bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+‐ or Mg2+‐Stimulated ATPase Activity in Bullfrog Spinal Nerve: Relation to Ca2+ Requirements for Fast Axonal Transport
Authors: Hammerschlag Richard , Bobinski Judith A.
Published: October 2006

Abstract: Adenosine triphosphatase (ATPase) activity stimulated by Ca2+ or Mg2+ was characterized in spinal nerve and spinal sensory ganglion of bullfrog. Enzyme activity of homogenates from both sources reached a maximum at a 1‐2 mM concentration of either cation, although the level of maximal activity in nerve trunks was approximately twice that in ganglia. Enzyme activation was not observed with 2 mM‐Sr2+ or Ba2+, Co2+ or Mn2+, at 2 mM, depressed Ca2+ activation of the enzyme by 50‐60% in nerve but had no inhibitory effect on ganglia activity. In intact spinal gangliodspinal nerve preparations, incubated for 20 h in medium containing 0.2 mM‐Co2+, no effect was detected on Ca2+/Mg2+ ATPase activity in ganglia or nerve trunks whereas fast axonal transport was inhibited by 80%. Incubation in medium containing 0.02 mM‐Hg2+ depressed enzyme activity in ganglia by 64% and in nerve trunks by 44%, whereas fast transport was again inhibited by 80%. When only nerve trunks were exposed to these ions, Hg2+ but not Co2+ was observed to slow the rate of fast axonal transport. The divalent cation specificity of the Ca2+/Mg2+ ATPase activity is distinct from the ion specificities, determined in previous work, of the Ca2+ requirement during initiation of fast axonal transport in the soma, and of the Ca2+ requirement during translocation in the axon. Thus, previous observations of Ca2+‐dependent events in fast axonal transport cannot be taken per se to suggest the involvement of Ca2+/Mg2+ ATPase in the transport process.
Dopamine Receptors in Subcellular Fractions from Bovine Caudate: Enrichment of [3H]Spiperone Binding in a Postsynaptic Membrane Fraction
Authors: Near Joseph A. , Mahler Henry R.
Published: October 2006

Abstract: Specific binding of tritiated dopamine, spiperone, and N‐propylnorapomorphine was examined in subcellular fractions from bovine caudate nucleus. All fractions contained at least two sets of specific binding sites for [3H]spiperone (KD 1aPP= 0.2 nM, KD 2aPP= 2.2 nM), the higher affinity sites accounting for one‐third to one‐eighth of the total. [3H]Spiperone binding was slightly enriched over the total particulate fraction in P2, P3, SPM, and a crude fraction of synaptic mitochondria. A microsomal subfraction (P3B2) exhibited the highest specific binding capacity obtained, representing a fourfold enrichment over the total particulate fraction. [3H]Dopamine exhibited apparent binding to a single class of high‐affinity sites in all fractions examined (KDaPP= 4.0 nM). A greater than twofold enrichment was observed in all fractions except myelin and P3, with a fivefold enrichment in SPM and P3B2. At least two classes of receptors were labeled by [3H]‐N‐propylnorapomorphine (KD 1aPP= 0.55 nM, KD 2aPP= 20 nM), using 50 nM‐spiperone together with 100 nM‐dopamine to define nonspecific binding. Although binding to the higher affinity site was displaced by spiperone, and lower affinity binding by dopamine, comparison of receptor densities with values obtained by using [3H]spiperone and [3H]dopamine directly suggested that [3H]‐N‐propylnorapomorphine labeled additional sites. We have also examined a postsynaptic membrane (PSM) fraction obtained from SPM by successive extraction with salt and EGTA followed by sonication and separation on a density gradient. [3H]Spiperone binding in PSM was enriched two‐ to threefold over unfractionated SPM with a concomitant decrease in [3H]dopamine binding. The enrichment in spiperone receptors was almost entirely due to an increase in the number of lower affinity binding sites, suggesting that these sites may be associated with the postsynaptic membrane.
Lectin‐Induced Inhibition of Nerve Growth Factor Binding by Receptors of Sympathetic Ganglia
Authors: Costrini Nicholas V. , Kogan Melanie
Published: October 2006

Abstract: Nerve growth factor (NGF) initiates a pleiotypic response in numerous tissues derived from the neural crest by binding to specific plasma membrane receptors. In sympathetic ganglia this receptor has been characterized as a highly asymmetric, minimally hydrophobic, intrinsic membrane protein with a molecular weight of 135,000 (Costrini et al., 1979b). To further characterize this moiety we assessed the effects of lectins on 125I‐NGF specific binding to preparations of particulate and nonionic detergent‐extracted micro‐somal receptors of rabbit superior cervical ganglia (SCG). Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean agglutinin or Ulex europaeus I, induced a concentration‐related, carbohydrate‐specific decrease in 125T‐NGF binding. Following Con A exposure, 125I‐NGF specific binding to particulate SCG receptors was maximally reduced to 23% of control values. WGA similarly reduced NGF binding to particulate microsomal receptors to 37% of control values. Scatchard analysis of growth factor binding following Con A exposure indicated that this lectin effect was principally due to a sixfold reduction in maximum receptor affinity. Lectin‐associated impairment of NGF binding was also demonstrated by using a Triton X‐100 solubilized receptor preparation. These results provide evidence that the high‐affinity‐state NGF receptor of SCG is a glycoprotein containing N‐acetylglucosamine and α‐D‐mannopyranoside residues. These residues are probably located in close proximity to the growth factor binding region of the NGF receptor.
Free Fatty Acid Composition of Human and Rat Peripheral Nerve
Authors: Yao Jeffrey K. , Dyck Peter James , VanLoon Jon A. , Moyer Thomas P.
Published: October 2006

Abstract: The free fatty acid (FFA) composition of peripheral nerve resembles that of erythrocytes but the composition of both is different from that of brain and other tissues. Approximately 75% of FFAs of nerve and erythrocytes are saturated and <5% are polyunsaturated whereas in brain and other tissues, 30‐45% of FFAs are saturated and 25‐50% are polyunsaturated. Approximately 10‐15% of the total FFA of nerve have very long chain lengths [C24, C26, C28, and C30]. The presence of these very long‐chain FFAs in endoneurium cannot be accounted for by the retention of erythrocytes or by lipid degradation. During Wallerian degeneration a significant increase of 18:1, associated with a decrease of saturated FFAs, was found in rat sciatic endoneurium, but normal values were approached when fiber regeneration was well under way. The FFA composition with chain length ≥C26 were not, however, significantly altered with degeneration or repair of nerves. The metabolic significance of this striking difference between nerve and brain FFA composition is unknown but may reflect different functional properties.
Comparison of the Effect of Intravenous Administration of d‐Lysergic Acid Diethylamide on Free and Membrane‐Bound Polysomes in the Rabbit Brain
Authors: Heikkila John J. , Brown Ian R.
Published: October 2006

Abstract: The intravenous administration of LSD to young adult rabbits resulted in the disaggregation of both free and membrane‐bound classes of brain polysomes. Based on the analysis of LSD dosage and the time course of the LSD‐induced brain polysome shift, it was found that free polysomes were more sensitive to the drug than the membrane‐bound polysome fraction. LSD‐induced hyperthermia may be involved in the disaggregation of free and membrane‐bound polysomes, since a correlation was found between the extent of LSD‐induced hyperthermia and the degree of brain polysome shift. Prevention of LSD‐induced hyperthermia by maintaining the animal at 4°C blocked the disaggregation of both polysome classes. Induction of hyperthermia by elevation of ambient temperature also resulted in a shift in free and membrane‐bound polysomes. In all cases the disaggregation of polysomes to monosomes was not caused by RNase activation. During polysome disaggregation, polyadenylated mRNA associated with both free and membrane‐bound polysomes was not degraded but was relocalized from polysomes to monosomes.
Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of the Methylation Cycle and Cystathionine β‐Synthase
Authors: Rassin David K. , Sturman John A. , Gaull Gerald E.
Published: October 2006

Abstract: The subcellular distributions of the enzymes associated with the methylation and cystathionine‐synthesizing portion of the sulfur amino acid metabolic pathway have been determined in the occipital lobe of the rhesus monkey. 5‐Methyltetrahydrofolate‐homocysteine methyltransferase and 5, 10‐methylenetetrahydrofolate reductase activities are located mainly in the soluble compartment. Serine hydroxymethyltransferase activity is located primarily in mitochondria. Cystathionine β‐synthase is a soluble enzyme with a significant component occluded within the nerve endings. Glycine, serine, and cystathionine increase per gram of tissue during development. Glycine and serine are approximately 30% occluded within the nerve endings. These data are consistent with a localization of sulfur amino acid metabolism that supports a differential compartmentation of potential neurotransmitter function and methylation function in the primate.
Distribution of Cyclic Nucleotide Phosphodiesterase in Mouse Brain
Authors: Yang Mildred S. , Passonneau Polly , Lowry Oliver H.
Published: October 2006

Abstract: Seventy‐one regions of mouse brain, and many subdivisions of some of these, were analyzed for cyclic nucleotide phosphodiesterase. The samples were dissected from lyophilized frozen sections. Since the average sample weighed only 25 ng (20 × 75 × 75 μ3), regions as small as the locus ceruleus could be analyzed. Activities in gray areas ranged 40‐fold from a high in the pars reticulata of the substantia nigra to a low in the deep cerebellar nuclei. The activity in fiber tracts also vaned about 40‐fold, and on a lipid‐free dry weight basis was similar to the activity in the gray matter where the fibers originated. The rank order for gray regions was basal ganglia, amygdala, hippocampus, cerebral cortex, most of the diencephalic nuclei, nuclei of the pons, cerebellum, and nuclei of the medulla.
The Structural Specificity of Choline Transport into Cholinergic Nerve Terminals
Authors: Collier B.
Published: October 2006

Abstract: The accumulation of choline, homocholine, and 4‐hydroxybutyl‐trimethylammonium by rat brain synaptosomes was measured; the choline uptake mechanism transported homocholine but not hydroxybutyltrimethylammonium, which, in addition, did not block choline accumulation. In cats’superior cervical ganglia, preganglionic nerve stimulation increased the accumulation of homocholine, but not that of hydroxybutyltrimethylammonium. It is concluded that the substrate specificity of the choline transport mechanism is such that increasing the N‐O atom distance by one methylene group retains affinity, but increasing this distance by two methylene groups does not.
Oxidation of β‐Phenylethylamine by Both Types of Monoamine Oxidase: Examination of Enzymes in Brain and Liver Mitochondria of Eight Species
Authors: Suzuki O. , Katsumata Y. , Oya M.
Published: October 2006

Abstract: β‐Phenylethylamine (PEA) was characterized as a substrate for type A and type B monoamine oxidase (MAO) in brain and liver mitochondria of eight species at different substrate concentrations. In all species, at 10.0 μM, PEA was almost specific for type B MAO. At 1000 μM, however, the amine was common for both types of MAO in rat brain and liver, human brain and liver, mouse brain, guinea pig brain and liver, and bovine brain, while it was specific for type B MAO in mouse liver, rabbit brain and liver, bovine liver, pig brain and liver, and chicken brain and liver. From the present study, when PEA is used as a type B substrate, it is recommended that the substrate concentration should be sufficiently low to avoid the effects of species and tissue differences.
Effect of Wallerian Degeneration on Histamine Concentration of the Peripheral Nerve
Authors: MacDonald S. M. , Mezei M. , Mezei C.
Published: October 2006

One sciatic nerve of a White Leghorn hen was severed and the distal portion was allowed to undergo Wallerian degeneration. The change in histamine and DNA concentration and mast cell number was measured at different times following nerve sectioning in the proximal regenerating, distal degenerating, and intact, contralateral nerves. The experimental results revealed a significant accumulation of histamine in the proximal desheathed segment and in the contralateral “functional nerve,” whereas the biogenic amine in the distal desheathed nerve significantly decreased. The pattern of change of histamine in the distal and proximal nerve sheaths was different: it dropped at 2 h and then rose in the later stages of Wallerian degeneration. In the distal desheathed nerves and in both the proximal and distal nerve sheaths DNA increased significantly by 14 days. The number of mast cells appeared to be highest in the 14‐day distal nerve and in the 7‐day proximal nerve sheaths. These results support a dual localization of histamine in the peripheral nerve, and are consistent with the interpretation that the amine has either some role in neurotransmission or in the process of growth and regeneration.
Mammalian Brain Alkaline Phosphatase: Expression of Liver/Bone/Kidney Locus Comparison of Fetal and Adult Activities
Authors: Goldstein D. J. , Harris Harry
Published: October 2006

The alkaline phosphatases (EC 3.1.3.1) are determined by at least three gene loci, which can be sharply distinguished one from another by their sensitivity to inhibition with various amino acids and peptides and by ther‐mostability. Alkaline phosphatase is present in the brains of guinea pig, rat, mouse, hamster, squirrel, rabbit, cat, sheep, cow, tamarin, baboon, and man. The gene locus coding for alkaline phosphatase in all these brains is the liver/ bone/kidney locus, as indicated by thermostability studies and by inhibition studies with L‐phenylalanine, L‐homoarginine, and L‐phenylalanylglycylglycine. The average brain alkaline phosphatase activity is about 35% of the average for the livers and only 7.2% and 4.4% of the average kidney and placental activities, respectively. During growth and development, brain alkaline phosphatase activity decreases in the mammals studied. The amount of change is tissue‐ and species‐dependent.
Calcium Transport by Primary Cultured Neuronal and Glial Cells from Chick Embryo Brain
Authors: Barnes Eugene M. , Mandel P.
Published: October 2006

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM‐Ca2+ and 5.4 mM‐K+. The rate of calcium entry into neurons was increased 2.7‐fold by elevating medium potassium to 60 MM. The effect of high external potassium was to increase the Vmax value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium‐dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM‐D‐600 (a verapamil derivative) or by 1 mM‐CoCl2, but 0.5 μM‐tet‐rodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight‐of‐protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization‐sensitive, calcium‐specific channel. A similar calcium transport activity was not detected in cultured glial cells.
Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions
Authors: Vrana Kent E. , Allhiser Carin L. , Roskoski Robert
Published: October 2006

Tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA. Cyclic AMP‐dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short‐term pre‐incubation (3 min) of extracts under phosphorylating conditions (Mg. ATP, cAMP) increases enzyme activity two‐ to tenfold over control as measured during a subsequent 15‐min assay. We now report that pre‐incubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg. ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible de‐naturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 ± 0.1). Although the apparent Km, of the enzyme for the 6‐methyltetrahydropterine (6‐MPH4) cofactor is reduced (0.86 mM, control; 0.32 MM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki, for dopamine is increased from 4.5 μM for the control to 28 μM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 μM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation‐inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km, for 6‐MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low‐voltage paper electrophoresis.
Identification and Distribution of m‐ and p‐Hydroxyphenylacetic Acids in the Brain of the Rat
Authors: Durden David A. , Boulton Alan A.
Published: October 2006

The m and p isomers of hydroxyphenylacetic acid have been identified and quantitated in whole rat brain and in several regions using a capillary column high resolution gas chromatography–mass spectrometry procedure. Their concentrations were: for m‐hydroxyphenylacetic acid (mean ± S.E., number of determinations in parentheses)—whole brain, 2.3 ± 0.3 ng/g (7); hypothalamus, 1.2 ± 0.3 ng/g (5); caudate nucleus, 5.5 ± 0.6 ng/g (5); brain stem, 1.8 ± 0.1 ngig (5); cerebellum, 1.2 ± 0.1 ng/g (5) and the “rest,” 1.7 ± 0.1 ng/g (5); and for p‐hydroxyphenylacetic acid–whole brain, 10.6 ± 0.7 ng/g (7); hypothalamus, 4.5 ± 0.1 ng/g (4); caudate nucleus, 28.3 ±1.6 ng/g (5); brain stem, 8.6 ± 0.6 ng/g (5); cerebellum, 8.1 ± 0.4 ng/g (9, and the “rest,” 5.3 ± 0.5 ng/g (5). This heterogeneous distribution parallels closely that exhibited by their respective precursor amines, m‐ and p‐tyramine.
Characterization and Comparison of Neurofilament Proteins from Rat and Mouse CNS
Authors: Brown Beverly A. , Nixon Ralph A. , Strocchi Paola , Marotta Charles A.
Published: October 2006

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co‐purifying lower‐molecular‐weight CNS proteins, including α and β tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co‐workers and were demyelinated with Triton X‐100. On one‐dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower‐molecular‐weight proteins (63,000‐16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two‐dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) α and β tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co‐purify by the axon flotation procedure also have nonidentical peptide maps; and (4) the corresponding NFPs from rat and mouse have similar peptide maps. The co‐purifying proteins examined in detail (63,000–49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.
In Vitro and In Vivo Binding of S‐Adenosyl‐L‐Homocysteine to Membranes from Rat Cerebral Cortex
Authors: Fonlupt P. , Rey C. , Pacheco H.
Published: October 2006

Membranes from rat cerebral cortex are able to bind S‐adenosyl‐L‐homocysteine (SAH) with a KD of 5. 10‐7 M and n of 170 pmol/g fresh tissue (i.e. 20 mg protein). The binding is enhanced by Mg2+ and Ca2‐ but not K+ and Na+, γ‐Aminobutyric acid, diazepine, noradrenaline and a antagonists are without any effect; S‐adenosyl‐L‐methionine, adenosine and adenosine triphosphate inhibit SAH binding. Linkage with an adenosine receptor has not been expressly demonstrated by our method. SAH binding proteins are more abundant in the crude synaptosomal pellet (P2). A similar fixation seems to occur on brain membranes after [3H]SAH administration to rat. The binding might be linked to a methylase activity or an adenosine receptor.
Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites
Authors: Leysen Josée E. , Gommeren Walter
Published: October 2006

I. Binding of [3H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris‐HCl buffer, pH 7.5, containing 1 mM‐EDTA and 0.01% ascorbic acid, using a washed total‐membrane fraction. The receptor binding was stereospecifically inhibited by (+)‐butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [3H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [3H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [3H]spiperone and [3H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high‐ionic‐strength Tris buffer with salts added and low‐ionic‐strength Tris buffer with EDTA. Apparent KD, values of spiperone decreased with decreasing tissue concentrations. KD, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the 3H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist‐antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high‐affinity agonist and antagonist binding.
Changes in Rabbit Brain Cytosolic and Membrane‐Bound Gangliosides During Prenatal Life
Authors: Sonnino Sandro , Ghidoni Riccardo , Masserini Massimo , Aporti Franco , Tettamanti Guido
Published: October 2006

The present study deals with the developmental profile of cytosolic and membrane‐bound gangliosides in rabbit whole brain from the 21st day of pregnancy, the time at which brain could be macroscopically recognized and handled, till birth. In this period of prenatal life the content of membrane‐bound gangliosides showed a 2.5‐fold increase, referred to fresh and dry brain weight and to membrane‐bound protein; the content of cytosolic gangliosides reached a maximum at 21‐22 days of pregnancy, and then underwent to birth a threefold diminution. The qualitative pattern of membrane‐bound gangliosides, in the same period of life, was characterized by an increase of GD1a and GM1 (more marked for GD1a), a decrease of GT1a, GT1b and GQ1b and a constant level of GD3 and GD1b. At 21 days of pregnancy the most abundant gangliosides were GT1b, and GQ1b, followed by GD1a and GD1b; at birth it was GD1a followed by GT1b GD1b, and GM1 The qualitative pattern of cytosolic gangliosides closely resembled, during the entire period of prenatal life examined, that of membrane‐bound gangliosides.
Distribution of Soluble Proteins Within Spinal Motoneurons: A Quantitative Two‐Dimensional Electrophoretic Analysis
Authors: Weil David E. , McIlwain D. L.
Published: October 2006

Soluble protein fractions obtained from bovine lumbar spinal motoneuron cell bodies, ventral gray matter, and ventral and dorsal roots were analyzed by two‐dimensional gel electrophoresis. Each extract was separated into Coomassie blue‐stained patterns of up to 350 polypeptides ranging in isoelectric point from pH 4 to 8 and in molecular weight from 10,000 to 200,000. Visual inspection of the protein pattern of the isolated cell bodies showed it to be substantially different from those of ventral gray matter and the spinal roots, while the patterns obtained from ventral and dorsal roots were indistinguishable. Computer‐assisted densitometry of the major soluble proteins from spinal roots showed no quantitative difference between the predominant proteins in ventral and dorsal root extracts. Differences of 10‐fold or more were common when the major proteins of the isolated perikarya were compared with those of the other fractions. Since most of the soluble proteins extracted from ventral and dorsal roots were probably derived from the axoplasm of motor and sensory nerves, respectively, these results are interpreted to mean that large differences exist in the distribution of individual soluble proteins between the cell body and axon of spinal motoneurons, while the major soluble proteins of spinal motor and sensory axons are highly similar.
Influence of Sodium‐Independent γ‐Aminobutyric Acid Binding on the Assay of Sodium‐Dependent γ‐Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain
Authors: Kurioka Susumu , Matsuda Makoto
Published: October 2006

A large amount of [3H]GABA was bound to crude synaptic membrane fractions of rat. by sodium‐independent process in a medium that contained 100 μM [3H]GABA used for assaying GABA uptake site. This [3H]‐GABA binding was different from receptor binding of GABA. It was confirmed that this sodium‐independent [2H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium‐independent GABA binding had a negligible influence on sodium‐dependent GABA binding.
Inhibition by Methylmalonate of Glycine Uptake by Synaptosomes from Rat Spinal Cord
Authors: Lopez‐Lahoya J. , Garcia M. L. , Benavides J. , Ugarte M.
Published: October 2006

Methylmalonate is accumulated in the genetically linked deficiency of methylmalonyl‐CoA mutase (methylmalonic acidemia). In this condition is also observed an elevation of the glycine levels. This communication reports the inhibition of the synaptosomal glycine uptake by methylmalonate, when present at similar concentrations to those found in methylmalonic acidemia. This inhibition could be responsible, at least in part, for the neurological damage characteristic of this disease, by increasing the glycine levels in the synaptic cleft and thus interfering with the normal function of the inhibitory glycinergic synapsis in the spinal cord.
Endogenous Modulator of Benzodiazepine Binding in Rat Cortex
Authors: Chiu T. H. , Rosenberg H. C.
Published: October 2006

Benzodiazepine binding sites, solubilized with 1% digitonin, were used to study specific [3H]flunitrazepam ([3H]FNP) binding. Specific binding increased nonlinearly with increasing amounts of digitonin extract in the assay. Specific binding was increased, and the relationship to amount of extract became linear. in the presence of 2% polyethylene glycol 6000 (PEG). Heat treatment destroyed binding activity of the extract, but not ability to inhibit [3H]FNP binding. Kinetic analysis showed inhibition to be noncompetitive. The inhibitory activity was sensitive to trypsin. Extracts of repeatedly frozen, thawed, and washed membrane preparations still possessed inhibitory activity. It is suggested that digitonin solubilizes a membrane protein that inhibits benzodiazepine binding. PEG apparently removes this substance from the binding sites.
Neuronal Membrane D2‐Protein During Rat Brain Ontogeny
Authors: Jørgensen Ole Steen
Published: October 2006

Abstract The D2‐protein exists as a sialylated form in fetal and in perinatal rat brain, and as a desialylated form in adult rat brain. By crossed Immunoelectrophoresis the concentrations and amounts of these forms were investigated during ontogeny of both forebrain and cerebellum. The concentration of sialylated D2‐protein reached two peaks during ontogeny. The first peak occurred in forebrain around embryonic day 13, and in cerebellum just after birth. In both brain areas it coincided with the periods of major neuronal migration. The second peak occurred in forebrain around postnatal day 6 and in cerebellum around postnatal day 20, during the initial period of synaptogenesis in both brain areas. Moreover, the desialylated form of D2‐protein was found only in postnatal rats and it increased to a slight maximum at postnatal day 25 in forebrain, and postnatal day 35 in cerebellum. The findings are discussed in relation to the possible role of D2‐protein as an adhesion molecule.
Effect of Freezing on γ‐Aminobutyric Acid Levels in Human Cerebrospinal Fluid
Authors: Abbott R. J. , Keidan J. , Pye I. F. , Nahorski S. R.
Published: October 2006

Abstract Using a radioreceptor assay, the concentration of γ‐aminobutyric acid (GABA) in human cerebrospinal fluid (CSF) was found to be elevated significantly following a single deep‐freeze to –70°C and thaw. Mean CSF GABA (± SD) in unfrozen CSF was 173 ± 73 pmol/ml (n= 24). After a single deep‐freeze, the mean level was 243 ± 106 pmol/ml (p < 0.02). Subsequent freeze‐thaw cycles resulted in further irregular and unpredictable elevations in CSF GABA. Mean level after two freezes was 379 ± 125 pmol/ml and after three freezes 654 ± 411 pmol/ml. These changes could result in the incorrect interpretation of results in patients suffering from neurological diseases.
Presence of Conjugated 5‐Hydroxytryptamine in In Vivo Superfusates of Rat Spinal Cord
Authors: Hammond Donna L. , Yaksh Tony L. , Tyce Gertrude M.
Published: October 2006

Abstract The presence of an acid‐hydrolyzable conjugate of 5‐hydroxytryptamine, presumably 5‐hydroxytryptamine‐O‐sulfate, was demonstrated in in vivo superfusates of rat spinal cord by HPLC with electrochemical detection. In untreated rats, the concentration of the 5‐hydroxytryptamine conjugate measured during the basal efflux of 5‐hydroxytryptamine did not differ from that measured during the release of 5‐hydroxytryptamine evoked by dl‐ρ‐chloroamphetamine. Pretreatment of the rats with clorgyline, an inhibitor of the enzyme monoamine oxidase (EC 1.4.3.4), or with probenecid did not alter the concentrations of conjugated 5‐hydroxytryptamine measured during the basal efflux of 5‐hydroxytryptamine, but did elevate the concentrations of conjugate measured during the evoked release of 5‐hydroxytryptamine.
Solubilization of Collagen‐Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures
Authors: Gómez‐Barriocanal Javier , Barat Ana , Escudero Elena , Rodríguez‐Borrajo Corona , Ramírez Galo
Published: October 2006

Abstract— We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen‐tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one‐third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt‐EDTA‐extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes “insoluble.” However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X‐100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X‐100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.
Some Biochemical Properties of the Rapid Adenosine Uptake System in Rat Brain Synaptosomes
Authors: Bender A. S. , Wu P. H. , Phillis J. W.
Published: October 2006

Abstract: The rapid uptake of adenosine into rat brain cortical synaptosomes is mediated by a facilitated diffusion process. The carrier mediated uptake is sensitive to pH and temperature. The average Q10 value for the system is approximately 1.77 and the necessary activation energy (Ea) is estimated to be 8870 cal/mol. These values are essentially in agreement with values reported for adenosine uptake carriers of other tissues. Substrate specificity of the uptake system in the CNS demonstrates that nucleotides do not interact with the carrier until they have been hydrolyzed to nucleosides. Structural modification of the purine moiety at the “2” position did not have a profound effect on the ability of the molecule to serve as a substrate for the uptake system. Competitive inhibition by sulfhydryl reagents, p‐chloromercuribenzoate, and N‐ethylmaleimide on adenosine uptake suggests a direct involvement of sulfhydryl group(s) in the uptake mechanism. Other purines and pyrimidines also inhibited adenosine uptake, suggesting that a variety of nucleosides can interact with a common carrier system.
Some Properties of an Astrocytic Protein Fraction That Binds Serotonin
Authors: Hertz Leif , Tamir Hadassah
Published: October 2006

Abstract: A protein fraction that binds serotonin was obtained from astrocytes in primary cultures. It was found to differ in some of its physical, chemical, and pharmacological properties from neuronal serotonin binding protein.
Affinities of Histamine H1‐Antagonists in Guinea Pig Brain: Similarity of Values Determined from [3H]Mepyramine Binding and from Inhibition of a Functional Response
Authors: Hill S. J. , Daum P. , Young J. M.
Published: October 2006

Abstract: Affinity constants for five antagonists at histamine H1‐receptors in guinea pig brain have been determined from inhibition of the potentiation by histamine of the adenosine‐induced accumulation of cyclic AMP in cerebral cortical slices. This action of histamine appeared to be mediated solely through H1‐receptors. The affinity constants obtained were similar to those determined on peripheral H1‐receptors and from inhibition of high‐affinity [3H]mepyramine binding. This provides strong evidence that at least some of the [3H]mepyramine binding sites in guinea pig brain can be identified with functional H,‐receptors.
Effect of β‐Bungarotoxin on the Release of Endogenous Amino Acids from the Sensorimotor Cortex
Authors: Abdul‐Ghani A.‐S. , Coutinho‐Netto J. , Bradford H. F. , Summers B. A. , Thompson E. J.
Published: October 2006

Abstract: β‐Bungarotoxin, a snake neurotoxin purified from the venom of Bungarus multicinctus, caused a significant increase in the in vivo release of glutamate from the superfused sensorimotor cortex of awake animals. A smaller effect on GABA release was observed, but no change was detected in the release of six other amino acids measured. The effects on glutamate and GABA release were entirely blocked by tetrodotoxin (1 μM) and were reversible when the cortical tissue was washed with saline.
Interactions of Catecholestrogens with Cytoplasmic and Nuclear Estrogen Receptors in Rat Pituitary Gland and Hypothalamus
Authors: Kirchhoff Josef , Hornung Elisabeth , Ghraf Rüdiger , Ball Peter , Knuppen Rudolf
Published: October 2006

Abstract: The affinity of a series of catecholestrogens for 7S cytoplasmic receptor proteins from hypothalamus and pituitary gland of ovariectomised rats was assessed in vitro by a competitive charcoal binding assay at 4°C. The equilibrium dissociation constants (Ki) of catecholestrogens 4‐hydroxyestradiol, 4‐hydroxyethynylestradiol, 2‐hydroxyestradiol, 2‐hydroxyethynylestradiol, and 4‐hydroxyestrone were of the same order (Ki 0.3–0.6 nm) as those of estradiol and ethynylestradiol (Ki: 0.1 nm). Methylation of 2‐hydroxyestradiol led to a substantial loss of binding affinity. Tritium‐labelled receptor complexes were demonstrated in KCl extracts of purified nuclei from pituitary and hypothalamic tissue 1 h after intravenous injection of 0.1 mCi tritiated 2‐ or 4‐hydroxyestradiol. These macromolecular complexes sedimented in the 5‐6S region of 5–20% (w/v) sucrose gradients containing 0.4 m‐KCl. Further evidence for the translocation of estrogen receptors by catecholestrogens into the nuclei of rat pituitary and hypothalamus was the increase in nuclear receptor concentrations, measured by exchange assay, 1 h after the intraperitoneal injection of 0.1 mg unlabelled catecholestrogen. Administration of 4‐hydroxyestradiol and 4‐hydroxyethynylestradiol increased nuclear receptor concentrations to the same maximal levels as those following application of the same dose of estradiol or ethynylestradiol, whereas the respective 2‐hydroxylated compounds exhibited only 60–70% of the maximal translocating capacity. The in vivo translocating capacities of the various catecholestrogens tested at this dose correlated well with their binding affinities for cytosol receptors determined in vitro.
Thymidine Kinase Activity Is Reduced in the Developing Staggerer Cerebellum
Authors: Messer Anne , Savage Michael , Carter Thomas P.
Published: October 2006

Abstract: In the mouse cerebellar mutant staggerer, thymidine kinase levels do not increase developmentally at ages when the wild‐type level is high. Mixing experiments show that this effect is not due to an endogenous inhibitor of the enzyme. Both the Km and the susceptibility of the thymidine kinase to nucleotide inhibitors are unaltered in the mutant animals, suggesting that the enzyme is not induced in the mutant.
α‐N‐Acetyl β‐Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1‐13)
Authors: Weber Eckard , Truscott Roger J. W. , Evans Christopher , Sullivan Sue , Angwin Pamela , Barchas Jack D.
Published: October 2006

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1‐13) treated with a water‐soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β‐endorphin and dynorphin(1‐13) as well as by CDI‐treated dynorphin(1‐13), but only weakly by authentic dynorphin(1‐13). Neither nonacetylated β‐endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI‐treated dynorphin(1‐13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1‐13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI‐treated dynorphin(1‐13) used as a trace and as a standard. Competition studies showed 0.5‐1% cross‐reactivity with α‐N‐acetyl β‐endorphin(1‐31), α‐N‐acetyl β‐endorphin(1‐27), and totally acetylated β‐endorphin(1‐31). Nonacetylated β‐endorphins did not cross‐react. Posterior‐intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior‐intermediate lobe extracts were also fractionated by cation‐exchange chromatography. Fractionated extracts were analyzed by RIAs for β‐endorphin, CDI‐treated dynorphin(1‐13), and authentic dynorphin(1‐13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β‐endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β‐endorphins in the pituitary.
Variations in Brain Cortical Polysome Translation Products Among Rats of the Same Strain
Authors: Strocchi Paola , Gilbert Jeffrey M. , Marotta Charles A.
Published: October 2006

Abstract: Studies were undertaken to determine whether there exist variations among the translation products of polysomes from different brains of animals of the same strain. Polysomes were prepared from individual rat cortices and translated in a reticulocyte protein‐synthesizing system containing rabbit reticulocyte factors and l‐[35S]methionine; the resulting radioactive proteins were analyzed by two‐dimensional polyacrylamide gel electrophoresis autoradiography. Comparison of the autoradiographs revealed that two acidic proteins, A and B, of apparent 54,000 M. W. occur as three phenotypes: A only, B only, or A plus B. These proteins were not detectable by Coomassie brilliant blue staining of two‐dimensional electrophoretograms of brain protein preparations. Messenger RNA was extracted from pooled cortices and translated in a wheat germ extract, and both A and B proteins were detected among the products of translation. Cyclic AMP affinity chromatography of the translation products of cortical polysomes showed that both A and B proteins bind to cyclic AMP. Our data are consistent with the conclusion that there are qualitative differences in the polysome translation products that bind to cyclic AMP among individual cortices of rats of the same strain.
Species Specificity of Brain Cyclic AMP Receptor Proteins
Authors: Panter S. Scott , Butley Martin S. , Malkinson Alvin M.
Published: October 2006

Abstract: The cyclic AMP binding proteins present in mouse, rat, bovine, and sheep brains were compared. Extracts were isotopically labeled with 8‐azido‐cyclic [32P]AMP, a photoaffinity analog specific for cyclic AMP binding sites, and then subjected to two‐dimensional gel electrophoresis. The resulting autoradiographic patterns were generally similar, but showed consistent species variations. Proteins identified by their size and phosphorylatability as regulatory subunits of Type II protein kinase isozymes were present in all species, but with slight variations in pi. A series of charge variants identified as regulatory subunits of Type I kinase isozymes on the basis of their size was also ubiquitously present, as were several smaller proteins postulated to be proteolytic fragments derived from the regulatory subunits. The major species difference was a series of labeled proteins found only in rodent brains and not in the brains of any ruminant, or in other rodent tissues. These proteins had a molecular weight of 54,000 and a pi range of 5.89‐6.26, and could not be endogenously phosphorylated. The identities of these proteins and their relationship to the protein kinase regulatory subunits are unknown.
Neurochemical Characterization of an Antimony‐Choline Analog in Rat Cortical Synaptosomes
Authors: Meyer Edwin M. , Barrnett Russell J. , Cooper Jack R.
Published: October 2006

Abstract: An analog of choline, in which nitrogen was replaced by antimony, was neurochemically characterized in rat cortical synaptosomes. It was found to be a substrate for several cholinergic enzymes, transported by a Na+‐dependent, hemicholinium‐3‐sensitive process, acetylated, and subsequently released by depolarization in a calcium‐dependent manner. Sb‐choline also competed with choline for Na+‐dependent uptake and for acetylation by [14C]acetyl‐CoA in synaptosomes. These results suggest that Sb‐choline and its acetylated product should be useful substrates for the x‐ray microanalytical localization of cholinergic pools in intact nerve terminals.
Induction of Glutamine Synthetase by Dibutyryl Cyclic AMP in C‐6 Glioma Cells
Authors: Browning Edward T. , Nicklas William J.
Published: October 2006

Abstract: Glutamine synthetase was found to be increased in C‐6 glioma cells as a result of increasing culture passage and N‐6,2′‐O‐dibutyryl cyclic AMP (dbcAMP) treatment. At low passage dbcAMP produced a 2.5‐fold increase in glutamine synthetase activity per unit of cellular protein. At high passage control glutamine synthetase was approximately double that seen at low passage, but dbcAMP produced an additional 65% increase. Lactate dehydrogenase activity was also increased by dbcAMP treatment at both low and high passage, but culture passage produced no change in the lactate dehydrogenase. With increasing culture passage, the ratio of cellular protein to DNA doubled. Therefore, expression of data per unit of protein tended to minimize the apparent changes in activity. The maximum increase in glutamine synthetase activity produced by both dbcAMP and increasing culture passage and expressed on a DNA basis was 5.6‐fold. The increase in glutamine synthetase activity was generally linear during the first 20 h of drug treatment, after which enzyme activity remained nearly constant up to 72 h. Ninety percent or more of the dbcAMP remained in the medium at the end of 48‐h exposure of cells to dbcAMP. 8‐Br‐Cyclic AMP also increased glutamine synthetase activity of C‐6 cells, but n‐butyrate did not. Isoproterenol, which increases cyclic AMP in C‐6 cells, increased glutamine synthetase activity. The effect of isoproterenol on glutamine synthetase was inhibited by the β‐adrenergic blocking agent sotalol. Cyclo‐heximide (10 μg/ml) inhibited the dbcAMP effect on glutamine synthetase activity and also decreased the control enzyme activity by 60%.