bluuebunny/crossref_metadata_2025 huggingface dataset:
Dose Response for Amphetamine‐Induced Changes in Dopamine Levels in Push‐Pull Perfusates of Rat Striatum
Authors: Kuczenski Ronald
Published: October 2006

Abstract: Levels of dopamine were determined in push‐pull perfusates of striatum in chloral hydrate‐anesthetized rats as a function of increasing systemic doses of amphetamine over the range 0.5–5.0 mg/kg. In the absence of amphetamine administration, basal dopamine levels remained stable for at least 6 h. Perfusate levels of dopamine responded in a quantitatively predictable fashion to increasing doses of amphetamine: (1) the maximal increase in perfusate levels of dopamine after amphetamine, relative to predrug levels, was directly proportional to the dose of the drug up to 3 mg/kg (fivefold after 0.5 mg/kg to 30‐fold after 3 mg/kg); (2) the duration over which perfusate levels of dopamine were significantly elevated, with respect to preamphetamine levels, was proportional to the dose of amphetamine up to 5 mg/kg; and (3) each successively higher dose of amphetamine significantly increased the perfusate level of dopamine over that observed at the next lower dose up to 3 mg/kg amphetamine. However, maximal levels of dopamine in striatal perfusates were achieved following 3 mg/kg amphetamine and were not increased further at higher doses of the drug. The data suggest that, at higher doses of amphetamine, extraneuronal metabolism of dopamine may be of sufficient capacity to limit increases in synaptic levels of dopamine. The absence of further increases in perfusate levels of dopamine as the dose of amphetamine is increased beyond 3 mg/kg is discussed in terms of potential relevance to mechanisms of amphetamine‐induced stereotyped behaviors.
Influence of Monoclonal Antibodies on Microtubule Assembly
Authors: Matus Andrew , Riederer Beat , Huber Gerda
Published: October 2006

Abstract: The influence on microtubule assembly in vitro of monoclonal antibodies against microtubule‐associated proteins (MAPs) was studied. Light scattering was used for measuring net polymer formation and electron microscopy for determining the influence of antibodies on microtubule morphology. Control experiments showed that nonimmune mouse IgG had no effect on either the assembly or appearance of microtubules. The same was true for monoclonal antibodies against MAPI. At low levels, antibodies against MAP2 caused the aggregation of microtubules into bundles, an effect that did not occur with antibodies against any other MAP type studied. At increasing concentrations, anti‐MAP2 progressively inhibited tubulin polymerization, producing irregular, shortened filaments. Anti‐MAP5 produced a striking fragmentation of microtubules into very short pieces that were otherwise morphologically identical to control microtubules. The different effects of these antibodies show the potential of monoclonal antibodies for investigating MAP function and form an important adjunct to cellular microinjection experiments.
Affinity Purification of Synenkephalin‐Containing Peptides Including a Novel 23.3‐Kilodalton Species
Authors: Metters Kathleen M. , Rossier Jean
Published: October 2006

Abstract: Affinity chromatography has been used for rapid and high‐yield purification of synenkephalin (proenkephalin 1 ‐70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN‐Br‐activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mMammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low‐molecular‐weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high‐molecular‐weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6‐ and 12.6‐ kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met5]‐enkephalin‐Arg6‐Gly7‐Leu8. The former column retains the 23.3‐kilodalton species, whereas the latter column retains the 18.2‐kilodalton species. The 23.3‐ kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin‐1–206), containing [Leu5]‐ enkephalin at the C‐terminus.
Isolation of NILE Glycoprotein‐Related cDNA Probes
Authors: Sajovic P. , Ennulat D. J. , Shelanski M. L. , Greene L. A.
Published: October 2006

Abstract: The nerve growth factor (NGF)‐induced large external (NILE) glycoprotein is an NGF‐inducible surface component of PC 12 cells that is also widely distributed in the nervous system. It has recently been shown to be indistinguishable from the high‐molecular‐weight species of the brain antigens L1 and neuron‐glia cell adhesion molecule (Ng‐CAM) and may have a function in regulating cell adhesion in the developing nervous system. We have used polyclonal anti‐NILE antisera to screen a λgt11 cDNA library made from NGF‐treated PC12 cells. Four molecular probes have been isolated that encode parts of the apoprotein, related proteins, or both. These probes are 1,500, 800, 330, and 300 base pairs long, respectively, and in Northern blots they recognize a family of messages having lengths of ∼5.9, 3.4, 2.4, and 1.9 kilobases. The two smaller messages are modestly but reproducibly up‐regulated by NGF in PC 12 cells, as is the NILE glycoprotein; however, only the two larger species would appear to be large enough to encode it. These messages are prominent in brain but not in nonneuraltissues, in accordance with the observed levels of the protein. The recombinant phages produce fusion proteins that share specific epitopes with the NILE glycoprotein but not with other proteins. In these experiments, filters coated with recombinant fusion protein were prepared. Antibodies bound to and eluted from these filters specifically immunoprecipitate NILE glycoprotein, but not other proteins, from PC12 cell extracts. Other activities present in the original sera are not specifically retained by recombinant fusion proteins, and Escherichia coll lysates made with nonrecombinant λgt11 do not absorb the anti‐NILE activity. The probes described here will be of use in studying the function, biochemistry, and molecular biology of the NILE glycoprotein and related species.
Poly‐N‐Acetyllactosamine Glycans of Cellular Glycoproteins: Predominance of Linear Chains in Mouse Neuroblastoma and Rat Pheochromocytoma Cell Lines
Authors: Spillmann Dorothe , Finne Jukka
Published: October 2006

Abstract: To study the properties of protein‐bound oligosaccharides in neuronally differentiating cells, two model systems were used: murine N1E‐115 and N‐18 neuroblastoma cells inducible by serum starvation and rat PC12 pheochromocytoma cells inducible by nerve growth factor. Glycopeptides were prepared from cells metabolically labeled with [3H]glucosamine and analyzed by gel nitration. The properties of the high‐molecular‐weight glycopeptides were studied using enzymatic digestion with neuraminidase and endo‐β‐galactosidase. In contrast to other cell lines analyzed, the neuroblastoma and pheochromocytoma lines contained predominantly glycopeptides completely cleavable with endo‐β‐galactosidase, which indicated that they were linear‐type poly‐N‐acetyllactosamine glycans. The proportion of these linear chains in the high‐molecular‐weight fraction increased during neuronal differentiation in both cell systems. The linear nature of the glycans was also correlated with positive anti‐i and negative anti‐I reactivity of the cells in immunofluorescence microscopy. Specific cell surface labeling for poly‐N‐acetyllactosamine glycans and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed several glycoprotein components, some of which showed changes during neuronal differentiation. The high proportion of linear poly‐N‐acetyllactosamine chains in these neuronal cell lines and its increase during neuronal differentiation suggests that these glycans may be a characteristic feature of neuronal or neuronally differentiating cells.
In Vivo Microdialysis Studies on the Effects of Decortication and Excitotoxic Lesions on Kainic Acid‐Induced Calcium Fluxes, and Endogenous Amino Acid Release, in the Rat Striatum
Authors: Butcher Steven P. , Lazarewicz Jerczy W. , Hamberger Anders
Published: October 2006

Abstract: The in vivo effects of kainate (1 mM) on fluxes of 45Ca2+, and endogenous amino acids, were examined in the rat striatum using the brain microdialysis technique. Kainate evoked a rapid decrease in dialysate 45Ca2+, and an increase in the concentration of amino acids in dialysates in Ca2+‐free dialysates. Taurine was elevated six‐to 10‐fold, glutamate two‐to threefold, and aspartate 1.5‐to twofold. There was also a delayed increase in phosphoethanolamine, whereas nonneuroactive amino acids were increased only slightly. The kainic acid‐evoked reduction in dialysate 45Ca2+ activity was attenuated in striata lesioned previously with kainate, suggesting the involvement of intrinsic striatal neurons in this response. The increase in taurine concentration induced by kainate was slightly smaller under these conditions. Decortication did not affect the kainate‐evoked alterations in either dialysate 45Ca2+ or amino acids. These data suggest that kainate does not release acidic amino acids from their transmitter pools located in corticostriatal terminals.
Effect of Repeated Treatment with a γ‐Aminobutyric Acid Receptor Agonist on Postnatal Neural Development in Rats
Authors: Meier Eddi , Jørgensen Ole Steen , Schousboe Arne
Published: October 2006

Abstract: The effect of treatment with the γ‐aminobutyric acid (GABA) agonist tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP) on neural development was monitored in rats by following the expression of the neuron‐specific proteins neural cell adhesion molecule (NCAM), D1, and D3 as well as the enzymes glutamate decarboxylase (GAD) and glutamate dehydrogenase (GLDH). As judged from the effect of the treatment on the expression of NCAM and GAD, GABA agonists have the capacity to accelerate and enhance neuronal development during the early postnatal period. However, as judged from the expression of D1‐and D3‐protein some adverse late effects may result from prolonged treatment with high doses of GABA agonists. The decrease in GLDH specific activity observed in THIP‐treated rats during their late postnatal development possibly indicates a repression of glutamatergic neurons.
Carboxylmethylation of Calmodulin in Cultured Pituitary Cells
Authors: Vincent Pamela L. , Siegel Frank L.
Published: October 2006

Abstract: We have used fast protein liquid chromatography (FPLC) and reverse‐phase HPLC to rapidly resolve carbox‐ylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein frac‐tionation. GH3 cells were incubated with [methyl‐3H]‐methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3cells by gel permeation FPLC, anion‐exchange FPLC, and reverse‐phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3cells was incubated with S‐adenosyl‐l‐[methyl‐3H] methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.
Histamine Affects Release and Biosynthesis of Opioid Peptides Primarily via H1‐Receptors in Bovine Chromaffin Cells
Authors: Bommer Michael , Liebisch Daniel , Kley Nikolai , Herz Albert , Noble Ernest
Published: October 2006

Histamine is a potent secretagogue for opioid pentapeptides (Met‐ and Leu‐enkephalin) in adrenal chromaffin cells in vitro. This effect is dependent on extracellular Ca2+ and is reduced by Ca2+ channel blockers such as Co2+, D 600, and nifedipine. Moreover, histamine also produced a profound compensatory increase in cellular peptide content after 48 h of exposure, most likely caused by a four‐ to fivefold increase in the mRNA levels coding for the proenkephalin A precursor. All the histamine‐in‐duced effects (acute release, changes in peptide cell content, proenkephalin A mRNA levels) are antagonized by the H1‐receptor antagonist, clemastine, whereas the H2‐receptor antagonists, ranitidine and cimetidine, were less effective (∼20% inhibition).
Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro
Authors: Panzetta Pedro , Gravotta Diego , Maccioni Hugo J. F.
Published: October 2006

Cells from neural retina from 7‐day chick embryos were cultured on polylysine‐coated dishes up to 7 days. The small, round‐shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GDI a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was to and. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl‐N‐acetyl‐galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.
Cerebrospinal Fluid Proteins Studied by Two‐Dimensional Gel Electrophoresis and Immunoblotting Technique
Authors: Wiederkehr Felix , Ogilvie Allan , Vonderschmitt Dieter J.
Published: October 2006

The proteins of lumbar CSF have been investigated by two‐dimensional gel electrophoresis, and their patterns have been compared with the corresponding serum protein patterns. Serum proteins in CSF have been identified by electroblotting and immunoreaction with antiserum against total human serum proteins. Proteins derived from brain have been identified with antiserum against human brain proteins. The most prominent CSF protein group has been identified as a multiple form of apolipoprotein E. The correct position of the glial fibrillary acidic protein has also been determined. The prefractionation of CSF proteins by size exclusion chromatography or by affinity chromatography followed by two‐dimensional electrophoresis has facilitated the detection of trace components in CSF and the corresponding serum.
Reversible and Irreversible Inhibition of High‐Affinity Choline Transport Caused by Ethylcholine Aziridinium Ion
Authors: Pittel Zipora , Fisher Abraham , Heldman Eliahu
Published: October 2006

The effect of ethylcholine aziridinium ion (AF64A) on choline transport in hippocampal, striatal, and cerebrocortical synaptosomes was studied. Synaptosomes prepared from these three brain regions were equally sensitive to AF64A. Low concentrations of AF64A produced a reversible inhibition (IC50 values = 1.35–2.25 μM), whereas higher concentrations produced an irreversible inhibition (IC50 values = 25–30 μM), which started as competitive. The irreversible component of the inhibition was independent of extracellular Na+ concentration, a finding suggesting that the choline transporter is alkylated at its outward position. The kinetics of the inhibition were rapid and similar in the three brain regions examined. The high‐affinity choline transport was more sensitive to the toxin than the low‐affinity choline transport. Based on these results, we propose a kinetic model that explains the reversible and the irreversible inhibitions induced by AF64A. The possible relationships between the concentrations that in vitro produce reversible and irreversible inhibition and those that in vivo produce selective and nonselective cholinergic hypofunc‐tion are discussed.
Effect of Lanthanum on the Release of Acetylcholine from the Myenteric Plexus and on Its Activation by Ouabain and Electrical Stimulation
Authors: Doležal Vladimir , Somogyi Gyorgy T. , Bernath Sandor , Tuček Stanislav , Vizi E. Sylvester
Published: October 2006

The effect of lanthanum ions (La3+) on the release of acetylcholine (ACh) from longitudinal muscle strips of the guinea pig ileum with the myenteric plexus attached was investigated. After an exposure of the tissue to 2 mM LaCl3 for 18 min the rate of ACh release was increased approximately eightfold and the increased release lasted for more than 100 min. The augmented release of ACh was accompanied by enhanced synthesis. At the end of the experiments (102 min after LaCl3 had been removed), when the release of ACh was still more than six times higher than in controls, the content of ACh was the same in La3+‐treated and un treated tissues. Electrical field stimulation failed to cause a further increase in the release of ACh from La3+‐pretreated preparations whereas ouabain released considerable more ACh when compared to controls. It is concluded from this difference that electrical stimulation and ouabain release ACh from different pools.
Neurotensin‐Metabolizing Peptidases in Rat Fundus Plasma Membranes
Authors: Checler Frédéric , Barelli Hélène , Kwan Chiu‐Yin , Kitabgi Patrick , Vincent Jean‐Pierre
Published: October 2006

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8‐Arg9, Pro10‐Tyr11, and Ile12‐Leu13 peptidyl bonds. Hydrolysis at the Arg8‐Arg9 bond was fully abolished by the use of N‐[1 (R,S)‐carboxy‐2‐phenylethyl]‐alanyl‐alanyl‐phenylalanine‐p‐aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10‐Tyr11 bond was totally resistant to N‐benzyloxycarbonyl‐prolyl‐prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT‐degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12‐Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin‐sensitive aminopeptidases and post‐proline dipeptidyl aminopeptidase. The content in NT‐metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.
Effects of Reserpine and Tetrabenazine on Catecholamine and ATP Storage in Cultured Bovine Adrenal Medullary Chromaffin Cells
Authors: Caughey Byron , Kirshner Norman
Published: October 2006

The in vivo storage relationship between catechol‐amines and ATP in chromaffin vesicles of cultured bovine adrenal medulla cells was investigated using drugs that block vesicular catecholamine uptake. Three‐day treatments with reserpine and tetrabenazine causing 85–90% depletion of catecholamines resulted in 41–46% reductions in cellular ATP content. Subcellular fractionation of reserpine‐treated cells indicated that the ATP is lost from the chromaffin vesicle pool. This was confirmed in experiments using metabolic inhibitors to differentiate the vesicular and extravesicular ATP pools. The vesicular ATP loss was not proportional to that of catecholamines, resulting in a reduction by 50% in the chromaffin vesicle mole ratio of catecholamines to ATP after 48 h of treatment. In metabolic labeling studies, it was found that reserpine treatment reduced the incorporation of [3H]adenosine into vesicular ATP selectively, but it reduced the incorporation of 32Pi into both the vesicular and extravesicular pools. The reduction of the [3H]adenosine incorporation was not due to diminished vesicular nucleotide uptake resulting from low catecholamine levels, because when the catecholamines were depleted by tetrabenazine pretreatment followed by removal of the drug before labeling, no reduction in [3H]adenosine incorporation was observed. When present during the labeling, tetrabenazine was found to be a reversible inhibitor of plasma membrane adenosine uptake. The observed loss of adenine nucleotides from catecholamine‐depleted chromaffin vesicles in vivo provides evidence that interactions between ATP and catecholamines are important in the vesicular storage of high concentrations of these compounds.
Myelin‐Associated Glycoprotein in the Central and Peripheral Nervous System of Quaking Mice
Authors: Inuzuka Takashi , Johnson David , Quarles Richard H.
Published: October 2006

The myelin‐associated glycoprotein (MAG) was quantitated in the CNS and PNS of quaking mice and the levels compared to the levels of myelin basic protein (MBP) and 2′:3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) activity. In the brainstems of 36‐day‐old quaking mice, MBP, MAG, and CNPase were reduced to 12, 16, and 29% of control levels, respectively. In the sciatic nerves of the 36‐day‐old quaking mice, MBP and CNPase were 38 and 75% of control levels, respectively, whereas the concentration of MAG was unchanged or slightly increased. Similar quantitative results were obtained for the sciatic nerves and spinal roots of 7‐month‐old quaking mice. Immunoblots showed that the principal MAG band from the brainstems, sciatic nerves, and spinal roots of the quaking mice had a higher than normal apparent Mr. In addition, there was a minor component reacting with anti‐MAG antiserum in the brain‐stems of the quaking mice that had a slightly lower Mr than control MAG and was not detected in the normal mice. The results for the quaking mice are compared with those from similar studies on other mutants with dysmyelination of the CNS and PNS.
Effect of Quinolinic Acid in the Nucleus Basalis Magnocellularis on Cortical High‐Affinity Choline Uptake
Authors: Metcalf R. H. , Boegman R. J. , Quirion R. , Riopelle R. J. , Ludwin S. K.
Published: October 2006

A transient 45% increase in cortical high‐affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific (3H]hemicholinium‐3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.
Temperature Dependence of Drug Interaction with the Platelet 5‐Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox
Authors: Segonzac Alain , Schoemaker Hans , Langer Salomon Z.
Published: October 2006

Abstract: Although [3H]imipramine is a selective radioli‐gand for the 5‐hydroxytryptamine (5‐HT) transporter in human platelets, its affinity for binding to the 5‐HT transporter complex at 0°C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5‐HT uptake at the physiological temperature of 37°C (Ki= 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5‐HT transporter at assay temperatures between 0°C and 37°C, using as radioligands [3H]imipramine (0°C and 20°C) and [3H]paroxetine (20°C and 37°C), a newly available probe for the 5‐HT transporter. At 20°C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p < 0.001), validating the use of these two radioligands to study the 5‐HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5‐HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5‐HT transporter at 0°C. At 37°C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5‐HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5‐HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5‐HT transporter similar to that of imipramine. On the other hand, the temperature sensitivity of nontricyclic 5‐HT uptake inhibitors was much less pronounced. Moreover, Ki values for drug inhibition of [3H]5‐HT uptake into human platelets were significantly (p < 0.05) better correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37°C (r = 0.97) than with their Ki values for inhibition of [3H]imipramine binding at 0°C (r = 0.87). Therefore, drug inhibition of [3H]paroxetine binding to the 5‐HT transporter at 37°C may be more representative of 5‐HT uptake inhibitory activity than its inhibition of [3H]imipramine binding to the 5‐HT transporter as usually measured at 0°C.
Day‐Night Rhythm of Rat Pineal Tyrosine Hydroxylase Activity as Determined by HPLC with Amperometric Detection
Authors: Abreu Pedro , Santana Celsa , Hernandez Guadalberto , Calzadilla Carlos H. , Alonso Rafael
Published: October 2006

Abstract: Tyrosine hydroxylase activity in the rat pineal gland was measured by means of HPLC determination of the amount of l‐3,4‐dihydroxyphenylalanine formed. Enzyme activity showed a clear day‐night rhythm, paralleling that of plasma melatonin levels in the same animals, with values being high during the dark period apparently because of changes in Vmax. In animals maintained under constant illumination for 3 days, tyrosine hydroxylase activity and plasma melatonin level rhythms were completely abolished, a result indicating that both are under photoperiodic control.
Polyamines Stimulate Mitochondrial Calcium Transport in Rat Brain
Authors: Jensen John R. , Lynch Gary , Baudry Michel
Published: October 2006

Abstract: The effects of the polyamines spermine and spermidine on rat brain mitochondrial calcium transport were examined using a variety of techniques for measuring the kinetics of calcium uptake and the buffering capabilities of isolated mitochondria. Spermine both increased the rate of calcium accumulation and decreased the set‐point to which isolated mitochondria buffer free calcium concentration. In the presence of physiological concentrations of sodium and magnesium, spermine lowered the extramitochondrial calcium level to ∼0.3 μM, a value close to the resting intracellular calcium concentration. The effect of polyamines was concentration dependent, with a half‐maximal effect of spermine observed at ∼0.1–0.4 mM (respiratory substrate dependent), whereas spermidine was ∼ 10 times less potent. Calcium transport by hippocampal mitochondria was stimulated markedly more by spermine than was calcium transport by mitochondria isolated from brainstem. The stimulatory effect of spermine was not due to an increase in the transport of respiratory substrates inside the mitochondria nor to an effect on the enzymes using these respiratory substrates. An examination of the effect of spermine on the kinetics of calcium uptake indicated that spermine increased calcium uptake maximally at low calcium concentrations. Beyond that level, the stimulatory effect of spermine decreases, and spermine can even inhibit calcium uptake. These results are in good agreement with previous reports on the effects of polyamines on calcium transport in mitochondria from peripheral tissue. They support the hypothesis that spermine increases the rate of calcium uptake by mitochondria by increasing the affinity of the uniporter for calcium. Because polyamine levels exhibit large variations under a wide range of conditions (postnatal development, injury, and intense electrical activity), these results indicate that polyamines might play a critical role in the regulation of the calcium‐buffering ability of cells in the brain.
Antigenic Pattern of Human Brain Glycoproteins as Described by Monoclonal Antibodies
Authors: Berglund Erik , Almqvist Per , Lindroos Hans , Carlsson Sven , Stigbrand Torgny
Published: October 2006

Abstract: A battery of monoclonal antibodies was raised against a preparation of lentil lectin‐binding membrane glycoproteins from human brain. Out of 26 established hybrid‐omas, nine produced antibodies against the human Thy‐1 antigen. For the remaining 17 lines, reactivity with at least six other antigens could be identified after immunoprecipi‐tation and immunoblotting. Several of the antigens were di‐or trimeric, mainly in the molecular weight range of 60–120 kDa. Two of the antibodies were reactive with high‐molecular‐weight aggregates and four targets for the antibody reactivity were not identifiable by immunoprecipita‐tion of iodinated antigens. Three of the identified antigens were shown by quantitative enzyme‐linked immunosorbent assay tests on various human tissues to be specifically expressed in the brain.
Comparison of 125I‐SCH 23982 and [3H]SCH 23390 as Ligands for the D‐1 Dopamine Receptor
Authors: Manik Christopher P. , Molinoff Perry B. , McGonigle Paul
Published: October 2006

Abstract: 125I‐SCH 23982, an antagonist with high affinity and selectivity for the D‐l subtype of dopamine receptors, has recently been synthesized. Densities of D‐l receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5‐ to 10‐fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal ho‐mogenates confirmed this discrepancy. One explanation for this difference is that 125I‐SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I‐SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I‐SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in 5max values is that an unlabeled inhibitor is present in commercial preparations of 125I‐SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I‐SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.
Predicted Amino Acid Sequence of Bovine Tyrosine Hydroxylase and Its Similarity to Tyrosine Hydroxylases from Other Species
Authors: Saadat Siawusch , Stehle August D. , Lamouroux Annie , Mallet Jacques , Thoenen Hans
Published: October 2006

Abstract: The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1–157) and 91% in the carboxy terminal (amino acids 158–491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.
Identification of 5‐Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex
Authors: Takeuchi Yutaka , Yang Wu , Shih Jean C.
Published: October 2006

Abstract: 5‐Hydroxytryptamine1A (5‐HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5‐HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P‐40, and bound effectively to l‐[2‐(4‐aminophenyl)ethyl]‐4‐(3‐trifluoromethylphenyl)piperazine (PAPP)‐coupled Affi‐Gel 10 in a time‐dependent manner. PAPP was shown previously to be a selective ligand for the 5‐HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5‐HT or 1 μM [3H]l‐[2‐(4‐azidophenyl)ethyl]‐4‐(3‐trifluoromethyI‐phenyl)piperazine ([3H]p‐azido‐PAPP). [3H]p‐Azido‐PAPP is a selective photoaffinity labeling probe for the 5‐HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p‐azido‐PAPP into these two proteins decreased significantly when the solubilized fraction was prein‐cubated with excess 5‐HT or PAPP (saturating all 5‐HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5‐HT1A receptor protein. The isoelectric points of the photolabeled 5‐HT1A receptor proteins were 6.0 and 6.5.
Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes
Authors: Whitton Peter S. , Nicholson Russell A. , Strang Robin H. C.
Published: October 2006

Abstract: The effect of taurine on the release of [3H]acetylcholine ([3H]ACh) and [3H]‐γ‐aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 μM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration‐dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration‐dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.
Aging and Brain Cholinergic Muscarinic Receptors: An Autoradiographic Study in the Rat
Authors: Biegon A. , Duvdevani R. , Greenberger V. , Segal M.
Published: October 2006

Abstract: Cholinergic muscarinic receptors in aged and young rat brains were studied by quantitative autoradiography of tritiated quinuclidinyl benzilate. A selective pattern of decreased binding density was observed in the aged rat. A large number of regions showed no effect of aging; these include subdivisions of the hippocampal formation and most tha‐lamic and hypothalamic nuclei. Small but significant decreases were found in cortical regions and in the striatum. The largest effects were seen in ventral forebrain cholinergic nuclei, where 40‐60% depletions were found in the diagonal band, nucleus basalis magnocellularis, ventral pallidum, and substantia innominata.
Studies on DNA Damage and Repair in the Mammalian Brain
Authors: Walker A. P. , Bachelard H. S.
Published: October 2006

Abstract: Methods for studying breaks in DNA strands and their repair, originally developed for prokaryotes and cultured cell lines, have been applied to preparations from rat brain. The relative sensitivities of these methods, which include alkaline sucrose density gradient sedimentation, nucleoid sedimentation, and ADP‐ribosyltransferase assay, are compared.
Developmental and Age‐Related Changes in Rat Brain Glycosaminoglycans
Authors: Jenkins H. G. , Bachelard H. S.
Published: October 2006

Abstract: The quantities of each major class of glycosaminoglycan were determined in rat cerebrum from postnatal day 5 to 30 months of age. Chondroitin sulphate, dermatan sulphate, heparan sulphate, heparin, and hyaluronate were found, but no keratan sulphate was detected. Large and rapid changes in glycosaminoglycan content were observed during the period of brain maturation, and thereafter relatively steady levels were maintained until after the age of 12 months. The most remarkable change in the aged rat cerebrum was the ratio by weight of hyaluronate to chondroitin sulphate, which was ∼ 1:1 from postnatal day 10 to 18 months but increased to 2.6:1 by the age of 30 months. In immature rats, the proportion of nonsulphated and 6–sulphated disaccharides derived from chondroitinase AC digests of brain glycosaminoglycans was much greater than in adults. In mature rats, chondroitin sulphate was composed almost entirely of 4–sulphated disaccharide subunits. The possibility that these changes could affect the permeability properties of the cerebral extracellular space and ionic equilibria in the brain is discussed.
Metabolism of Functional Groups Modifying the CNS Myelin‐Associated Glycoprotein
Authors: Toews Arrel D. , White Francis V. , Morell Pierre
Published: October 2006

Abstract: We have examined the metabolic turnover of the peptide backbone of the CNS myelin‐associated glycoprotein (MAG) and of the fucose and sulfate groups modifying this protein. Rats (20 or 90 days old) were injected intracranially with mixtures of [3H]fucose and [14C]glycine, [3H]glycine and [35S]sulfuric acid, or [3H]fucose and [35S]sulfuric acid. At times ranging from 30 min to 4 weeks later, myelin was isolated, and radioactivity in MAG was determined following electrophoretic separation. Following the peak of incorporation, glycine‐derived radioactivity in the MAG peptide backbone declined several‐fold during the first week and was then metabolically stable (half‐life ≫ 1 month). Declines with time in [3H]fucose‐ and [35S]sulfate‐derived radioactivity in MAG were similar to that of [3H]glycine, an observation indicating that the fucose and sulfate groups modifying MAG are metabolized together with the peptide backbone as a single metabolic entity. These results were confirmed by experiments involving selective immunoprecipitation of MAG. The rates of incorporation of labeled glycine, fucose, and sulfate into MAG all decreased ∼ 12–fold between 20 days of age and adulthood, a finding providing further evidence for concerted turnover of the entire molecule. Because of this concerted turnover, we suggest that functional groups modifying MAG serve some permanent structural role in protein configuration.
Uptake of Amino Acids by Brain Micro vessels Isolated from Rats with Experimental Chronic Renal Failure
Authors: Cangiano Carlo , Cardelli‐Cangiano Patrizia , Cascino Antonia , Ceci Fabrizio , Fiori Anna , Mulieri Massimo , Muscaritoli Maurizio , Barberini Claudio , Strom Roberto , Fanelli Filippo Rossi
Published: October 2006

Abstract: The neurological disorders seen in patients with chronic renal failure and liver cirrhosis are analogous. Previous in vivo studies have shown that the impaired blood‐brain amino acid transport seen in rats with chronic renal failure is similar to that of rats with portocaval anastomosis. To elucidate whether a comparable underlying pathogenic mechanism plays a role in both pathological conditions, blood and brain amino acid levels together with amino acid transport by isolated brain microvessels have been studied in rats with chronic renal failure and in sham‐operated rats. Brain microvessels isolated from rats with experimental chronic renal failure showed that the uptake of labeled large neutral amino acid, i.e., leucine or phenylalanine, but not of lysine or a‐methylaminoisobutyric acid, was significantly increased with respect to sham‐operated rats; conversely, the uptake of glutamic acid in rats with chronic renal failure was significantly lower compared with values in controls. Kinetic analysis indicated that this was mainly due to increased exchange transport activity (Vmax) of the L‐system, rather than to changes in the affinity (Km) of the carrier system for the relative substrate. These data, together with the significant rise of brain glutamine levels and an increased brain‐to‐plasma ratio of the sum of large neutral amino acids, are analogous to what was previously observed in rats with portocaval anastomosis. Because increased brain influx of large neutral amino acids, especially the neurotransmitter precursor amino acids, is thought to be responsible for the altered brain neurotransmission observed in chronic liver failure, these data support the hypothesis that the neurological disorders seen in chronic renal failure may result from functional modifications to the L‐system's transport activity at the blood‐brain barrier, similar to those observed in chronic liver failure.
1‐Oleoyl‐2‐Acetylglycerol and A23187 Potentiate Axolemma‐and Myelin‐Induced Schwann Cell Proliferation
Authors: Saunders Royal D. , DeVries George H.
Published: October 2006

Abstract: The effects of 1‐oleoyl‐2‐acetylglycerol (OAG) and the calcium ionophore A23187 on the proliferation of Schwann cells stimulated with either a myelin‐enriched membrane fraction (MEF) or an axolemma‐enriched membrane fraction (AEF) have been examined. Using incorporation of [3H]thymidine as an index of proliferation, 16% of the cells became labeled after incubation with MEF (20 μg protein/ml) and AEF (40 μg protein/ml) for 72 h. Only 0.5% of the cells became labeled in cultures which were not exposed to the membrane fractions. Addition of OAG (10‐500 μM) or A23187 (1.9‐190 nM) in the absence of the membrane mitogens had no effect on the proliferative response of quiescent cultures of Schwann cells. When added simultaneously, however, OAG and A23187 were able to induce proliferation of the cells, although the response was only 30% of the response achieved with maximal doses of either AEF or MEF. Both OAG and A23187 were able to potentiate the mitogen‐icity of AEF or MEF, but only when AEF and MEF were added at submaximal concentrations. When Schwann cells were prelabeled with [3H]glycerol and then stimulated to proliferate with AEF or MEF, the amount of [3H]diacylglyc‐erol was increased two‐ to threefold above that in control cultures for time periods up to 1 h. These results suggest that the proliferation of Schwann cells induced by either AEF or MEF is partially mediated through the combined effects of diacylglycerol and an increase in intracellular calcium.
Regulation of Brain and Cerebrospinal Fluid Calcium by Brain Barrier Membranes Following Vitamin D‐Related Chronic Hypo‐ and Hypercalcemia in Rats
Authors: Murphy Vincent A. , Smith Quentin R. , Rapoport Stanley I.
Published: October 2006

Abstract: Male Fischer‐344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36C1 transfer coefficients (KCa and KC1) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. ATCa values at CSF, parietal cortex, and pons‐medulla were negatively correlated with plasma ionized [Ca], whereas KC1 values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo‐ or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25‐dihydroxyvitamin D3.
Cross‐Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA‐Produced Human Butyrylcholinesterase Peptides
Authors: Dreyfus Patrick , Zevin‐Sonkin Dina , Seidman Shlomo , Prody Catherine , Zisling Rivka , Zakut Haim , Soreq Hermona
Published: October 2006

Abstract: To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three‐dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone‐produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone‐produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tet‐ramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody‐enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti‐cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I‐Protein A were subjected to emulsion autoradiography, followed by cyto‐chemical ChE staining. The anti‐cloned BuChE antibodies, as well as anti‐Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.