bluuebunny/crossref_metadata_2025_split huggingface dataset:
Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site
Authors: Lomneth Rich , Martin Thomas F. J. , DasGupta Bibhuti R. 
Published: October 2006

AbstractBotulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described “cell cracking” procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell “ghosts” which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca2+‐activated [3H] nuorepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150‐kDa NT and the 50‐kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca2+‐activated [3H] norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100‐kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by “cell cracking.” The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]‐ norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.
Biochemical and Autoradiographical Determination of Protein Synthesis in Experimental Brain Tumors of Rats
Authors: Widmann R. , Kocher M. , Ernestus R.‐I. , Hossmann K.‐A. 
Published: October 2006

AbstractThe rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5–3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma‐derived) and endogenous (proteolysis‐derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60–70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 ± 16.0 nmol/g of tissue/min in brain tumor, and 17.2 ± 4.2 and 9.7 ± 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic interventions.
Specific Expression of N‐Acetylaspartate in Neurons, Oligodendrocyte‐Type‐2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro
Authors: Urenjak Jutta , Williams Stephen R. , Gadian David G. , Noble Mark 
Published: October 2006

AbstractTo test the specificity of N‐acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type‐1 astrocytes, meningeal cells, oligodendrocyte‐type‐2 astrocyte (O–2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type‐1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O‐2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors.
Reduced Neuropeptide Y Concentrations in Suicide Brain
Authors: Widdowson Peter S. , Ordway Gregory A. , Halaris Angelos E. 
Published: October 2006

AbstractNeuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY‐immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY‐immunoreactivity were significantly lower in postmortem frontal cortex (−14%) and caudate nucleus (−27%) from suicide victims compared with age‐matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY‐immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY‐immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age‐matched controls. We suggest there is a deficit in the brain NPY system leading to region‐specific reductions in peptide concentrations in subjects who have a history of depression.
Glutamate, of the Endogenous Primary α‐Amino Acids, Is Specifically Released from Hair Cells by Elevated Extracellular Potassium
Authors: Drescher Marian J. , Drescher Dennis G. 
Published: October 2006

AbstractA hair cell (octavolateralis mechanoreceptor cell) sheet preparation from the trout saccular macula was superfused with bicarbonate‐based physiological saline. Among the primary amine‐containing compounds resolved by cation‐exchange HPLC, glutamate alone was released in a statistically significant manner with elevation of extracellular [K+] from 3.5 to 14 mM in the presence of 1.8 mM calcium. Release of glutamate averaged 10.9 ± 2.5 pmol (mean ± SEM) over a 10‐min period for a hair cell sheet preparation representing 20 μg of cell protein. No potassium‐evoked release of glutamate was observed in 0 mM calcium/ 10 mM magnesium saline, suggesting calcium dependency. Because the sheet preparation, by the method of its isolation, contained only the hair cell as the intact cell type, release of glutamate, induced by relatively small increases in extracellular potassium, can be attributed directly to the receptor cell. The specific release of glutamate and its block by magnesium are consistent with the hypothesis that glutamate is one neurotransmitter/neuromodulator mediating receptoneural transmission in the octavolateralis periphery.
Identification of an Activator of the Microtubule‐Associated Protein 2 Kinases ERK1 and ERK2 in PC12 Cells Stimulated with Nerve Growth Factor or Bradykinin
Authors: Ahn Natalie G. , Robbins David J. , Haycock John W. , Seger Rony , Cobb Melanie H. , Krebs Edwin G. 
Published: October 2006

AbstractTreatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal‐regulated kinases ERK1 and ERK2, two isozymes of microtubule‐associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyro‐sine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42‐ (ERK1) and 44‐kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide‐binding protein‐coupled receptors are both capable of regulating these pathways.
Ethanol‐Induced Increase in Endogenous Dopamine Release May Involve Endogenous Opiates
Authors: Widdowson Peter S. , Holman R. Bruce 
Published: October 2006

AbstractThe effect of opiate peptides on basal and potassium‐stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the striatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the δ‐opiate receptor agonists Met5‐enkephalin, [D‐Ala2,D‐Leu5]enkephalin (DADLE), and [D‐Ser]Leu‐enkephalin‐Thr (DSLET), increased the basal DA release without affecting potassium‐stimulated release in a dose‐dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the μ‐opioid receptor agonist [D‐Ala2, N‐MePhe4,Gly‐ol5]enkephalin (DAGO) and the e‐opioid agonist β‐endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium‐stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incubation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose‐dependent, selective increase in basal DA release. Naloxone and the selective δ‐opiate antagonist ICI 174864 inhibited the eth‐anol‐induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol‐in‐duced increase in basal DA release is an indirect effect involving an endogenous δ‐opiate agonist.
Screening of Thiol Compounds: Depolarization‐Induced Release of Glutathione and Cysteine from Rat Brain Slices
Authors: Zängerle Leo , Cuénod Michel , Winterhalter Kaspar H. , Do Kim Quang 
Published: October 2006

AbstractSuperfusates from rat brain slices were screened for thiol compounds after derivatization with monobromo‐bimane by reversed‐phase HPLC. Only glutathione and cysteine were detected. The Ca2+‐dependent release of these compounds from slices of different regions of rat brain was investigated, applying a highly sensitive and reproducible quantification method, based on reduction of superfusates with dithiothreitol, reaction of thiols with iodoacetic acid, precolumn derivatization with o‐phthalaldehyde reagent solution, and analysis with reversed‐phase HPLC. This methodology allowed determination of reduced and total thiols in aliquots of the same superfusates. Mostly reduced glutathione and cysteine were released upon K+ depolarization and the Ca2+ dependency suggests that they originate from a neuronal compartment. The GSH release was most prominent in the mesodiencephalon, cortex, hippocampus, and striatum and lowest in the pons‐medulla and cerebellum. This underscores a physiologically significant role for glutathione in CNS neurotransmission.
In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain
Authors: Masana Monica I. , Bitran Jose A. , Hsiao John K. , Potter William Z. 
Published: October 2006

AbstractIn vivo microdialysis of cyclic AMP from prefron‐tal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 ±0.05 mM; n= 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin‐catalyzed ADP ribosyla‐tion of αi (and αo) was increased by 20% in prefrontal cortex from lithium‐treated rats, but the αi and αs contents (as determined by immunoblot) as well as the cholera toxin‐catalyzed ADP ribosylation of αs were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin‐stimulated adenylate cyclase activity.
Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy
Authors: Corbett Ronald J. T. , Laptook Abbot R. , Garcia Damian , Ruley Joan I. 
Published: October 2006

AbstractCerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6‐6.0, compared to 7.0‐6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1‐month‐old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.
Effect of Intracellular Glutamine on the Uptake of Large Neutral Amino Acids in Astrocytes: Concentrative Na+‐Independent Transport Exhibits Metastability
Authors: Brookes Neville 
Published: October 2006

AbstractTo examine whether the concentration gradient of glutamine (Gln) drives concentrative Na+‐independent uptake of neutral amino acids (NAA) in mouse cerebral astrocytes, uptake was compared in “Gln‐depleted” and “Gin‐replete” cultures. Uptake (30 min in Na+‐free buffer) of histidine, kynurenine, leucine, tyrosine, and a model substrate for System L transport was 70–150% greater in Gin‐replete cultures. Phenylalanine uptake was not affected. All of these NAA trans‐stimulated the export of Gln from astrocytes. However, the increase in NAA uptake was sustained even though the Gln content of Gin‐replete cultures declined. Also, uptake of Gln itself was enhanced in Gln‐re‐plete cultures. Thus, countertransport of Gln was insufficient to explain the enhancement of NAA uptake. Enhanced uptake was restored, and could be magnified, by reloading Gin‐depleted cultures either with Gln or with histidine. It is suggested that substrate‐induced asymmetry and molecular hysteresis in the Na+‐independent carrier could account for the sustained enhancement of NAA uptake. Only histidine and kynurenine were concentrated comparably to Gin (15‐ to 29‐fold at 1 mM in Na+‐free buffer). The other NAA were four to six times less concentrated. At least two Na+‐dependent transport systems also supported the concentration gradient of Gln in regular buffer.
In Vivo Partial Inactivation of Dopamine D1 Receptors Induces Hypersensitivity of Cortical Dopamine‐Sensitive Adenylate Cyclase: Permissive Role of α1‐Adrenergic Receptors
Authors: Trovero Fabrice , Hervé Denis , Blanc Gérard , Glowinski Jacques , Tassin Jean‐Pol 
Published: October 2006

AbstractAs shown by autoradiography, peripheral injections of N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) induced a dose‐dependent decrease of [3H]SCH 23390 and [3H]prazosin high‐affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin‐sensitive α1‐adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA‐sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA‐sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA‐sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (‐20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ‐induced supersensitivity of DA‐sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%. Demonstrating further a role of noradrenergic transmission of the α1 type in the regulation of the cortical D1 receptor sensitivity, the blockade of α1‐adrenergic receptors by prazosin prevented the appearance of the EEDQ‐induced supersensitivity of DA‐sensitive adenylate cyclase in groups of rats exhibiting similar decreases in the density of D1 binding sites (‐20%). The DA‐sensitive adenylate cyclase activity was significantly lower in cortices of animals pretreated with prazosin than in those not treated with the α1 antagonist. These results are discussed in relation to previous studies indicating that prazosin pretreatment also abolishes the locomotor hyperactivity induced by bilateral electrolytic lesions of the ventral teg‐mental area.
Regulated Expression of the Neurofibromin Type I Transcript in the Developing Chicken Brain
Authors: Baizer Lawrence , Ciment Gary , Hendrickson Susan K. , Schafer Gwen L. 
Published: October 2006

AbstractNeurofibromatosis type 1 (NF‐1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF‐1 gene is similar in sequence to the ras‐GTPase activator protein (ras‐GAP), and investigations have confirmed that the NF‐1 gene product (now known as neurofibromin) stimulates ras‐GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63‐bp exon inserted in the GAP‐related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon‐intron boundaries. RNase protection and RNA‐polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is enriched in neurons.