bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the Interaction of Ca2+Ions with Some Fractions of the Neurospecific S‐100 Protein
Authors: Starostina M. V. , Malup T. K. , Sviridov S. M.
Published: October 2006

Abstract:Fractions of neurospecific S‐100 protein were purified from bovine brain and their physicochemical properties were studied. Conformational changes caused by the binding of calcium to S‐100 protein fractions were detected by means of differential and fluorescence spectroscopy. Fractions demonstrating opposite shifts of their spectra also differ in the distribution in double‐phase system. The number of calcium‐binding centers and their association constants were determined by means of equilibrium dialysis and gel filtration. The nature of the differences in the interaction of various S‐100 protein fractions with calcium is discussed.
Evidence that Lithium Alters Phosphoinositide Metabolism: Chronic Administration Elevates Primarily d‐myo‐Inositol‐1‐Phosphate in Cerebral Cortex of the Rat
Authors: Sherman William R. , Leavitt Alan L. , Honchar Michael P. , Hallcher Loretta M. , Phillips Bruce E.
Published: October 2006

Abstract: The administration of LiCl (3.6 mequiv./kg/day) to adult male rats for 9 days results in an increase in the cerebral cortex level of myo‐inositol‐1‐phosphate (M1P) to 4.43 ± 0.52 mmol/kg (dry weight) compared with a control level of 0.24 ± 0.02 mmol/kg. This establishes that the previously observed acute effect of lithium on M1P (Allison et al., 1976) is both prolonged and augmented by repeated doses of lithium. Larger doses of LiCl over a 3–5 day period result in even larger increases in M1P and a 35% decrease in myoinositol. In each case, 90% of the increase is due to the d‐enantiomer, evidence that lithium is largely producing this effect via phospholipase C‐mediated phosphoinositide metabolism. Data are presented showing that lithium is an uncompetitive inhibitor of the hydrolysis of both d‐ and l‐M1P by M1P’ase.
Purification and Mode of Action of Synexin: A Protein Enhancing Calcium‐Induced Membrane Aggregation
Authors: Morris Stephen J. , Hughes John M. X. , Whittaker Victor P.
Published: October 2006

Abstract: Synexin, a protein from the cytosol of the adrenal medulla, selectively increases the ability of Ca2+ to aggregate chromaffin granules and other membrane‐bound particles. The ability of synexin to self‐aggregate in the presence of Ca2+ can be employed in the purification of the protein by monitoring purification with parallel assays that utilize the aggregation of both chromaffin granule membranes and phosphatidylserine liposomes. It is shown that the enhancement of the Ca2+‐induced aggregation of both liposomes and chromaffin granule membranes is a property associated with a 47,000 Mr protein. Trypsin inactivated synexin. We found that if granule membranes were well washed after trypsin treatment, they were still excellent substrates for synexin aggregation. This finding cannot be explained by extinction changes owing to synexin self‐aggregation. The 47,000 Mr protein enhanced Ca2+ aggregation of phosphatidylserine liposomes containing up to 40% phosphatidylcholine, liposomes made from lipids extracted from chromaffin granule membranes, and trypsin‐treated chromaffin granule membranes, thus suggesting that synexin activity in vivo may be independent of specific membrane proteins but dependent on the presence of acidic phospholipids in the membrane.
Tissue, Subcellular, and Submitochondrial Distributions of Semidehydroascorbate Reductase: Possible Role of Semidehydroascorbate Reductase in Cofactor Regeneration
Authors: Diliberto Emanuel J. , Dean Grace , Carter Charles , Allen Pamela L.
Published: October 2006

Abstract: The immediate product of ascorbate oxidation coupled to dopamine‐β‐hydroxylation is not dehydroascorbate, as previously thought, but rather semidehydroascorbate. For this reason, the possible participation of the enzyme semidehydroascorbate reductase (SDR) in cofactor regeneration was investigated. In the adrenal medulla, the primary subcellular localization of this reductase was shown to be in the mitochondria. Submitochondrial fractionation studies indicated that SDR is an outer membrane protein. Thus, although dopamine‐β‐hydroxylase and SDR have different subcellular localizations, a physiological role for SDR in β‐hydroxylation still appears plausible through reduction of cytosolic semidehydroascorbate. The specific activities of SDR in various rat and guinea pig tissues appear to parallel their ascorbate contents, suggesting a similar participation of SDR in ascorbate metabolism in other tissues.
Substance P Hydrolysis by Human Serum Cholinesterase
Authors: Lockridge Oksana
Published: October 2006

Abstract: Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear was Arg1‐Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min−1 mg−1 of protein, which corresponded to a turnover number of 0.6 min−1. A second cleavage yielded Lys3‐Pro4. A third cleavage occurred at the C‐terminal, where the amide was removed from Met11 to yield a peptide containing residues 5–11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropyl‐fluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.
Microvessels from the Spinal Cord: Isolation Procedure and Characterization of the Fraction
Authors: Joó Ferenc , Dux Ernö , Szücs Attila
Published: October 2006

Abstract: A simple method for the preparation of microvessels from the spinal cord is described. The fraction being enriched in the microvessels is characterized by morphological techniques and compared biochemically with the original suspension. The method makes possible detailed studies on the metabolism of microvessels and can be complementary to procedures that have been developed for separating the capillaries from other brain tissues.
Norepinephrine Metabolism in Man Using Deuterium Labeling: Turnover of 4‐Hydroxy‐3‐Methoxymandelic Acid
Authors: Mårdh Göran , Sjöquist Birgitta , Änggård Erik
Published: October 2006

Abstract: 4‐Hydroxy‐3‐methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 μmol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography‐mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 × 10% (mean ± SD). No conversion of HMMA t o 4‐hydroxy‐3‐methoxyphenyl glycol (HMPG) occurred. The biological half‐life of HMMA was 0.54 ± 0.22 h. The apparent volume of distribution was 0.36 ± 0.11 L/kg. The production rate or body turnover was 1.27 ± 0.51 μmol HMM/h and urinary excretion rate was 0.82 ± 0.22 μmol/h. These results show that HMMA is turning over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.
On the Role of Calcium Ions in the Regulation of Glycogenolysis in Mouse Brain Cortical Slices
Authors: Ververken D. , Van Veldhoven P. , Proost C. , Carton H. , De Wulf H.
Published: October 2006

Abstract: Using mouse brain cortical slices, we investigated the relative roles of cyclic AMP and of calcium ions as the intracellular messengers for the activation of glycogen phosphorylase (EC 2.4.1.1; α‐1,4‐glucan:orthophosphate glucosyltransferase) induced by noradrenaline and by depolarization. Activation of phosphorylase by 100 μM noradrenaline is mediated by β‐adrenergic receptors and does not require the copresence of adenosine. The role of the concomitant small increase in cyclic AMP is questioned. Short‐term treatment with EGTA or LaCl3 abolishes the noradrenaline activation of phosphorylase, pointing to a critical role of extracellular calcium. Depolarization by 25 mM K+ or 100 μM veratridine produces a rapid and large (fourfold) activation of phosphorylase. Only veratridine increases the cyclic AMP levels; exogenous adenosine deaminase essentially blocks this cyclic AMP accumulation but not the phosphorylase activation. A halfmaximal activation of phosphorylase occurs at about 12 mM K+. Addition of EGTA or LaCl3, reduces the effect of both depolarizations to a slight and transient activation of phosphorylase. These results indicate that activation of glycogen phosphorylase by K+ or veratridine occurs by a cyclic AMP‐independent and calcium‐dependent mechanism. The calcium dependency of brain phosphorylase kinase renders this kinase the prime target enzyme for regulation of glycogenolysis by calcium ions.
Effects of the Putative GABAergic Agonists, Muscimol and THIP, upon Local Cerebral Glucose Utilisation
Authors: Kelly Paul A. T. , McCulloch James
Published: October 2006

Abstract: The dose‐dependent effects of two putative γ‐aminobutyric acid (GABA)‐ergic agonists, muscimol and 4,5,6,7‐tetrahydroisoxazolo(4,5‐e)‐pyridin‐3‐ol (THIP), upon local cerebral glucose utilisation in 60 discrete regions of the CNS have been examined in conscious rats using the [14C]2‐deoxyglucose quantitative autoradiographic technique. The intravenous administration of muscimol (0.15‐5 mg/kg) and THTP (1‐10 mg/kg) resulted in a heterogeneous pattern of significantly reduced glucose utilisation throughout the CNS. The regional hierarchy of changes in glucose utilisation was similar for both muscimol and THIP in all regions (with the exception of the superior colliculus), with muscimol being approximately six times more potent in all regions investigated. The regions in which glucose utilisation was extremely sensitive to change, displaying reductions of approximately 40% following muscimol (1.5 mg/kg) or THIP (10 mg/kg) administration, included all layers of the neocortex (frontal, sensory motor, posterior parietal, primary auditory and visual cortices), the lateral portion of the caudate nucleus, and some thalamic nuclei (lateral geniculate body, mediodorsal and ventrolateral nuclei). Regions displaying more modest reductions in glucose utilisation, approximately 20% following muscirnol (1.5 mg/kg) and THTP (10 mg/kg) administration, included most extrapyramidal regions (substantia nigra, pars cornpacta and reticulata, globus pallidus, subthalamic nucleus, medial portion of the caudate nucleus), a number of cortical and subcortical limbic areas (cingulate and olfactory cortices, hippocampus, nucleus accumbens, anterior thalamus), and medial raphe nucleus. In contrast, in a large number of regions (including cerebellum and related nuclei such as the inferior olivary, red and vestibular nuclei, white matter, pontine reticular formation, hypothalamus, lateral habenula and amygdala), there were only minimal (approximately 10%) reductions in glucose utilisation following muscimol (1.5 mg/kg) and THIP (10 mg/kg) administration. In no region of the CNS was a significant increasc in glucose utilisation observed with any concentration of either muscirnol or THIP. The regional distribution of alterations in glucose utilisation following muscimol and THIP administration, which does not correspond to the known topography of GABAergic neurones and receptors, provides a comprehensive description of the functional alterations, as reflected in rates of glucose utilisation, that occur in conscious rats after systemic administration of these two putative GABAergic agonists.
Ascorbic Acid in Neural Tissues
Authors: Kratzing C. C. , Kelly J. D. , Oelrichs B. A.
Published: October 2006

Abstract: Large amounts of ascorbic acid were readily removed from neural tissue by washing with warmed saline solutions. In areas where the original level was highest, such as cortex and cerebellum, a higher percentage was removed than from areas of lower concentration, such as pons‐medulla. The residual level in both types of tissue was similar. During scurvy, the ascorbic acid retained in the guinea pig brain is more readily removed by washing than is that of the normal brain.
Adenosine Receptor Agonists Inhibit K+‐Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes
Authors: Wu P. H. , Phillis J. W. , Thierry D. L.
Published: October 2006

Abstract: The uptake of Ca2+ by a K+‐depolarized rat brain cerebral cortical crude synaptosomal preparation (P2 fraction) was investigated. The characteristics of the Ca2+ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca2+ uptake and neurotransmitter release, as it is generally accepted that K+‐evoked Ca2+ uptake is intimately related to depolarization‐induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine‐evoked inhibition of K+‐evoked Ca2+ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A2‐receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re‐uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K+‐ evoked uptake of Ca2+ by the synaptosomal fraction.
Calcium‐Activated ATPases in Presynaptic Nerve Endings
Authors: Lin Shih‐chia C. , Way E. Leong
Published: October 2006

Abstract: We studied the properties of calcium‐activated ATPases present in preparations of isolated presynaptic nerve ending (synaptosome) and its sub‐fractions from mouse brain. ATPase activity in the preparation was stimulated by Ca2+ and by Mg2+, but not by Na+ and K+, when each was added alone. The substrate specificities were found to be similar. The ATPases hydrolyzed only the high‐energy phosphate bond and similar activity was exhibited for all nucleoside triphosphates tested (ATP, CTP, GTP, UTP). Moreover, the enzymes were insensitive to mitochondrial markers and to ouabain, but were inhibited by La3+. La3+ produced uncompetitive inhibition of Ca2+‐ATPase in intact synaptosomes. Inhibition by La3+ was greatly increased after lysis of the synaptosomes, suggesting that the active sites of the enzymes may be on the cytosolic face of the membranes. The Ca2+‐ATPase activity in synaptosomes was increased by increasing concentrations of external K+, suggesting that Ca2+ influx may be involved. The Ca2+‐ATPase in synaptosomal plasma membranes and synaptic vesicles had higher specific activities than those of intact synaptosomes and were activated, both in the presence and the absence of Mg2+, by Ca2+ concentrations approximating the intracellular level (10−7M). It is concluded that the nonmitochondrial synaptosomal Ca2+‐ATPase may play an important role in the regulation of intracellular Ca2+.
Acidosis‐Induced Enhanced Activity of the Na‐K Exchange Pump in the In Vivo Choroid Plexus: An Ontogenetic Analysis of Possible Role in Cerebrospinal Fluid pH Homeostasis
Authors: Pershing Lynn K. , Johanson Conrad E.
Published: October 2006

Abstract: Changes in cellular [K] and [Na] in the choroidal epithelium (as a reflection of Na‐K pump activity) were analyzed in Sprague‐Dawley rats subjected to acute systemic acidosis. In the lateral and 4th ventricle choroid plexus (CP) of adult rats in which metabolic acidosis was induced for 1 h, cell [K] and [Na] increased and decreased by 35 and 15 mm/kg water, respectively, indicating marked stimulation of the Na‐K exchange pump in the CSF‐facing membrane; in contrast, this striking response of the CP to acidosis could not be elicited in immature animals (1 week old). Since the effects of respiratory acidosis on CP cell [K] and [Na] were similar to those of metabolic acidosis, the reduction in plasma pH (rather than in [HCO3]) is likely the mechanism underlying the enhanced turnover of Na and K across the CP in adults. The concentration of Na and K in the cerebral cortex, medulla, and CSF was generally not altered during acute acid‐base distortions in both mature and immature animals. The striking difference in the response of CNS tissue protected by the blood‐CSF barrier (i.e., CP) and the blood‐brain barrier (BBB) to systemic acidosis emphasizes a unique role, presumably homeostatic, for the plexus. Since propranolol substantially attenuated the acidosis‐induced changes in choroidal cell [K] and [Na], it is possible that there is β‐receptor modulation of the Na,K‐ATPase (Na‐K pump) in the CP. We postulate that the generally observed enhanced electropositivity in the CSF in systemic acidosis is brought about, at least in part, by facilitation of Na‐K pumping in the CP, although induced changes in membrane permeability may also be a factor.
Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties
Authors: Pettersson Hans , Tottmar Olof
Published: October 2006

Abstract: Kinetic studies suggested the presence of several forms of NAD‐dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low‐ and high‐Km activities with acetaldehyde as well as the substrate‐specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low‐Km activity was also present in the cytosol (<20%). The low‐Km activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two Km values were obtained with both acetaldehyde (0.2 and 2000 μm) and 3,4‐dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μm), and one Km value with succinate semialdehyde (5 μm). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low‐Km activity with acetaldehyde and DOPAL, but not the high‐Km activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg2+ and Ca2+ in phosphate buffer. The low‐ and high‐Km activities with acetaldehyde showed different pH optima in pyrophosphate buffer.
Uptake of Adenosine by Isolated Rat Brain Capillaries
Authors: Wu P. H. , Phillis J. W.
Published: October 2006

Abstract: Adenosine uptake by isolated rat brain capillaries is a carrier‐mediated, temperature‐ and pH‐sensitive process. The Km value for adenosine uptake is 4.74 μm and the Vmax is 21.7 picomol/mg protein/10 min. This is a high‐affinity uptake system that can be cross‐inhibited by several nucleosides and by the adenosine analogs tubercidin and 5′‐deoxyadenosine. The uptake is very sensitive to inhibition by papaverine, hexobendine, and dipyridamole. These results confirm the existence of a nucleoside transport system associated with the blood‐brain barrier observed during in vivo studies.
Deamination of 5‐Hydroxytryptamine by Both Forms of Monoamine Oxidase in the Rat Brain
Authors: Fowler Christopher J. , Tipton Keith F.
Published: October 2006

Abstract: K
m and Vmax values of monoamine oxidase (MAO) A and B towards 5‐hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l‐deprenyl. Km values of 178 and 1170μm, and Vmax values of 0.73 and 0.09 nmol·mg protein−1·min−1 towards 5‐hydroxytryptamine were found for MAO‐A and ‐B, respectively. The K1 for 5‐hydroxytryptamine as a competitive inhibitor of β‐phenethylamine oxidation by MAO‐B was found to be 1400 μm. The significance of these findings is discussed.
Postnatal Action of Growth and Thyroid Hormones on the Retarded Cerebral Myelinogenesis of Snell Dwarf Mice (dw)
Authors: Noguchi Tetsuya , Sugisaki Tetsuro , Tsukada Yasuzo
Published: October 2006

Abstract: Snell dwarf mice (dw) showed a lower CNPase activity (59% of the normal controls) only in the cerebrum among different parts of the CNS, and a strikingly reduced level of spontaneous locomotion activity with an indistinct diurnal periodicity in a 24‐h record at 40 days of age. Daily administration of bGH and T4 to the dwarfs during the first 40 days of postnatal life restored CNPase activity to the level of the normal controls, and was accompanied by normalization of the pattern of spontaneous locomotion activity. Daily administration of bGH alone also restored CNPase activity and spontaneous locomotion, but to a lesser extent. The daily administration of thyroid stimulating hormone (TSH) alone, however, failed to restore CNPase activity, in spite of the fact that the thyroid glands of the TSH‐treated dwarfs were indistinguishable from the normal controls in organization and appearance. These results indicate that the restoration of both the retarded myelinogenesis and abnormal behavior of the Snell dwarf mice might essentially depend upon GH levels and the synergistic effects of T4.
Metabolic Fate of 14C‐Labeled Glutamate in Astrocytes in Primary Cultures
Authors: Yu Albert C. , Schousboec Arne , Hertz Leif
Published: October 2006

Abstract: The metabolic fate of L‐[U‐14C]‐ and L‐[l‐14C]glutamate was studied in primary cultures of mouse astrocytes. Conversion of the uniformly labeled compound to glutamine and aspartate was followed by determination of specific activities after dansylation with [3H]dansyl chloride and subsequent thin layer chromatography of the dansylated amino acids. Metabolic fluxes were calculated from the alterations of specific activities and the pool sizes, which were likewise measured by a dansylation method. Formation of 13CO2 from [l‐14C]glutamate was determined by the trapping of CO2 in hyamine hydroxide in a gas‐tight chamber, which is, in the known absence of glutamate decarboxylase activity in the cultured astrocytes, an unequivocal expression of the metabolic flux via α‐ketoglutarate to CO2 and succinyl‐CoA. The metabolic fluxes determined by these procedures amounted to 2.4 nmol/midmg protein for glutamine synthesis, 1.1 nmolimidmg protein for aspartate production, and 4.1 nmollmidmg protein for formation and subsequent decarboxylation of α‐ ketoglutarate. The latter process was unaffected by virtually complete inhibition of glutamate‐oxaloacetic transaminase with aminooxyacetic acid, indicating that the formation of α‐ketoglutarate occurs as an oxidative deamination rather than as a transamination. This suggests that the formation of α‐ketoglutarate from glutamate represents a net degradation, not an isotopic exchange.
Molecular Properties of Drosophila Acetylcholinesterase
Authors: Zingde S. , Rodrigues V. , Joshi S. M. , Krishnan K. S.
Published: October 2006

Abstract: Two distinct classes of acetylcholinesterase (AChE) from the fruit fly Drosophila melanogaster are reported: a soluble species that shows heterogeneity of forms and a particulate species. The subunit composition of the particulate enzyme was studied using the active site label [3H]diisopropylfIuorophosphate. Comparison of the electrophoretic patterns on nondenaturing gels using the activity stain and the active site label shows that the label is specific to AChE. The smallest active site‐containing subunit of the enzyme is a monomer of —60,000 daltons MW. Two such units are linked by disulphide bonds to produce a dimer of about 110,000 daltons. Another monomeric form of MW —64,000 daltons, although present, does not participate in the dimerisation. The particulate enzyme when solubilised exists as a 9–10S species as determined by sucrose gradient centrifugation. This species has a MW > 200,000, as shown by its behaviour on a coarse‐bead Sephadex‐G200 column. Electrophoretic analysis suggests a MW of nearly 250,000 daltons for this form. Thus, this species is likely to be a tetramer. One possibility is that this tetramer is made up of two units of 64,000 daltons each and a dimer of 110,000 daltons. Preliminary data on mutant enzymes that support such a possibility are also presented.
Passive Avoidance Training Increases Fucokinase Activity in Right Forebrain Base of Day‐Old Chicks
Authors: Lössner Bernd , Rose Steven P. R.
Published: October 2006

Abstract: Fucokinase (EC 2.7.1.52) activity was estimated in supernatants of homogenate from day‐old chick forebrain. Enzyme kinetic studies gave a Km of 4.5 × 10‐6M and Vmax of 3.72 nmol fucose converted into fucose‐1 phosphate/mg prot/h. The pH optimum was 7.5. The enzyme is thus considerably more active than was reported for other species and tissues. There were no differences in enzyme activity between the four forebrain regions studied. One hour after chicks were trained on a one‐trial passive avoidance learning paradigm, enzyme activity in the right forebrain base increased 14% over control values (p < 0.02). The 11.3% increase in activity in the left forebrain base and 10.3% increase in the left roof were not statistically significant. The relationship of this change to the increased fucose incorporation into glyco‐proteins known to occur over a similar time period and the significance of the lateralization of the increase are discussed.
The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism
Authors: Butler Madeline , Morell Pierre
Published: October 2006

Abstract: In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP‐ethanolamine and its transfer to diglyceride, or by base‐exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base‐exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L‐[G‐3H] serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L‐[U‐14C]serine and [13H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole‐brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.
Effects of Temperature on Arachidonic Acid‐Induced Cellular Edema and Membrane Perturbation in Rat Brain Cortical Slices
Authors: Chan Pak Hoo , Fishman Robert A. , Chen Sylvia , Chew Susan
Published: October 2006

Abstract: The effects of temperature on arachidonic acid‐induced cellular edema in the first cortical brain slices of rats were studied. Incubation of the cortical slice in arachidonic acid at 37°C induced cellular swelling, and increased intracellular Na+ and lactic acid contents concomitant with decreased intracellular K+ When the incubation temperature was reduced these changes were reduced in severity. The uptake of [3H]arachidonic acid in cortical slices was temperature‐dependent. The incorporation of [3H]arachidonic acid into various lipid fractions was further studied by HPLC. The majority of [3H]arachidonic acid was incorporated into triacylglycerol and phosphatidylinositol (PI), but the incorporation of [3H]arachidonic acid into PI was temperature‐dependent, unlike that into other phospholipids and neutrolipids. Further, cortical (Na++ K+)‐ATPase activity was inhibited whereas its subunit K+‐activated p‐nitrophenyl‐phosphatase was activated by arachidonic acid at various incubation temperatures. The effects of arachidonic acid on these enzymes is similar to that of thimerosal, a lipid removal agent. These data suggest that both temperature and arachidonic acid play an important role in the development of cellular edema associated with membrane perturbation and inactivation of (Na++ K+)‐ATPase activity.
Catecholamine‐Derived Tetrahydroisoquinolines: O‐Methylation Patterns and Regional Brain Distribution Following Intraventricular Administration in Rats
Authors: Collins Michael A. , Origitano Thomas C.
Published: October 2006

Abstract: The metabolism of 6,7‐dihydroxy (catecholic) ‐1,2,3,4‐tetrahydroisoquinoIines (TIQs) is of interest because the heterocyclic substances may form in mammals normally or during certain diseases via condensations of catecholamines (CAs) with aldehydes or α‐keto acids. With a specific capillary gas chromatography procedure and confirmatory liquid chromatographic assays, we have determined the structural isomers and relative amounts of mono‐O‐methylated (phenolic) TIQ metabolites in several rat brain regions 40 min following the acute intracerebroventricular injection of four structurally related catecholic TIQs. In sharp contrast with the established selective m‐O‐methylation of dopamine (DA) by catechol‐O‐methyltransferase in brain, the two simple TIQs derived from DA produced predominantly or even exclusively the metabolic isomer arising from methylation of the original p‐hydroxyl group (7‐O‐methylation). In three catecholaminergic brain regions examined, the 7‐O‐methyl isomer was the only detectable phenolic metabolite of (±) salsolinol‐1‐carboxylic acid (a condensation product of DA and pyruvic acid) and, as first noted by Bail et al. (1980), constituted 95% of the two possible isomeric mono‐O‐methyl metabolites of (±) salsolinol (TIQ derivative of DA and acetaldehyde). Though less, the 7‐O‐methyl isomers still were a significant proportion (40–55%) of the two mono‐O‐methylated metabolites of (±) 4‐hydroxy‐desmethylsalsolinol (a TIQ derived from norepinephrine and formaldehyde), or of the DOPA/acetaldehyde‐derived TIQ, (cis) salsolinol‐3‐carboxylic acid. In the time frame of the study, all four administered TIQs showed higher levels in hypothalamus than in striatum or hippocampus, with the two carboxylated alkaloids displaying the greatest differences. However, for a given TIQ, the proportion present as mono‐O‐methyl metabolite(s) and the O‐methyl isomer distribution did not differ across the three brain regions, indicating that O‐methylation involved a single homogenous enzyme that was not saturated in vivo at the concentrations attained by the heterocyclic catechols. The most notable findings, that of exclusive methylation on the original p‐hydroxyl group of a physiologically relevant CA derivative, and an O‐methylation pattern that appeared dependent on the substituents on the heterocyclic ring, are explained on the basis of an important hydrophobic binding site in catechol‐O‐methyltransferase in vivo.
Multiple Molecular Forms of Enkephalins in the Guinea Pig Hippocampus
Authors: Hoffman Douglas W. , Altschuler Richard A. , Gutierrez Joanne
Published: October 2006

Abstract: The combined techniques of HPLC and radioimmunoassay were used to identify and quantitate enkephalin‐related peptides in the guinea pig hippocampus. Both met‐ and leu‐enkephalin were identified, in approximately a 2:1 ratio, as well as a third enkephalin‐like molecule that is neither met‐ nor leu‐enkephalin. The third enkephalin elutes earlier than met‐ or leu‐enkephalin from a reversed‐phase column, has a molecular weight similar to the other enkephalins, and is as active as these enkephalins are in inhibiting binding of labeled opiates to rat brain membranes. All regions of the hippocampus (dentate gyrus, CA1–2, CA3–4, and subiculum) contain all three immunoreactive peptides. Immunocytochemical techniques, using antisera raised against met‐enkephalin, show with one antiserum immunoreactivity in the granule cell‐mossy fiber system, and with the other scattered immunoreactive cells mostly in the CA2 region. Enkephalins are not confined to the mossy fiber system, as previously suggested, but may be a component of another hippocampal innervation.
[3H]Bicuculline Methochloride Binding to Low‐Affinity γ‐Aminobutyric Acid Receptor Sites
Authors: Olsen Richard W. , Snowman Adele M.
Published: October 2006

Abstract: The binding of [3H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [3H]γ‐aminobutyric acid ([3H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris‐citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN−. An apparent single class of binding sites for [3H]BMC (KD= 30 nM) was observed in 0.1 M SCN− for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The Bmax was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [3H]GABA. Frozen membranes, however, showed decreased levels of [3H]BMC binding with no decrease or an actual increase in [3H]GABA binding sites. [3H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [3H]GABA binding. Kinetics experiments with [3H]GABA binding revealed that low‐ and high‐affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high‐affinity [3H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low‐affinity [3H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist‐ and antagonist‐preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [3H]BMC may relatively selectively label low‐affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [3H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.
Cellular Localization of Gangliosides in the Developing Mouse Cerebellum: Analysis Using the Weaver Mutant
Authors: Seyfried T. N. , Miyazawa N. , Yu R. K.
Published: October 2006

Abstract: The distribution of gangliosides was studied in the weaver (wv/wv) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the +/wv heterozygotes. By correlating changes in ganglioside composition with the well‐documented histological events that occur during cerebellar development in the normal (+/+), heterozygous (+/wv), and weaver (wv/ wv) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside GM1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of GM1, which may result from altered metabolism, occurred in the adult wvlwv mice. GD3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. GD1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas GT1a appeared enriched in Purkinje cell synaptic spines. A possible fucose‐containing ganglioside was quantitated only in the wvlwv mice. Ganglioside GT1b became enriched in granule cells during synaptogenesis, whereas GQ1b became enriched in these cells after synaptogenesis. The concentrations of GT1b and especially GQ1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.
Phosphorylation of B‐50 Protein by Calcium‐Activated, Phospholipid‐Dependent Protein Kinase and B‐50 Protein Kinase
Authors: Aloyo Vincent J. , Zwiers Henk , Gispen Willem Hendrik
Published: October 2006

Abstract: B‐50 is a brain‐specific phosphoprotein, the phosphorylation state of which may play a role in the regulation of (poly)phosphoinositide metabolism. Several kinases were tested for their ability to phosphorylate purified B‐50 protein. Only calcium‐activated, phospholipid‐dependent protein kinase (kinase C) and B‐50 protein kinase were able to use B‐50 protein as a substrate. Furthermore, kinase C specifically phosphorylates B‐50 when added to synaptic plasma membranes. We further characterized the sensitivity of kinase C and B‐50 kinase to ACTH (and various fragments), phospholipids, chlorpromazine, and proteolytic activation. Since the sensitivities of both kinases were similar, we conclude that B‐50 protein kinase is a calcium‐dependent, phospholipidstimulated protein kinase of the same type as kinase C.
Synaptic Chemistry Associated with Aberrant Neuronal Development in the Reeler Mouse
Authors: Singer Harvey S. , Weaver Debbie , Tiemeyer Michael , Coyle Joseph T.
Published: October 2006

Abstract: Pre‐ and postsynaptic neurochemical markers for several afferent and intrinsic neuronal systems were measured in the mouse mutant, reeler. In the neocortex of the reeler, the relative positions of the polymorphic and pyramidal cells were inverted but this was not associated with alterations in the content/mg protein of synaptic markers for noradrenergic [tyrosine hydroxylase (TH), norepinephrine (NE), NE uptake], cholinergic [choline acetyltransferase (ChAT), quinuclidinyl benzilate (QNB) binding], γ‐aminobutyric acid (GABA)ergic (glutamate decarboxylase, GABA uptake, GABA receptors, GABA) or glutamatergic (glutamate uptake, receptors, glutamate) neurons. The laminar distributions of the hippocampal neurons were disrupted and associated with mild hypoplasia; consistent with this alteration, the content/mg protein of some GABAergic (GABA uptake) and glutamatergic (glutamate receptors) markers were slightly increased. The reeler cerebellum was characterized not only by misalignment of neurons but also by a marked loss of granule cells. Commensurate with the degree of cerebellar hypoplasia, the total amount of glutamate content, [3H]l‐glutamate uptake activity, [3H]muscimol, and [3H]QNB ligand binding were reduced in the reeler cerebellum. In contrast, presynaptic markers for the noradrenergic (TH, NE) climbing fibers and the cholinergic (ChAT) mossy fibers were significantly increased/mg protein but their total content/cerebellum was near normal. Our data support suggestions that cerebellar granule cells use glutamate as their neurotransmitter and contain GABA and cholinergic receptors. The findings also suggest that misplaced cortical and cerebellar neurons retain normal neurochemical characteristics and that the morphologic alterations do not markedly affect the quantitative development of aminergic afferent systems.
Suriclone: A New Cyclopyrrolone Derivative Recognizing Receptors Labeled by Benzodiazepines in Rat Hippocampus and Cerebellum
Authors: Blanchard Jean‐Charles , Julou Louis
Published: October 2006

Abstract: Suriclone (RP 31,264), like zopiclone (RP 27, 267), belongs to the family of cyclopyrrolones and is chemically entirely different from the benzodiazepines (BZDs). However, it possesses a pharmacological profile close to that of the BZDs and proved to be useful in therapeutics as an anxiolytic agent. In the present paper it is shown that suriclone possesses a high affinity for flunitrazepam binding sites and that tritiated suriclone binds specifically with high affinity in rat hippocampus (Kd= 0.44 ± 0.03 nM) and rat cerebellum (Kd= 0.53 ± 0.12 nM. Further, suriclone binding sites are recognized by BZDs or zopiclone, similarly in the two regions. The affinities of four BZD derivatives–nitrazepam, flunitrazepam, diazepam, and chlordiazepoxide–are similar for suriclone and flunitrazepam binding sites. Suriclone binding sites are, like flunitrazepam sites, protected from thermal inactivation by γ‐aminobutyric acid (GABA) (10 μM), but only flunitrazepam binding is enhanced by GABA. It could be postulated from this that suriclone interacts with a subpopulation of receptors that might be modulated differently from flunitrazepam binding sites. Our results indicate that suriclone could be a new probe for investigating the so‐called BZD receptors.
Complex Inhibition of [3H]Imipramine Binding by Serotonin and Nontricyclic Serotonin Uptake Blockers
Authors: Sette M. , Briley M. S. , Langer S. Z.
Published: October 2006

Abstract: Tricyclic antidepressant drugs inhibit [3H]imipramine binding to the rat brain cortex in a competitive manner, giving linear Hofstee plots and Hill coefficients of approximately 1.0. Serotonin, the only neurotransmitter to inhibit [3H]imipramine binding, does so in a complex manner, exhibiting a Hill coefficient of 0.40‐0.50. Nontricyclic inhibitors of serotonin uptake such as fluoxetine, paroxetine, norzimelidine, and citalopram inhibit [3H]imiprarnine binding in the same complex manner as serotonin. These results are interpreted as suggesting that [3H]imipramine binds to a site associated with the serotonin uptake system but different from either the substrate recognition site for serotonin or the site of action of the nontricyclic inhibitors of neuronal uptake of serotonin.
Formation of a Persistent Inhibitory State of Brain Adenylate Cyclase by GTP Analogs
Authors: Yamamoto Toshifumi , Shimizu Hirotoshi
Published: October 2006

Abstract: Addition of 10μM guanyl‐5′‐ylimidodiphos phate at 30° or 0° to guinea pig brain particulates instantaneously evoked nearly 50% inhibition of adenylate cyclase activity as determined after removal of the GTP analog by washing of the particulates. The inhibitory state, once formed, persisted for at least 60 min as long as the preparation was kept either in a medium devoid of the analog (0‐30°) or in its presence at 0°. During incubation at 30° in the presence of the analog, however, the inhibited or nontreated enzyme showed a gradual increase in enzyme activity. Both the inhibitory and the activating effects of the analog were saturable, with a half‐maximal concentration of about 1.0 μM, and were antagonized by simultaneous addition of GTP, GDP, and GMP (in decreasing order). The persistently inhibited enzyme enabled the detection of marked stimulation by norepinephrine and histamine, whereas these amines showed only marginal stimulation of the enzyme before treatment with the analog. Formation of such a persistent inhibitory state appears to be specific to brain cyclase.
Hippocampal Electrical Activity and γ‐Aminobutyrate Metabolism in Brain Tissue Following Administration of Homocysteine
Authors: Dewhurst Ian C. , Hagan James J. , Morris Richard G. M. , Griffiths Roger
Published: October 2006

Abstract: The concentration of γ‐aminobutyrate (GABA) and the activity of glutamate decarboxylase and GABA‐transaminase were measured in extracts of mouse brain before the onset and during the course of generalized seizures induced by systemic administration of homocysteine thiolactone. The results indicate that whole brain GABA metabolism is unaffected by subconvulsive and convulsive doses of homocysteine at all stages of the generalized seizure. Electroencephalographic monitoring of rat brain electrical activity via hippocampal electrode implantation allowed the course of homocysteine‐induced seizures to be followed and afforded a means of quantifying such seizures.
On the Significance of Tyrosine for the Synthesis and Catabolism of Dopamine in Rat Brain: Evaluation by HPLC with Electrochemical Detection
Authors: Westerink B. H. C. , Wirix E.
Published: October 2006

Abstract: A chemical assay of tyrosine (Tyr) in nervous tissue is described. The method is based on a rapidly performed isolation of Tyr on small Sephadex G 10 columns, followed by reverse‐phase HPLC in conjunction with amperometric detection. The method permitted the additional quantification of 3,4‐dihydroxyphenylalanine, dopamine (DA), and its acidic metabolites. The method was applied to a study of the effects of γ‐butyrolactone, haloperidol, haloperidol in combination with amfonelic acid, morphine, NSD 1015, and tyrosine methylester on the concentration of Tyr in the striatum, frontal cortex, hypothalamus, and cerebellum of rat brain. The effect of tyrosine methylester on DA and its acidic metabolites was investigated in the striatum and frontal cortex. Morphine and NSD 1015 were found to increase Tyr levels. γ‐Butyrolactone, haloperidol, and haloperidol combined with amfonelic acid decreased the Tyr content in a manner related to their stimulatory effect on DA biosynthesis. These effects were restricted to DA‐rich brain areas. It was concluded that during conditions of increased DA biosynthesis, the Tyr pool still possesses a considerable reserve capacity. The results bring into question the concept that brain Tyr is an important additional factor controlling catechol synthesis during increased tyrosine hydroxylase activity.
Effect of Electroconvulsive Shock on the Uptake and Release of Noradrenaline and 5‐Hydroxytryptamine in Rat Brain Slices
Authors: Minchin M. C. W. , Williams J. , Bowdler J. M. , Green A. R.
Published: October 2006

Abstract: The uptake and release of [3H]noradrenaline and [3H]‐5‐hydroxytryptamine (5‐HT) were studied in cerebral cortex slices from rats 30 min and 24 h after a single electroconvulsive shock (ECS) and 24 h after a series of five shocks given over 10 days. Both the Km and Vmax for 5‐HT uptake were lower than controls 24 h after a single ECS, whereas after 5 ECS spread over 10 days both parameters remained depressed, though only the fall in Vmax was significant. Noradrenaline uptake was not altered after a single ECS, but the Vmax and Km were elevated following chronic ECS treatment. Neither ECS treatment schedule had any effect on the potassium‐stimulated release of either transmitter. It is possible that the changes in monoamine uptake seen following ECS are an adaptive response to alterations in the synaptic cleft concentration of these transmitters.