the neonatal superior colliculus. This extract can support 95% of ganglion cells over 24 h in culture; in addition it promotes the expression of neurites from these cells. This report describes the purification of a neurotrophic factor from the superior colliculus which supports the survival of 80% of retinal ganglion cells over 24 h in vitro. The purification procedure involves a combination of dye‐ligand, anion‐exchange, and molecular sieve chromatography. The purified neurotrophic factor has a Stokes radius of approximately 200 Å using molecular sieve chromatography in the presence of a chaotropic agent. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified factor indicates that it is a glycoprotein that migrates with a molecular mass >400 kDa. Further characterization of this high‐molecular‐mass glycoprotein by enzymatic digestion demonstrated that it is a chondroitin sulfate pro‐teoglycan. This factor is clearly distinguishable from other neurotrophic factors that have an effect on retinal ganglion cells such as brain‐derived neurotrophic factor and fibroblast growth factor. The chondroitin sulfate proteoglycan from the neonatal superior colliculus is the first proteoglycan to be identified as a neurotrophic factor.
Infection with Murine Retrovirus Confers Resistance to the Neurotoxin 1‐Methyl‐4‐Phenylpyridinium Ion in PC12 Cells
Authors: Kadan Michael J. , Lo Mathew M. S.
Published: October 2006

Abstract: High concentrations of the dopaminergic neurotoxin l‐methyl‐4‐phenylpyridinium ion (MPP+) are toxic to the catecholaminergic cell line PC 12, derived from rat pheochromocytoma. Prolonged exposure of wild‐type PC 12 cells to 500 μM MPP+ yields toxin‐resistant colonies at a frequency of 2 ± 10‐‐4. These spontaneously arising MPP+‐resistant cells are morphologically quite distinct from wild‐type PC 12 cells, and are lacking in most of their characteristic catecholaminergic properties. In contrast, among PC12 cells infected with the murine retrovirus ZIPNEOSV(X), 20% are resistant to the toxin MPP+, a resistance frequency approximately 1,000 times higher than for uninfected cells. The morphology and catecholaminergic phenotype of the virus‐infected MPP+ resistant cells are quite similar to those of wild‐type PC 12 cells. The results presented in this study suggest a unique mechanism of MPP+ resistance in the infected PC 12 cells which may be conferred by the presence and/or expression of the retrovirus ZIPNEOSV(X).