bluuebunny/crossref_metadata_2025_split huggingface dataset:
An In Vivo Study of Dopamine Release and Metabolism in Rat Brain Regions Using Intracerebral Dialysis
Authors: Sharp T. , Zetterström T. , Ungerstedt U. 
Published: July 2010

AbstractIntracerebral dialysis was used with a specifically designed HPLC with electrochemical detection assay to monitor extracellular levels of endogenous 3,4‐dihydroxyphenylethylamine (dopamine, DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in brain regions of the halo‐thane‐anesthetized rat. Significant amounts of DA, DOPAC, and HVA were detected in control perfusates collected from striatum and n. accumbens whereas the medial prefrontal cortex showed lower monoamine levels. The ratio of DA in perfusate to DA in whole tissue suggests that in f. cortex, compared to n. accumbens and striatum, there is a greater amount of DA in the extracellular space relative to the intraneuronal DA content. The DOPAC/HVA ratio in control perfusates varied between regions in accordance with whole tissue measurements. This ratio was highest in n. accumbens and lowest in f. cortex. The monoamine oxidase inhibitor pargyline (100 mg/kg i.p.) caused an exponential decline in DOPAC, but not of HVA, in regional perfusates, an effect that was associated with an increase in DA. The data indicated a higher turnover of extracellular DOPAC in n. accumbens than in striatum and the lowest DOPAC turnover in f. cortex. The rate of decline in extracellular DA metabolite levels was slow compared to whole tissue measurements. In the perfusates there was no statistical correlation between basal amounts of DA in the perfusates and DOPAC and HVA levels or DOPAC turnover for any of the areas, indicating that measurement of DA metabolism in the brain under basal conditions does not provide a good index of DA release. In summary, this study shows clear regional differences in basal DA release and metabolite levels, metabolite patterns, and DOPAC turnover rates in rat brain in vivo.
Regulation of Taurine Transport in Rat Skeletal Muscle
Authors: Iwata Heitaroh , Obara Takeo , Kim Bong Ki , Baba Akemichi 
Published: July 2010

AbstractTaurine concentration of soleus muscle (SL, slow‐twitch) was initially about twofold higher than that of extensor digitorum longus muscle (EDL, fast‐twitch). Taurine concentration in gastrocnemius muscle (GC) was intermediate between that of EDL and SL. Four days after sciatic nerve section, taurine concentration in the EDL but not in the SL was increased by 2.5‐fold. The increase was not due to the muscle atrophy and was observed 28 days after denervation. Tenotomy did not increase the total taurine content of the EDL. The increase in taurine concentration of the denervated EDL was prevented by simultaneous ingestion of guanidinoethane sul‐fonate, a competitive inhibitor of taurine transport. The initial and the maximal rates of [3H]taurine uptake were significantly higher in SL than in EDL. Denervation dramatically accelerated the initial and the maximal rates of the transport in EDL, whereas it significantly reduced those in SL. In contrast, the electrical stimulation of sciatic nerve accelerated the uptake of taurine by EDL and SL of the control but not of the curare‐treated rats. These results suggest that transport of taurine into rat skeletal muscles is regulated differently by neural information and by muscular activity, and that the regulation is dependent on the muscle phenotype.
Brain Adrenoceptor Binding Sites in Mice Susceptible (DBA/2J) and Resistant (C57 Bl/6) to Audiogenic Seizures
Authors: Jazrawi S. P. , Horton R. W. 
Published: July 2010

AbstractWe have examined if the age‐related susceptibility of DBA/2J mice to audiogenic seizures is the result of an abnormality in the number or sensitivity of brain adrenoceptors. The binding of α1‐‐, α2‐‐, and β‐adrenoceptor ligands to membranes prepared from whole brain or regions of brain of DBA/2J mice was measured at various ages, corresponding to the periods before, during, and after the maximal sensitivity to audiogenic seizures. For comparison, we have studied concurrently age‐matched C57 B1/6 mice, a strain resistant to audiogenic seizures at all ages. There was no difference in the binding of α2‐‐or β‐adrenoceptor ligands to whole brain membranes between the two strains of mice at any age. The maximal number of α1‐‐adrenoceptor binding sites was lower in whole brains of DBA/2J mice than of C57 B1/6 mice at all ages studied except 13–15 days of age. The differences were small (maximally 17%) but were statistically significant at 21–23 days of age, the time of maximal sensitivity of DBA/2J mice to audiogenic seizures. No difference between the two strains was found in the number or affinity of α1‐‐or α2‐‐adrenoceptor binding sites at any age in any of the brain regions studied. The age‐related susceptibility of DBA/2J mice to audiogenic seizures is not the result of an abnormality in number or sensitivity of α2‐‐or β‐adrenoceptor binding sites, but a reduced number of α1‐‐adrenoceptor binding sites may be involved.
Reevaluation of the Role of Gangliosides as Receptors for Tetanus Toxin
Authors: Critchley David R. , Habig William H. , Fishman Peter H. 
Published: July 2010

AbstractBinding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mMNaCl, 50 mMTris‐HCl (pH 7.4) than in 25 mMTris‐acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris‐saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100‐fold higher in Trissaline compared to Tris‐acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with125I‐labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GDlb≅ GTlb≅ GQ1b> GD2> GD3≫ GD1a≅ GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris‐saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low‐salt and ‐pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.
Studies of the Regulation of Basal Adenylate Cyclase Activity by Membrane Polyunsaturated Fatty Acids in Cultured Neuroblastoma
Authors: Murphy M. G. 
Published: July 2010

AbstractThe role of membrane polyunsaturated fatty acids (PUFAs) in the regulation of basal adenylate cyclase activity was examined in intact N1E‐115 neuroblastoma cells. Addition of linoleic acid (50 μM) to the culture medium for 48 h resulted in a significant increase in phospholipid PUFA content and in a two‐ to fivefold increase in basal accumulation of cyclic AMP (cAMP). Both phenomena were reversed on removal of linoleate from the medium. PUFA enrichment stimulated cell proliferation by —20% without altering the relative proportion of cellular protein. The supplemented cells synthesized significantly larger amounts of prostaglandin (PG) E and D than did the controls; however, blockade of PG synthesis by indomethacin or ibuprofen did not alter cAMP formation. Supplemented cells contained higher levels of malondialdehyde (MDA) than did controls, and MDA formation was reduced by coculture with α‐tocopherol; however, its inclusion in the medium did not affect cAMP accumulation. Linoleate‐supplemented cells responded to cyclase‐activating agonists to the same extent as did control cells. Responses to inhibitory agonists (e.g., isoproterenol and carbamylcholine) were altered, but not to a sufficient extent to account for the PUFA‐dependent increases in basal adenylate cyclase activity.
Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in the Aged Human Central Nervous System
Authors: Atack John R. , Perry Elaine K. , Bonham James R. , Candy John M. , Perry Robert H. 
Published: July 2010

AbstractThe distributions of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) molecular forms and their solubility characteristics were examined, using density gradient centrifugation, in various regions of the postmortem human CNS. Total AChE activity varied extensively (50‐fold) among the regions investigated, being highest in the telencephalic subcortical structures (caudate nucleus and nucleus of Meynert); intermediate in the substantia nigra, cerebellum, and spinal cord; and least in the fornix and cortical regions (hippocampus and temporal and parietal cortex). Total BChE activity was, in contrast, much more evenly distributed, with only a threefold variation between the regions studied. Although the patterns of molecular forms of each enzyme were broadly similar among the different areas, regional variations in the distribution and abundance of the various forms of AChE were much greater than those of BChE. Thus, although the tetrameric G4 form of AChE constituted the majority of the total AChE activity in all regions examined, the ratio of the G4 form to the monomeric Gl form, the latter of which constituted the majority of the remaining activity, varied markedly, ranging from 21 in the caudate nucleus to 1.7 in the tem poral cortex. In addition to the G4 and Gl forms of AChE, the dimeric G2 form was observed in the nucleus of Meynert and a fast‐sedimenting (16S) species was found in samples of both the parietal cortex and spinal cord. In contrast, the G4 and Gl forms of BChE were the only molecular species observed in the different areas and the G4.G1 ratio varied from 3.3 in the substantia nigra to 0.9 in the temporal cortex. Regarding the solubility characteristics of the individual AChE and BChE molecular forms, the majority of the G4 form of AChE was extractable only in the presence of detergent, indicating a predominantly membrane‐bound localization of this species. The smaller AChE forms (Gl and G2) and both the Gl and G4 forms of BChE were all relatively evenly distributed between soluble and membrane‐bound species. These findings are discussed in relation to neurochemical and neuroanatomical, particularly cholinergic, features of the regions examined.
Reduced Stability of Rat Brain Transketolase After Conversion to the Apo Form
Authors: Jeyasingham Marina D. , Pratt Oliver E. , Thomson Allan D. , Shaw George K. 
Published: July 2010

AbstractThe stability of rat brain transketolase, whether measured at 37 or 0°C, was reduced after conversion to the apo form by removal of thiamine diphosphate, as shown by a decline in the activity recovered when assayed in the presence of thiamine diphosphate. Both the shape of the breakdown curve and the failure to recover the full activity, even after incubation with thiamine diphosphate, showed that the breakdown of the apotrans‐ketolase was complex. The initial rate of breakdown of the apoenzyme was sharply pH dependent, being minimal at 37°C at a pH value of 7.6, close to that likely to exist in vivo. The rate rose sharply with deviation of the pH in either direction. The stability of the enzyme on storage at 0°C showed a similar pattern of pH dependence, provided that allowance is made for temperature effects on dissociation constants. These findings provide further support for the hypothesis that differences in brain transketolase may play a part in the etiology of Wernicke‐Korsakoff's syndrome.
Detection and Characterization of Some New Basic Proteins in Chicken Postsynaptic Densities
Authors: Yang Zan Wei , Babitch Joseph A. 
Published: July 2010

AbstractChicken brain postsynaptic density (PSD) polypeptides, obtained by treating synaptosomes with 0.5% Triton X‐100 and then further purified on a sucrose gradient, are demonstrated to contain four basic proteins of 76K (pi > 9.2), 58K (pi 8.1–8.8, heterogeneous), 40K (pI 9.0), and 24K (pi 8.9). Nonequilibrium pH gradient‐sodium dodecyl sulfate two‐dimensional gels further reveal six more basic proteins with pI values higher than 9.2: 76K, 52K, 47K, 45K, 36K, and 34K. These basic proteins are a major part of the total chicken PSD polypeptides appearing on the gels. Some of these basic proteins (58K, 52K, 47K, 36K, 24K, and two at 76K) are distinguishable from those of brain mitochondria, the major contaminant. The 40K and 34K proteins may be common mitochondrial polypeptides. The 45K protein is probably a mitochondrial contaminant. A number of proteins including 76K (synapsin I‐like protein) and 58K, along with some other minor ones, can be phosphorylated by endogenous protein kinase(s) in the presence of Ca2+, Mg2 +, and [γ‐32P]ATP. No PSD basic proteins bind Ca2+.
A Protein Modulator Stimulates C Kinase‐Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes
Authors: Kligman Douglas , Patel Jitendra 
Published: July 2010

AbstractWe have identified and partially purified an acidic, heat‐stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+‐dependent phosphorylation of an Mr90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+‐ and phospholipid‐dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mrform of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+‐dependent phosphorylation of both substrates is inhibited by the Ca2+‐binding proteins S‐100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr9K and 13K after limited proteolysis with V8 protease. Two‐dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic iso‐electric points (pI 5.0). MOP enhances Ca2+‐dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate‐polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase‐mediated phosphorylation in the nervous system.
Role of Adenylate Cyclase in Presynaptic α2‐Adrenoceptor‐ and μ‐Opioid Receptor‐Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices
Authors: Schoffelmeer Anton N. M. , Wierenga Eddy A. , Mulder Arie H. 
Published: July 2010

Abstract:Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ionophore A 23187 (10 μM) in the presence of 1.2 mMCa2+. Forskolin (10 μM), 8‐bromo‐cyclic AMP (300 μM), and dibutyryl‐cyclic AMP (300 μM) facilitated both the electrically evoked and A 23187‐induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3‐isobutyl‐1‐methylxanthine (IBMX, 300 μM) and 4‐(3‐cyclopentyloxy‐4‐methoxyphenyl)‐2‐pyrrolidone (ZK 62771, 30 μM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM–1 μM) and the facilitatory effect of phentolamine (0.01–10 μM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8‐bromo‐cyclic AMP. Clonidine (1 μM) reduced and phentolamine (3 μM) enhanced A 23187‐induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 μM) and [D‐Ala2‐D‐Leu5]enkephalin (DADLE, 0.3 μM), like that of the Ca2+antagonist Cd2+(15 μM), on the electrically evoked release of [3H]noradrenaline were not affected by 8‐bromo‐cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187‐induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic μ‐opioid receptors, α2‐adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed‐forward action of cyclic AMP on the secretion process.
Cerebrovascular Permeability Coefficients to Sodium, Potassium, and Chloride
Authors: Smith Quentin R. , Rapoport Stanley I. 
Published: July 2010

Abstract:CSF and regional brain concentrations of42K,22Na,36Cl, and [14C]mannitol were determined 3–45 min after intravenous injection of the tracers in pentobarbitalanesthetized rats. Rapid influx of36Cl and22Na into ventricular CSF immediately established concentration gradients from CSF to brain extracellular fluid. The CSF contribution to brain uptake of tracers was greatest in periventricular brain regions, where brain36Cl concentrations were up to ninefold higher than concentrations in regions distant from ventricular CSF. Acetazolamide (20 mg kg−1i.p.), an inhibitor of CSF formation, decreased36Cl uptake into CSF and into periventricular brain regions but not into frontal cortex.36Cl uptake into brain was unidirectional for 10 min after intravenous injection, and, during that period, diffusion from ventricular CSF did not contribute to uptake in the frontal cortex. Therefore, cerebrovascular permeability coefficients could be calculated from tracer concentrations in frontal cortex at 10 min and equaled, in cm s−1, 13.5 × 10−1for42K, 1.4 × 10−7for22Na, 0.9 × 10−7for36Cl, and 1.5 × 10−7for [14C]mannitol. The low cerebrovascular permeabilities to K, Na, and Cl, comparable to those of some cell membranes, and the permselectivity (K >> Na > Cl) suggest that a signficant fraction of ion transport across cerebral capillaries is transcellular, i.e., across the endothelial cell membrane.
Activation of the Voltage‐Sensitive Sodium Channel by a β‐Scorpion Toxin in Rat Brain Nerve‐Ending Particles
Authors: Bablito Joseph , Jover Emmanuel , Couraud François 
Published: July 2010

Abstract:Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of β‐scorpion toxin (β‐ScTx) on this preparation has not yet been described. In this report we show that a β‐ScTx induced an increase in22Na+uptake through synaptosomal voltage‐sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between β‐ScTx and veratridine, as is the case for α‐scorpion toxin (α‐ScTx) and veratridine. The effects of α‐ and β‐ScTx were additive and the concentration‐effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. β‐ScTx was shown to inhibit the uptake and to stimulate the release of [3H]γ‐aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltagesensitive sodium channels induced by the binding of β‐ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.
Highly Sensitive Immunoassay for Rat Brain‐Type Creatine Kinase: Determination in Isolated Purkinje Cells
Authors: Kato Kanefusa , Suzuki Fujiko , Shimizu Atsuko , Shinohara Haruo , Semba Reiji 
Published: July 2010

Abstract:Ultrasensitive enzyme immunoassay method for the measurement of rat brain‐type creatine kinase BB (CK‐BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK‐BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab' fragments labeled with β‐D‐galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK‐B), showing about 10% cross‐reactivity with CK‐MB, but it did not cross‐react with CK‐MM and neuron‐specific γγ enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK‐BB, being sufficiently sensitive for the measurement of CK‐B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK‐B in a single Purkinje cell was 1.64 pg. The CK‐B concentration in rat cerebellum (about 22 μg/mg protein) was about twofold higher than that (about 13 μg/mg protein) in the cerebrum. High levels (> 5 μ/mg protein) of CK‐B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK‐B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.
Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain
Authors: Colon Arlene D. , Plaitakis Andreas , Perakis Antonis , Berl Soll , Clarke Donald D. 
Published: July 2010

Abstract:Glutamate dehydrogenase (GDH) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low‐speed pellet (480gfor 10 min). Approximately 60% of the high‐speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similarKmvalues for the two GDH fractions for the substratesμ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences inKmvalues for NADH and NADPH. Although the allosteric stimulation by ADP and L‐leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mMADP, with apparentKivalues of 18.5 and 9.0 μMGTP for the soluble and particulate fractions, respectively. The HIll plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45°C and pH 7.4 in 50 mMphosphate buffer.