bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α‐N‐Acetyl Derivative
Authors: Boarder Michael R. , Weber Eckard , Evans Christopher J. , Erdelyi Elizabeth , Barchas Jack 
Published: October 2006

Abstract:A sensitive assay, which cross‐reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH2 terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ˜15 fmol of β‐endorphin per incubation tube, i.e., ˜ 100‐fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β‐endorphin) to 1.6 nM (Met‐enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH2 terminus corresponding to either a Leu‐ or Met‐enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.
Tyrosine Hydroxylase Inactivation Following cAMP‐Dependent Phosphorylation Activation
Authors: Vrana Kent E. , Roskoski Robert 
Published: October 2006

Abstract:Tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP‐dependent protein kinase (largely by decreasing the Km of the enzyme for its pterin co‐substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho‐diesterase, cAMP‐dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent Km for pterin co‐substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent Km, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP‐dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent Vmax with no change in the low apparent Km for pterin co‐substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low‐molecular‐weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos‐phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.
Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor
Authors: DeVries George H. , Anderson M. G. , Johnson David 
Published: October 2006

Abstract:Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose‐salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20‐40% (wt/wt) linear sucrose gradient in a Beckman Ti‐14 Zonal Rotor. After centrifu‐gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′‐cyclic nucleotide 3′‐phos‐phodiesterase (EC 3.1.4.37), myelin‐associated glycopro‐tein (MAG)]; microsomes [CDP‐choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho‐line hydrolase, EC 3.1.1.7), 5′‐nucleotidase (5′‐ribonu‐cleotide phosphohydrolase, EC 3.1.3.5), Na+,K+‐adeno‐sine triphosphatase (EC 3.6.1.3), [3H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin‐related markers were most enriched in the 20‐26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma‐related markers were most enriched in the 28‐32% region of the gradient, whereas the microsomal and mitochondrial‐related markers were enriched in the 35‐40% region of the sucrose density gradient. Mixing experiments utilizing 125I‐labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un‐ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20‐25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.
Comparative Hydrolysis of Sphingomyelin and 2‐N‐(Hexadecanoyl)‐Amino‐4‐Nitrophenyl‐Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH
Authors: Levade Thierry , Salvayre Robert , Douste‐Blazy Louis 
Published: October 2006

Abstract:Hydrolysis of sphingomyelin and 2‐N‐(hexade‐canoyl)‐amino‐4‐nitrophenyl‐phosphorylcholine (HDA‐PC), a synthetic analogue of sphingomyelin, by acid and Mg‐dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl2 nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA‐PC to sphingomyelin is ˜2). In contrast, Mg‐dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl2, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA‐PC to sphingomyelin is ˜0‐0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg‐dependent neutral sphingomyelinase.
Dopamine, Serotonin, and Acid Metabolites in Brain Regions from the Developing Offspring of Ethanol‐Treated Rats
Authors: Rathbun Wayne , Druse Mary J. 
Published: October 2006

Abstract:Female rats were pair‐fed control or ethanol liquid diets on a chronic basis prior to parturition. Six brain regions (hypothalamus, cerebellum, brain stem, cortex, corpus striatum, and hippocampus) were dissected from 19‐ and 35‐day‐old rat offspring for the determination of dopamine (DA), sertonin (5‐HT), 3,4‐dihydroxyphenylacetic acid (DOPAC), 5‐hydroxyindole‐acetic acid (5‐HIAA), and homovanillic acid (HVA). DA, 5‐HT, and the acid metabolites were separated simultaneously by reverse‐phase HPLC and were quantitated using electrochemical detection. Between 19 and 35 days of age in control rats we observed an increase in the concentration of 5‐HT and 5‐HIAA in the corpus striatum and hippocampus and a decrease in these compounds in the cerebellum. In addition, there was a development‐related decrease of 5‐HIAA in the hypothalamus and an increase in the brain stem. During the same age period the concentration of dopamine increased in the hypothalamus and corpus striatum. There was also a development‐related decrease in the concentration of DOPAC in the corpus striatum and an increase in the cortex as well as a decrease in HVA in the cerebellum and cortex. In comparison to age‐matched control animals the 19‐ and 35‐day‐old offspring of ethanol‐treated rats had a lower concentration of 5‐HT and/or 5‐HIAA in the cortex, cerebellum, and brainstem. In addition the 35‐day‐old offspring of ethanol‐treated rats exhibited a decrease in DA and HVA in the cortex. The results of the present study suggest that in utero ethanol exposure affects both the serotonergic and dopaminergic systems in brain.
Solubilization and Detergent Effects on Interactions of Some Drugs and Insecticides with the t‐Butylbicyclophosphorothionate Binding Site Within the γ‐Aminobutyric Acid Receptor‐Ionophore Complex
Authors: Seifert Josef , Casida John E. 
Published: October 2006

Abstract:Specific binding of [35S]t‐butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine‐fold by EDTA/water dialysis and 1.3‐ to 4.2‐fold by 50 nM ketosteroid R 5135, or 5 mM 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) or related piperazine‐N‐alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one‐fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water‐dialyzed RBM with 20 mM CHAPS. Similar KD values (64–86 nM) are obtained for the original EDTA/water‐dialyzed membranes and the CHAPS‐treated and/or ‐solubilized preparations. The Bmax of the EDTA‐treated RBM is reduced five‐fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: γ‐aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS; TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R,αS)‐cis‐cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor‐ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the α‐cyano pyrethroid and the barbiturate interact with TBPS binding.
Hydrolysis of Endogenous Phospholipids by Rat Brain Microsomes
Authors: Witter Brigitte , Kanfer Julian N. 
Published: October 2006

Abstract:Phosphatidylcholine of rat brain microsomes was labeled in vivo by intracerebral injection of either [3H]oleic acid or [methyl‐3H]choline chloride. These labeled microsomes served both as the enzyme source as well as a source of endogenously labeled substrate. Phospholipase D (PLD) activity was detected with these particles only in the presence of exogenous oleate, its activator. Ca2+ and the ionophore A 23187 inhibit PLD activity of oleate‐labeled microsomes. In oleate‐labeled particles, besides phosphatidic acid the product of PLD action radioactivity was also detected in diglyceride as a result of resident phosphatidate phosphohydrolase, which hydrolyzed the phosphatidic acid. The phosphatidate phosphohydrolase could not be completely inhibited by KF and propranolol. The release of endogenous fatty acids from labeled phospholipid by a mellitin‐stimulated phospholipase A2 also present in these particulates produced minimal stimulation of endogenous PLD. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are hydrolyzed by 50% in the presence of mellitin and 90% of the radioactivity was found in the lyso‐compounds. Mellitin and oleate together reduced the radioactivity found in lyso‐PC and increased that in lyso‐PE.
Separation of Recycling and Reserve Synaptic Vesicles from Cholinergic Nerve Terminals of the Myenteric Plexus of Guinea Pig Ileum
Authors: Ágoston Dénes V. , Kosh Joseph W. , Lisziewicz Julia , Whittaker Victor P. 
Published: October 2006

Abstract:Acetylcholine‐rich synaptic vesicles were isolated from myenteric plexus‐longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem.35, 993–1003 (1980)] using either unstimulated preparations or preparations field‐stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V ˙ cm−1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 μM) or [3H]acetate (2 μCi ˙ ml−1); d4 acetylcholine was measured by gas chromatography‐mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.
Further Studies on the Nature of Postsynaptic Dopamine Uptake and Metabolism in Rat Striatum: Sodium Dependency and Investigation of a Possible Role for Carrier‐Mediated Uptake into Serotonin Neurons
Authors: Schoepp Darryle D. , Azzaro Albert J. 
Published: October 2006

Abstract:The nature of postsynaptic sites involved in the uptake and metabolism of striatal 3,4‐dihydroxyphenylethylamine (dopamine, DA) was investigated. The accumulation of [3H]DA (10−7M) into slices of rat striatum was found to be greatly dependent (>99%) on the presence of sodium ion in the incubation medium. However, the formation of the [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) was only partially reduced in the absence of sodium (DOPAC, 27% of control; HVA, 47% of control). Inhibition of carrier‐mediated DA neuronal uptake with nomifensine (10−5M) significantly decreased DA accumulation (18% of control) and [3H]DOPAC formation (62% of control), but enhanced [3HVA production (143% of control), Inhibition of the 5‐hydroxytryptamine (5‐HT, serotonin) neuronal uptake system with fluoxetine (10−6M) or selective 5‐HT neuronal lesions with 5,7‐dihydroxytryptamine (5,7‐DHT) had no effect on [3H]DOPAC or [3H]HVA formed from [3H]DA in the presence or absence of nomifensine. These results demonstrate that the uptake and subsequent metabolism of striatal DA to DOPAC and HVA is only partially dependent on carrier‐mediated uptake mechanism(s) requiring sodium ion. These data support our previous findings suggesting a significant role for synaptic glial cell deamination and O‐methylation of striatal DA. Further, experiments with fluoxetine or 5,7‐DHT suggest that 5‐HT neurons do not significantly contribute in the synaptic uptake and metabolism of striatal DA.
Concentration of Free Amino Acids in Primary Cultures of Neurones and Astrocytes
Authors: Patel Ambrish J. , Hunt Anthony 
Published: October 2006

Abstract:The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes. In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo. In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate. In neuronal cell cultures, the concentrations of glutamate, aspartate, and γ‐aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes. By contrast, the amount of glutamine was $65% greater in astroglial cell cultures than in interneurone cultures. An unexpected finding was a very high concentration of glycine in astrocytes derived from 8‐day‐old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes. The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures. The present results, together with our previous findings, provide further support for the model assigning the “small” compartment of glutamate to glial cells and the “large” compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.
Expression of Hyaluronectin by C‐6 Glial Cells at High Density
Authors: Bhat Narayan R. , Brunngraber Eric G. , Delpech Bertrand 
Published: October 2006

Abstract:Hyaluronectin, a brain glycoprotein that has been localized to the nodes of Ranvier in vivo and to oligodendrocytes in primary cultures of neonatal rat brain cells, was shown by using an unlabeled immunoperoxidase method to be present in C‐6 glial cells grown to high density. The density‐dependent expression of this glycoprotein is in accordance with the known properties of the glial stem cells, i.e., induction of differentiated properties such as 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase, glutamine synthetase, S‐100 protein, and glial fibrillary acidic protein.
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

Abstract: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

AbstractThe 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

AbstractThe 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

AbstractThe 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.