bluuebunny/crossref_metadata_2025 huggingface dataset:
Aluminum Increases Agonist‐Stimulated Cyclic AMP Production in Rat Cerebral Cortical Slices
Authors: Johnson Gail V. W. , Li Xiaohua , Jope Richard S.
Published: October 2006

Abstract: The effects of A1C13 on basal and stimulated cyclic AMP production in rat cerebral cortical slices were studied. AICI3 (10–250 μM) had no effect on the cyclic AMP concentration in the absence of drugs that stimulate the synthesis of cyclic AMP. 2‐Chloroadenosine (25–200 μM) significantly stimulated the synthesis of cyclic AMP in a concentration‐dependent manner, and A1C13 significantly potentiated this response at 50 and 100 μM 2‐chloroadenosine. This effect of AICI3 was dependent on preexposure of the slices to A1C13 before addition of the agonist. The potentiation by A1C13 of the 2‐chloroadenosine‐induced increase in cyclic AMP level was concentration dependent, with significant enhancement by 100 (142% of the control) and 250 (150% of the control) μM AICI3. Lower concentrations of A1C13 had no significant effect on the production of cyclic AMP stimulated by 2‐chloroadenosine. AICI3 also potentiated the isoproterenol‐induced increase in cyclic AMP production. Forskolin‐induced production of cyclic AMP was unaltered by the presence of A1C13. These results demonstrate that A1C13 can potentiate agonist‐stimulated cyclic AMP production in a whole‐cell brain preparation without the addition of fluoride. This may account for the previously reported aluminum‐induced increase in cyclic AMP concentrations in rat brain in vivo.
Catalysis of Slow C‐Terminal Processing Reactions by Carboxypeptidase H
Authors: Smyth D. G. , Maruthainar K. , Darby N. J. , Fricker L. D.
Published: October 2006

Abstract: A hypothesis was examined that carboxypeptidase H (CpAse H), which is known to catalyse the release of lysine and arginine from the C‐terminus of peptides, can also release histidine, tyrosine, and phenylalanine. Synthetic peptides terminating in ‐His‐Lys or ‐Tyr‐Lys were used as model substrates for the enzyme and amino acid analysis was employed to detect release of the terminal amino acids. With N‐acetyl‐β‐Ala‐Asn‐Ala‐His‐Lys and N‐acetyl‐β‐Ala‐Asn‐Ala‐Tyr‐Lys, which correspond to intermediates in the processing of porcine and human β‐endorphin, lysine was removed rapidly and quantitatively but no release of histidine or tyrosine could be detected. To allow more sensitive analysis, radiolabelled substrates were employed and the amounts of the products formed on incubation with CpAse H were determined after separation by ion‐exchange chromatography. With 125I‐D‐Tyr‐Ala‐His‐Lys‐Lys as substrate at pH 5.7, very small amounts of D‐Tyr‐Ala were released; the main product was D‐Tyr‐Ala‐His. At pH 5.0 the release of histidine from 125I‐D‐Tyr‐AlaHis took place 6,000 times more slowly than the release of lysine from l25I‐D‐Tyr‐Ala‐Lys. When the tripeptides were incubated at pH 5 with porcine pituitary secretory granules, the lysine was released rapidly but no release of histidine could be detected. The results demonstrate that CpAse H catalyses the release of C‐terminal histidine with great difficulty. Evidence is also presented that CpAse H catalyses a similar slow reaction in converting α‐neo‐endorphin to β‐neo‐endorphin, which involves removal of lysine from a Pro‐Lys sequence. These very slow reactions provide an explanation for the incomplete processing that is observed with neuropeptides terminating in histidine, tyrosine, or phenylalanine or in prolyllysine.
Cultured Astrocytes and Neurons Synthesize and Secrete Carboxypeptidase E, a Neuropeptide‐Processing Enzyme
Authors: Vilijn Marie‐Hélène , Das Banasree , Kessler John A. , Flicker Lloyd D.
Published: October 2006

Abstract: Carboxypeptidase E (EC 3.4.17.10) is a carboxy‐peptidase B‐like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E‐like activity. Cultured astrocytes secrete ∼73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCI. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCI. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four‐ to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.
Extracellular ATP Stimulates Norepinephrine Uptake in PC12 Cells
Authors: Hardwick Jean C. , Ehrlich Yigal H. , Hendley Edith D.
Published: October 2006

Abstract: This study examined the effects of extracellular ATP on norepinephrine (NE) uptake, using PC12 cells as a model of noradrenergic neurons. Previous experiments with syn‐aptosomes led to the hypothesis that extracellular ATP can regulate NE uptake via an ecto‐protein kinase. In the present study, we examined the high‐affinity uptake of NE (referred to as uptake 1) in PC12 cells in the presence of varying concentrations of extracellular ATP. In the presence of Ca2+, low concentrations of ATP (0.1 μM) increased uptake 1 by approximately 36%. This increase could be mimicked by aden‐osine‐5′‐O‐(3‐thiotriphosphate) tetralithium salt (ATPγS), an analogue of ATP which can be utilized by protein kinases, and not by 5′‐adenylylimidodiphosphate tetralithium salt, a nonhydrolyzable analogue of ATP. GTP, ADP, and adenosine also had no effect on uptake 1. Preincubation of the cells with NE and ATPγS, followed by washing and assaying NE uptake 30 min later, resulted in a persistent increase in uptake 1. Similar pretreatment with ATP did not show this increase; however, simultaneous pretreatment with ATP and ATPγS blocked the activation produced by ATPyS alone. Kinetic analysis showed that ATPγS pretreatment produces an increase in the Vmax of uptake 1 without altering the apparent Km for NE. These results support the hypothesis that extracellular ATP can regulate NE uptake via an ecto‐protein kinase.
Solubilization and Characterization of σ‐Receptors from Guinea Pig Brain Membranes
Authors: Kavanaugh M. P. , Parker J. , Bobker D. H. , Keana J. F. W. , Weber E.
Published: October 2006

Abstract: The σ‐receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been sol‐ubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine‐treated glass fiber filters. The pharmacological specificity of the solubilized binding site for σ‐receptor ligands is nearly identical to the membrane‐bound form of the receptor, with the order of potencies for displacement of the selective σ‐ligand [3H]di‐o‐tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)‐benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD= 28 ± 0.5 nM) and (+)‐[3H]3‐(3‐hydroxyphenyl)‐N‐(l‐propyl)piperi‐dine {(+)‐[3H]3‐PPP} (KD= 36 ± 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p‐azido‐DTG, a σ‐selective photoaffinity label, resulted in labeling of a 29‐kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL‐6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the σ‐selective ligands (+)‐[3H]3‐PPP and (+)‐[3H]SKF‐10,047, supporting the hypothesis that all three ligands bind to the same macro‐molecular complex.
Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth
Authors: Mann Derek A. , Doherty Patrick , Walsh Frank S.
Published: October 2006

Abstract: The relative expression of the immunoglobulin su‐perfamily members Thy‐1 and L1 and the neural cell adhesion molecule (N‐CAM) in PC 12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140‐ and 180‐kilodalton subunits of N‐CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy‐1 and L1. Over longer periods of culture (3‐5 days), cholera toxin inhibited the NGF induction of N‐CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N‐CAM expression correlate best with the morphological differentiation status of PC12 cells.
Slow Axonal Transport of Structural Polypeptides in Rat, Early Changes in Streptozocin Diabetes, and Effect of Insulin Treatment
Authors: Larsen Jørn Rolighed , Sidenius Per
Published: October 2006

Abstract: The synthesis and transport of slowly transported polypeptides in sciatic nerves of rats was investigated by [35S]methionine pulse labeling and gel electrophoresis in control, diabetic, and insulin‐treated diabetic rats. To detect very early changes diabetes was induced by streptozocin only 5 days prior to the labeling of the dorsal root ganglion cells. Fourteen days were allowed for axonal transport. In this experimental system, the neurofilament triplet is transported at an apparent velocity of 1.1 ± 0.1 mm/day (mean ± SD). The actin‐related complex, including actin and two polypeptides of 87 kilodaltons and 37 kilodaltons, was transported at a velocity of 2.6 ± 0.2 mm/day. For α‐ and β‐tubulin we found an apparent transport velocity of 2.2 ± 0.1 mm/day, placing it between actin and the neurofilament triplet. The diabetic rats had a selective 32% decrease in the amount of the heaviest neurofilament subunit: 0.47 ± 0.19% of trichloroacetic acid‐insoluble radioactivity versus 0.69 ± 0.17% in controls; 2p < 0.05. This decrease was associated with a proximal accumulation of the two lighter neurofilament sub‐units. Insulin treatment of a diabetic group failed to normalize the changes of axonal transport and additional changes suggesting a hypoglycemic injury were observed.
Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N‐[3H]Methylcarbamylcholine Binding Sites in the Rat Brain
Authors: Lapchak P. A. , Araujo D. M. , Quirion R. , Collier B.
Published: October 2006

Abstract: It has been reported that N‐methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region‐specific up‐regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium‐dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC‐induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium‐evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested. These results suggest that chronic nicotine increases the nicotinic ([3H]MCC) binding site density in the frontal cortex, hippocampus, and striatum and results in the loss of nicotinic receptor‐mediated positive feedback in the cortex and hippocampus. The loss of presynaptic nicotinic receptor function is reversible, and the time course of this recovery differs from that of the change in [3H]MCC binding sites.
Highly Basic 30‐ and 32‐Kilodalton Proteins Associated with Synapse Formation on Polylysine‐Coated Beads in Enriched Neuronal Cell Cultures
Authors: Burry Richard W. , Hayes Diane M.
Published: October 2006

Abstract: Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two‐dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29‐kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysinecoated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two‐dimensional nonequilibrium pH gradient gel electrophoresis (2D‐NEPHGE), levels of a basic 32‐kD protein (pI 8) not detected in cultures alone, and increased levels of a 30‐kD protein (pI 10). When culture medium was examined with 2D‐NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine‐coated beads to identify basic 30‐kD and 32‐kD proteins that may be involved in synapse formation.
Reduction of K+‐Stimulated 45Ca2+ Influx in Synaptosomes with Age Involves Inactivating and Noninactivating Calcium Channels and Is Correlated with Temporal Modifications in Protein Dephosphorylation
Authors: Martínez‐Serrano Alberto , Bogónez Elena , Vitórica Javier , Satrústegui Jorgina
Published: October 2006

Abstract: The voltage‐dependent calcium uptake in rat brain synaptosomes was measured under conditions in which [Ca2+]o/[Na+]i exchange was minimized to characterize the voltage‐sensitive calcium channels from rats of different ages. In solutions of CaCl2 concentrations of <500 μM, the initial (5‐s) calcium uptake declined by ∼ 20–50% in 12‐ and 24‐month‐old rats relative to 3‐month‐old adults. Depolarization of synaptosomes from 3‐month‐old rats in a calcium‐free medium or in the presence of 0.5 mM CaCl2 led to an exponential decline of the calcium uptake rate after 20 s (voltage‐ or voltage‐and‐calcium‐dependent inactivation) to ∼ 66 and 34% of the initial value with a t1/2 of 1.6 or 0.7 s, respectively. The presence of 1 μM nifedipine resulted in a 15–25% reduction of 45Ca2+ uptake rates, which appeared to affect noninactivating calcium channels, but addition of the calcium channel agonist Bay K 8644 was without effect. In 24‐month‐old rats, inactivation of 45Ca2+ uptake in calcium‐free media was nondetectable, and in the presence of 0.5 mM CaCl2, the rate and extent of inactivation were also much lower than in 3‐month‐old animals (the t1/2 was 0.9 s, and the calcium uptake rate at 20 s was 55% of its initial value). Moreover, the presence of 1 μM nifedipine was without effect on initial calcium uptake or inactivation in synaptosomes from 24‐month‐old rats. These results indicate that the decrease in calcium channel‐mediated 45Ca2+ uptake involves an inhibition or block of both dihydropyridine‐resistant and ‐sensitive calcium channels. It is interesting that the large decrease in calcium uptake via calcium channels that occurs in 24‐month‐old rats is correlated with a slower dephosphorylation of the synaptosomal phosphoprotein P96, a process that is intimately related to the opening and ensuing calcium flow through voltage‐gated calcium channels.
Solubilization of Quisqualate‐Sensitive [3H]Glutamate Binding Activity from Rat Retina
Authors: Yoneda Yukio , Ogita Kiyokazu
Published: October 2006

Abstract: Binding activity of a putative central neurotransmitter, l‐glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P‐40. [3H]Glutamate binding activity increased linearly with increasing concentrations of the solubilized proteins up to 15 μg. The binding activity reached an equilibrium within 10 min at 2°C, while increasing with incubation time up to 60 min at 30°C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30°C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 μM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to l‐glutamate, and was abolished by quisqualate, l‐glutamate diethyl ester, and DL‐2‐amino‐3‐phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30°C to 2°C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation‐exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30°C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate‐sensitive membranous enzyme with a stereospecific and structure‐selective high affinity for the central neurotransmitter.
Characterization of Novel Fluorescent Ligands with High Affinity for D1 and D2 Dopaminergic Receptors
Authors: Monsma Frederick J. , Barton Anne C. , Chol Kang Hee , Brassard Diana L. , Haugland Richard P. , Sibley David R.
Published: October 2006

Abstract: We have synthesized and characterized a series of novel fluorescently labeled ligands with high affinity and specificity for D1 and D2 dopamine receptors. D1‐selective probes were synthesized using (R,S)‐5‐(4′‐aminophenyl)‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐[1H]‐3‐benzazepin‐7‐ol, the 4′‐amino derivative of the high‐affinity, D1‐selective antagonist SCH‐23390, whereas D2‐selective probes were synthesized using the high‐affinity, D2‐selective antagonist N‐(p‐aminophenethyl)spiperone (NAPS). These ligands were coupled via spacer arms of various lengths to the fluorophores fluorescein and bodipy, which fluoresce in the yellow–green region, and to tetramethylrhodamine, which is a red fluorophore. The interaction of these fluorescent ligands with dopamine receptors was evaluated by examining their ability to compete for the binding of the radiolabeled antagonists [3H]SCH‐23390 or [3H]methylspiperone to rat striatal D1 or D2 dopamine receptors, respectively. We report here that these novel fluorescent ligands exhibit very high affinity and specificity for either D1 or D2 dopamine receptors. The availability of various fluorescent ligands with different emission maxima and with high affinity and specificity for D1 and D2 dopamine receptors will now permit investigations involving the visualization and localization of these receptor subtypes at the single cell and intracellular levels in the CNS and on intact cells in culture.
Content and In Vitro Release of Endogenous Amino Acids in the Area of the Nucleus of the Solitary Tract of the Rat
Authors: Meeley Mary P. , Underwood Mark D. , Talman William T. , Reis Donald J.
Published: October 2006

Abstract: We sought to identify amino acid neurotransmitter candidates within the nucleus of the solitary tract in rats. Twenty endogenous amino acids were quantified by reverse‐phase HPLC with fluorescence detection (30‐fmol limit). Mi‐cropunches (1 mm) of the intermediate area of the solitary nucleus were prepared, and the amino acid content determined. Of all the components measured, the putative transmitters Glu, Gly, γ‐aminobutyric acid, taurine, Asp, and Ala appeared in greatest concentrations. Bilateral micropunches superfused in vitro with buffered medium containing 56 mM potassium released Glu, γ‐aminobutyric acid, and Gly in a significant manner (p < 0.05) compared with basal levels. With Glu, 78% was calcium‐dependent and, therefore, presumably from nerve endings; 99% of γ‐aminobutyric acid and 42% of Gly were dependent on calcium. After removal of the nodose ganglion, a bilateral decrease in the calcium‐dependent release of Glu and γ‐aminobutyric acid, but not Gly, was observed; decreases were significant ipsilateral to the site of ablation. We conclude that (a) Glu is a transmitter of primary afferents in the nucleus of the solitary tract; (b) glutamatergic afferents may interact with γ‐aminobutyric acid system(s) in this region; (c) Gly also may participate in the mediation and/or modulation of cardiovascular or other visceral reflexes; and (d) amino acid neurotransmission may play an integral role in the neurogenic control of arterial pressure
Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors
Authors: Gulat‐Marnay Christiane , Lafitte Andrée , Arrang Jean‐Michel , Schwartz Jean‐Charles
Published: October 2006

Abstract: The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with l‐[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+‐induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1 ‐receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with ki values of 1–6 × 10−8M. 11‐[{2‐t(Diethylamino)methyl]‐1‐piperidinyl}acetyl] ‐5,11 ‐ dihydro ‐ 6H ‐pyrido[2,3 ‐ b][1,4]benzo ‐ diazepine‐6‐one (AF‐DX 116), considered a preferential M2‐receptor antagonist, reversed the carbachol effect with a mean Ki of ∼2 × 10−7M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+‐induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H‐labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1‐receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF‐DX 116.
Phosphorylation of Myelin‐Associated Glycoprotein In Vivo and In Vitro Occurs Only in the Cytoplasmic Domain of the Large Isoform
Authors: Edwards Aled M. , Braun Peter E. , Bell John C.
Published: October 2006

Abstract: Myelin‐associated glycoprotein (MAG) was radioactively labelled with 32P both in intact brain and in myelin membrane preparations. Chemical deglycosylation of the phosphorylated products revealed that only one of the MAG isoforms (L‐MAG) is labelled in vitro. Furthermore, the phosphorylation events in vivo and in vitro are confined to the cytoplasmic portion of the L‐MAG isoform. Tryptic mapping of L‐MAG labelled both in vivo and in vitro revealed that the majority of the sites phosphorylated in intact brain are also phosphorylated in myelin membrane preparations; however, the extent of phosphorylation at individual sites is variable. The results demonstrate that partially purified myelin membrane preparations can be used to study the enzymes responsible for MAG phosphorylation and dephosphorylation events in vivo.
Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca2+‐Dependent Manner in Rat Brain Synaptic Plasma Membranes
Authors: Hollmann Michael , Seifert Wilfried
Published: October 2006

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl‐N‐acetylgalactosalminyl‐(N‐acetyl‐neuraminyl)‐galactosylglucosyl ceramide (GMl) on high‐affinity binding of L‐[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40–50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H‐labeled GM1 proved trypsin‐stable insertion of GM1 into SPM, with a maximum incorporation pf four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+‐stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 μl/mg of protein, in ganglioside‐treated as well as in control membranes. From the data, it is concluded that a special. Na+‐ and C1‐independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.
Temporal Profile of Levels of Monoamines and Their Metabolites in Striata of Rats Implanted with Dialysis Tubes
Authors: Reiriz J. , Mena M.A. , Bazán E. , Muradás V. , Lerma J. , Delgado J. M R. , De Yébenes J. G.
Published: October 2006

We have measured the levels of monoamines and their metabolites in rat striata implanted with a dialysis tube, in contralateral nonimplanted striata, and in diialysates obtained from the dialysis tube. The perfusion was done with Ringer solution. The animals were perfused either for a continuous period of 7 h at 1 day after implantation or for periods of 2 h on days 1, 4, and 7 after implantation] In animals perfused for 7 h, levels of monoamine metabolites in dialy‐sates remained stable for the first 4 h of perfusion, but a reduction was observed during the last 3 h. In Animals perfused for 2 h on days 1, 4, and 7 after implantation, we observed a progressive reduction in levels of metabolites in di‐alysates with respect to the first day of perfusion The levels of dopamine and its metabolites in the striata in which a dialysis cannula had been implanted showed a progressive reduction during the period postimplantation comparable to that observed in dialysates. The levels of 3‐methoxytyramine, 3,4‐dihydroxyphenylacetic acid, and homovanillic acid were elevated 24 h after implantation in the implanted striata with respect to the contralateral nonimplanted striata, but 7 days after implantation, the levels of dopamine were decreased in the implanted striata, and tne levels of metabolites were unchanged.
Direct Transfer into Nitrocellulose and Quantitative Radioautographic Anatomical Determination of Brain Tyrosine Hydroxylase Protein Concentration
Authors: Weissmann Dinah , Labatut René , Richard Françoise , Rousset Colette , Pujol Jean‐François
Published: October 2006

An improved quantitative immunochemical determination of brain tyrosine hydroxylase (TH) concentra‐tions was designed using direct transfer into nitrocellulose from 20‐μm thick brain sections, followed by immunodetection and quantitative radioautography in three reference brain structures (locus ceruleus, substantia nigra, and ventral tegmental area). Results obtained by this methodology were similar to those obtained after extraction and Western blotting of the TH protein in control and reserpine‐treated animals. Moreover, this methodology allows the combination of high sensitivity and high anatomical resolution in the study of the distribution of pharmacological effects. The locus ceruleus exhibited a significant posteroanterior distribution of TH protein concentration in control and reserpine‐treated animals.
Selective Inhibition of Nerve Growth Factor‐Stimulated Protein Kinases by K‐252a and 5′‐S‐Methyladenosine in PC12 Cells
Authors: Smith Deanna S. , King Carolyn S. , Pearson Eric , Gittinger Cynthia K. , Landreth Gary E.
Published: October 2006

K‐252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)‐stimulated phosphorylation of microtubule‐associated protein 2 (MAP2) and Kemptide (synthetic Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly) by blocking the activation of two independent kinases in PC 12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF‐stimulated activation of these kinases is inhibited in a dose‐dependent manner following treatment of the cells with K‐252a. Although these kinases also are activated by epidermal growth factoi (EGF) and 12‐O‐tetradecanoyl‐phorbol 13‐acetate, K‐252a has no inhibitory effect when these agents are used. Half‐maximal inhibition of the activation of both kinases was observed at 10–30 nM K‐252a. K‐252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added tc the phosphorylation reaction mixture in vitro; however, half‐maximal inhibition under these conditions was observed at ±50 nM K‐252a. These data suggest that K‐252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K‐252a are similar to those reported for 5′‐S‐methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF‐, but not EGF‐mediated activation of MAP2/pp250‐kinase (Ki± 500 μM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki= 50 μM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.
Catecholamine‐Sensitive Adenylate Cyclase in the White Perch (Roccus americanus) Retina: Evidence for β‐Adrenergic and Dopamine Receptors Linked to Adenylate Cyclase
Authors: O'Connor Patricia , Kropf Rita B. , Dowling John E.
Published: October 2006

We have examined the catecholamine‐sensitive adenylate cyclase in the retina of the white perch (Roccus americanus). Both dopamine and the β‐adrenergic agonist isoproterenol stimulate cyclic AMP accumulation in this retina, but serotonin, an indoleamine, and phenylephrine, an α‐adrenergic agonist, had no effect. The stimulation of adenylate cyclase by isoproterenol is more potent and effective than that of dopamine. The effects of dopamine and isoproterenol are mediated via independent dopamine and β‐adrenergic receptors. Haloperidol, a dopamine antagonist, blocks the stimulatory effect of dopamine but not of isoproterenol. Conversely, propranolol, a β‐adrenergic antagonist, blocks the stimulatory effect of isoproterenol but not of dopamine. The effects of dopamine and isoproterenol are not additive. In fractions of purified horizontal cells we found evidence for dopamine receptors linked to adenylate cyclase but did not find evidence for the presence of cyclase coupled β‐adrenergic receptors. The cellular location of the β‐adren‐ergic receptors is unknown. Our findings demonstrate the existence of both β‐adrenergic and dopamine receptors coupled to adenylate cyclase in the white perch retina. However, we did not find either epinephrine or norepinephrine, endogenous ligands of the β‐receptor, to be present in retinal extracts subjected to HPLC.
Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes
Authors: Goldin Ehud , Harel Shaul , Tomer Abraham , Yavin Ephraim
Published: October 2006

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short‐term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close‐to‐term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6‐keto‐PGF1 metabolites as detected by HPLC analysis. A significant increase of 20–30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2‐h restriction. The thromboxane B2 and 6‐keto‐PGF1 levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five‐ and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly high for 24 h. 6‐Keto‐PGF1 levels reached a peak value that was greater by 2.5‐ and 1.5‐fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9‐ and 1.1‐fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after ∼3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.
Transient Hypoxia Alters Striatal Catecholamine Metabolism in Immature Brain: An In Vivo Microdialysis Study
Authors: Gordon Kevin , Statman Daniel , Johnston Michael V. , Robinson Terry E. , Becker Jill B. , Silverstein Faye S.
Published: October 2006

Microdialysis probes were inserted bilaterally into the striatum of 7‐day‐old rat pups (n = 30) to examine extracellular fluid levels of dopamine, its metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5‐hydroxyindoleacetic acid (5‐HIAA). The dialysis samples were assayed by HPLC with electrochemical detection. Baseline levels, measured after a 2‐h stabilization period, were as follows: dopamine, not detected; DOPAC, 617 ± 33 fmol/min; HVA, 974 ± 42 fmol/min; and 5‐HIAA, 276 ± 15 fmol/min. After a 40‐min baseline sampling period, 12 animals were exposed to 8% oxygen for 120 min. Hypoxia produced marked reductions in the striatal extracellular fluid levels of both dopamine metabolites (p < 0.001 by analysis of variance) and a more gradual and less prominent reduction in 5‐HIAA levels (p < 0.02 by analysis of variance), compared with controls (n = 12) sampled in room air. In the first hour after hypoxia, DOPAC and HVA levels rose quickly, whereas 5‐HIAA levels remained suppressed. The magnitude of depolarization‐evoked release of dopamine (elicited by infusion of potassium or veratrine through the microdialysis probes for 20 min) was evaluated in control and hypoxic animals. Depolarization‐evoked dopamine efflux was considerably higher in hypoxic pups than in controls: hypoxic (n = 7), 257 ± 32 fmol/min; control (n = 12), 75 ± 14 fmol/min (p < 0.001 by analysis of variance). These data demonstrate that a brief exposure to moderate hypoxia markedly disrupts striatal catecholamine metabolism in the immature rodent brain.
Rat Brain Glutamic Acid Decarboxylase Sequence Deduced from a Cloned cDNA
Authors: Julien Jean‐François , Samama Philippe , Mallet Jacques
Published: October 2006

A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme. The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli. In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented. The predicted protein is 593 amino acids in length. The first 557 residues display a 95% identity when compared with the corresponding cat sequence. However, the deduced amino acid sequence of the carboxy‐terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.
A New Enzymatic Cycling Technique for Glutamate Determination in Brain Microdialysates
Authors: Mitani Akira , Kubo Hirohiko , Iga Kouzo , Imon Hitoshi , Kadoya Fumito , Kataoka Kiyoshi
Published: October 2006

A quantitative analysis of glutamate in brain dialysate was made by using an enzymatic cycling technique. This method made it possible to measure the concentration of glutamate in dialysate collected at 30‐s intervals. Dialysates were collected from Mongolian gerbil hippocampus before, during, and after two 90‐s ischemic insults at an interval of 5 min. An extracellular increase in levels of glutamate was already observed in samples collected during a 30–60 s period after the onset of each ischemia, and the levels of glutamate were maximal at the end of each period of ischemia (approximately a fourfold increase). The increased levels of glutamate rapidly returned almost to preischemic levels by 30 s of recirculation. This method will provide more precise information about temporal changes in the extracellular glutamate concentration in the brain during ischemia.
Phaco‐trabeculotomy combined with deep sclerectomy, a new technique in combined cataract and glaucoma surgery: complication profile
Authors: Lüke Christoph , Dietlein Thomas S. , Lüke Matthias , Konen Walter , Krieglstein Günter K.
Published: October 2006

Purpose:  Combined phacoemulsification, intraocular lens implantation and trabeculectomy (PTE) is currently the standard procedure for most ophthalmic surgeons for treating uncontrolled open‐angle glaucoma and cataract at the same time. The present pilot study was designed to prospectively evaluate outcomes in glaucoma patients who underwent a new technique of phaco‐trabeculotomy plus deep sclerectomy, with particular attention to the complication profile.

Methods:  A consecutive series of 15 patients with uncontrolled open‐angle glaucoma and cataract underwent combined glaucoma and cataract surgery. The procedure started as a two‐site approach with phacoemulsification and intraocular lens (IOL) implantation through a temporal incision in clear cornea. Trabeculotomy and deep sclerectomy were performed in the superior quadrant. Postoperative examinations were performed daily for 1 week. Follow‐up visits were carried out at 1, 3, 6 and 12 months after surgery.

Results:  At 1 day post‐surgery, mean intraocular pressure (IOP) was significantly reduced to 14.2 mmHg (SD 4.4). At 12 months post‐surgery, the complete success rate, defined as IOP < 22 mmHg without medication, was 60%. Qualified success was achieved in 93.3% of patients. At 12 months post‐surgery, the mean number of antiglaucoma medications had fallen to 0.4 (SD 0.6) (p < 0.001). Visual acuity improved by a mean value of 1.6 lines (SD 2.4) over baseline (p = 0.021). Specific complications such as choroidal deroofing, inadvertent perforation of the trabeculo−descemetic membrane, and non‐identification of Schlemm's canal were seen among the patients in our trial. The incidence of complications due to overfiltration was low. A relatively high incidence of hyphaemas (53%) was noted in this series. All the hyphaemas were trivial and resolved quickly. No severe complications, such as bleb infection, endophthalmitis or choroidal haemorrhage were seen in this series.

Conclusions:  Phaco‐trabeculotomy plus deep sclerectomy offered significant IOP reduction and a success rate that may be comparable with that of the current standard, PTE. Intra‐ and postoperative complications specific to deep sclerectomy and trabeculotomy were seen in our series, although the overall rate of postoperative complications proved low. Prospective comparative trials are needed to assess which of PTE and phaco‐trabeculotomy plus deep sclerectomy is more successful.
Leucine 7−proline 7 polymorphism in the signal peptide of neuropeptide Y is not a risk factor for exudative age‐related macular degeneration
Authors: Kaarniranta Kai , Holopainen Juha M. , Karvonen Matti K. , Koulu Markku , Kallio Jaana , Pesonen Ullamari , Teräsvirta Markku , Uusitalo Hannu , Immonen Ilkka
Published: October 2006

Purpose:  Because of the regulatory role of neuropeptide Y (NPY) in angiogenesis, we set out to determine the presence of the leucine 7−proline 7 (Leu7Pro) polymorphism in exudative age‐related macular degeneration (AMD) patients and to analyse its implications.

Methods:  Genotype analysis of the Leu7Pro polymorphism in the signal peptide region of the human prepro‐NPY was performed in blood samples from exudative AMD patients (n = 240) and control subjects (n = 79).

Results:  In all, 11% of exudative AMD patients and 14% of control subjects exhibited the NPY signal peptide Leu7Pro polymorphism. There were no statistically significant differences in Leu7Pro polymorphism frequency between the exudative AMD and control cases, as analysed by Fisher's exact two‐sided test.

Conclusions:  Leu7Pro polymorphism in the signal peptide region of the human prepro‐NPY is not a risk factor for exudative AMD.
Longitudinal quantification of incipient carious lesions in postorthodontic patients using a fluorescence method
Authors: Aljehani Abdulaziz , Yousif Mirgani A. , Angmar‐Månsson Birgit , Shi Xie‐Qi
Published: October 2006

The aims of this study were to evaluate the effect of two caries‐preventive programs, and to apply the laser fluorescence method, DIAGNOdent, for longitudinal quantification of changes in incipient carious lesions. Twelve subjects with 127 test teeth exhibiting white spot lesions on the buccal surfaces after completed orthodontic therapy were enrolled in the study. Visual examination was performed at baseline and after 12 months. The subjects were divided into two groups: one group received repeated professional tooth cleaning combined with oral hygiene instruction; and the control group received repeated oral hygiene instruction only. The white spot lesions were measured by DIAGNOdent at baseline, and at 3, 6, 9, and 12 months thereafter. There was a significant difference in the DIAGNOdent readings between the first and the final evaluations. However, there was no statistically significant difference between the two treatment groups regarding changes of DIAGNOdent values over time. In conclusion, it may be possible to use DIAGNOdent for longitudinal quantification of carious lesions on smooth surfaces over a period of 1 yr under in vivo conditions. The combination of professional tooth cleaning and oral hygiene instruction had a similar efficacy to professional tooth cleaning only for promoting the remineralization of white spot lesions.
Smoking modulates interferon‐γ expression in the gingival tissue of patients with chronic periodontitis
Authors: César‐Neto João B. , Duarte Poliana M. , De Oliveira Maria Claudia G. , Casati Márcio Z. , Tambeli Claudia H. , Parada Carlos A. , Sallum Enilson A. , Nociti Francisco H.
Published: October 2006

Although interferon‐γ (IFN‐γ) plays a critical role in periodontitis, no information is available regarding the effect of smoking on this cytokine in the periodontium. Therefore, this study aimed to evaluate the effect of smoking on the IFN‐γ levels in gingival tissue from patients with chronic periodontitis. Sixty‐two patients were assigned to three groups: healthy [non‐smoking and periodontally healthy individuals (probing depth ≤ 3 mm and no bleeding on probing; n = 12)]; periodontitis [non‐smokers clinically diagnosed with moderate to severe chronic periodontitis (probing depth ≥ 5 mm and bleeding on probing; n = 25)]; and smoking [smokers (≥ 1 pack/day for at least 10 yr) diagnosed with chronic periodontitis (n = 25)]. Gingival biopsies were analyzed by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Regardless of smoking status, diseased patients presented higher levels of IFN‐γ than peridontally healthy patients. In sites with comparable types of periodontitis, smoking increased both protein and mRNA levels of IFN‐γ in gingival tissue. Within the limits of this study, it can be concluded that modulation of periodontal tissue destruction by smoking may involve its effect on IFN‐γ production.
Survival of the Mammary Tumor Milk Agents of Mice
Authors: Bittner John J. , Evans Charles A. , Green Robert G.
Published: October 2006

The mammary tumor milk agent has been recovered from a transplanted mammary carcinoma that was carried for 10 passages in mice that did not themselves originally have the agent. It is possible that the agent, carried in the transplanted mammary tumor cells, may be responsible for the genetic mutations which have been detected in transplantable tumors.

Preliminary results show that the agent can be recovered from the yolk sac of chick embryos 12 days after the injection into eggs of either tumor suspensions or cell-free filtrates of tissues containing the active agent.
Thyroid Allograft Immunogenicity Is Reduced After a Period in Organ Culture
Authors: Lafferty Kevin J. , Cooley Margaret A. , Woolnough James , Walker Karen Z.
Published: October 2006

The survival time of mouse thyroid, transplanted under the kidney capsule of an H-2 incompatible recipient, is extended by holding the thyroid in organ culture for 12 days prior to transplantation.
Thick and Thin Filaments in Postmitotic, Mononucleated Myoblasts
Authors: Holtzer H. , Strahs K. , Biehl J. , Somlyo A. P. , Ishikawa H.
Published: October 2006

Addition of cytochalasin B to primary muscle cultures allows the physical separation of postmitotic myogenic cells from replicating presumptive myoblasts and replicating fibroblasts. Mononucleated, postmitotic myoblasts proceed without fusion to synthesize myosin and actin and to assemble these proteins into thick and thin filaments. Although sarcomeres oriented in tandem are not evident and Z, H, and I bands are atypical in these mononucleated myoblasts, the irregularly scattered clusters of myofilaments are assembled into remarkably normal interdigitating arrays. These scattered clusters of stacked thick and thin filaments permit the cell to contact spontaneously in the presence of cytochalasin B.
Orientation of Gull Chicks Exposed to Project Sanguine's Electromagnetic Field
Authors: Southern William E.
Published: October 2006

Birds tested on clear days in the normal geomagnetic field showed a significant clustering of headings about a predicted bearing corresponding with the direction of migration. Individuals tested when a large antenna was energized dispersed randomly. Magnetic fields associated with such conductors may be sufficient to confuse orienting birds.