bluuebunny/crossref_metadata_2025 huggingface dataset:
Cerebral Phospholipid Content and Na+, K+‐ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil
Authors: Enseleit Wolfram H. , Domer Floyd R. , Jarrott David M. , Baricos William H.
Published: October 2006

Abstract: Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+, K+‐ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane‐bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to <50% of control values but returned to near‐normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15–45 min). Na+, K+‐ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30–240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (‐29%, p ≤ 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (‐51% at 60 min, p ≤ 0.05) and remained below preischemic values even after 180 min of reperfusion. The activity of cerebral Na+, K+‐ATPase increased to values slightly above preischemic values following reperfusion for either 60 or 180 min after 60 min of ischemia. These data show that major differences exist in the responses of neural and nonneural tissues to ischemia and raise important questions concerning the role of changes in membrane structure and function in irreversible ischemic injury.
Alterations in the Fatty Acid Composition of Rat Brain Cells (Neurons, Astrocytes, and Oligodendrocytes) and of Subcellular Fractions (Myelin and Synaptosomes) Induced by a Diet Devoid of n‐3 Fatty Acids
Authors: Bourre J‐. M. , Pascal G. , Durand G. , Masson M. , Dumont O. , Piciotti M.
Published: October 2006

Abstract: Rats were fed through four generations with a semisynthetic diet containing 1.0% sunflower oil (6.7 mg/ g n‐6 fatty acids, 0.04 mg/g n‐3 fatty acids). Ten days before mating, half of the animals received a diet in which sunflower was replaced by soya oil (6.6 mg/g n‐6 fatty acids, 0.8 mg/g n‐3 fatty acids) and analyses were performed on their pups. Fatty acid analysis in isolated cellular and subcellular material from sunflower‐fed animals showed that the total amount of unsaturated fatty acids was not reduced in any cellular or subcellular fraction (except in 60‐day‐old rat neurons). All material from animals fed with sunflower oil showed an important reduction in the docosahexaenoic acid content, compensated (except in 60‐day‐old rat neurons) by an increase in the n‐6 fatty acids (mainly C22:5 n‐6). When comparing 60‐day‐old animals fed with soya oil or sunflower oil, the n‐3/n‐6 fatty acid ratio was reduced 16‐fold in oligodendrocytes, 12‐fold in myelin, twofold in neurons, sixfold in synaptosomes, and threefold in astrocytes. No trienes were detected. Saturated and monounsaturated fatty acids were hardly affected. This study provides data on the fatty acid composition of isolated brain cells.
Relationship Between Cycles in Level of Serotonin N‐Acetyltransferase Activity and Cyclic GMP Content of Cultured Chick Pineal Glands
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: October 2006

Abstract: We examined effects of supplements on cycles in cyclic GMP content and serotonin N‐acetyltransferase (NAT) activity in cultured chick pineal glands. Increases in cyclic GMP content and NAT activity were stimulated by 1‐ethyl‐4(isopropylidene‐hydrazino)‐1H‐pyrazolo[3,4‐b]pyridene‐5‐carboxylic acid, ethyl ester, hydrochloride and isobutylmethyl xanthine under diurnal illumination and in constant darkness, but subsequent decreases were not inhibited. Hypoxanthine had little effect on NAT activity under all lighting conditions, or on the content of cyclic GMP in glands cultured in the dark. However, it markedly stimulated accumulation of cyclic GMP in illuminated cultures. EGTA or additional Ca2+had no effect on pineal NAT activity. However, EGTA markedly stimulated accumulation of cyclic GMP both in the light and in the dark. Supplementary Ca2+ slightly retarded accumulation of cyclic GMP in the dark but stimulated slightly in the light.
Genetic Study of Cationic ATPase Activities and Audiogenic Seizure Susceptibility in Recombinant Inbred and Congenic Strains of Mice
Authors: Palayoor Sanjeewani T. , Seyfried Thomas N.
Published: October 2006

Abstract: Total, Mg2+, and Na+, K+ ATPase activities were studied in fresh brain homogenates of the audiogenic seizure (AGS)‐resistant C57BL/6J (B6) and AGS‐susceptible DBA/2J (D2) inbred strains and in 13 B6 × D2 (BXD) recombinant inbred (RI) strains. These activities were also studied in the D2.B6‐Iasb congenic mice, that are similar genetically to D2 mice, except for the Iasb gene which inhibits the spread of AGS activity. The total and Mg2+ ATPase activities of the brainstem were significantly lower in the D2 than in the B6 mice at 21 days of age. No differences were found between these strains for Na+, K+ ATPase activity. The total, Mg2+, and Na+, K+ ATPase activities in the B6 brainstem did not change noticeably from 21 to 80 days of age. In the D2 brainstem, however, the Mg2+ activity increased with age, and the Na+, K+ ATPase activity decreased from 30 to 80 days of age. No genetic associations could be found between AGS susceptibility and total or Mg2+ ATPase activities in the D2.B6‐Iasb mice or among the 13 BXD RI strains. Hence, differences in genetic background, rather than differences in AGS susceptibility, can account for the lower ATPase activities in 21‐day‐old D2 mice. Further, the Mg2+ and Na+, K+ ATPase activities appear to be regulated by more than one gene. This study emphasizes the utility of RI and congenic strains for testing the biochemical basis of AGS susceptibility in mice.
Inhibition of Sphingolipid Synthesis by Cycloserine In Vitro and In Vivo
Authors: Sundaram K. Soma , Lev Meir
Published: October 2006

Abstract: d‐ and l‐cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3‐ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes, l‐Cycloserine was shown to be 100 times more inhibitory than the d‐isomer for the brain microsomal enzyme in vitro. In vivo, l‐cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of l‐cycloserine on a regimen of intraper‐itoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.
Glial Fibrillary Acidic Protein: Norepinephrine Stimulated Phosphorylation in Intact C‐6 Glioma Cells
Authors: Browning Edward T. , Ruina Monica
Published: October 2006

Abstract: Coelectrophoresis in two‐dimensional gels of rat glial fibrillary acidic protein (GFA) and 32P‐labeled whole cell extracts of rat C‐6 glioma cells showed that the GFA migrated in close proximity to a previously noted phosphoprotein, 50K‐6.1, of these cells. GFA electrophoresed as a 50K polypeptide with at least four charge variants, the most acidic of which coelectrophoresed with 50K‐6.1. Exposure of the C‐6 cultures to dibutyryl cyclic AMP (dbcAMP) for 48 h increased the relative abundance of the endogenous polypeptide associated with 50K‐6.1 by threefold, consistent with the hypothesis that 50K‐6.1 was GFA. Norepinephrine stimulated 50K‐6.1 phosphorylation 3.2‐fold in dbcAMP‐induced cultures. Peptide mapping with V8 protease and subtilisin was used to test the hypothesis that GFA and 50K‐6.1 were identical polypeptides. With V8 protease, the peptides generated from the [35S]methionine labeled putative GFA spot of the C‐6 cells were indistinguishable from the stained bands derived from authentic GFA in mixed samples of the two proteins. Likewise, the 35S‐labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. 32P‐labeled peptides derived from the 50K‐6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, 32P‐labeled 50K‐6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from 32P‐labeled C‐6 cells contained a 50K phosphoprotein. Pep‐, tide mapping with V8 protease produced a 32P‐peptide pattern which was a subset of that from authentic GFA. The pattern closely resembled the 32P‐peptide pattern for the 50K‐6.1 protein from 2‐dimensional gels of whole cell extract. It was concluded that the protein 50K‐6.1 is a phosphorylated form of GFA and that GFA is a phosphoprotein whose phosphòrylation is stimulated by norepinephrine in C‐6 glioma cells.
Carrier‐Mediated Transport of Chloride Across the Blood‐Brain Barrier
Authors: Smith Quentin R. , Rapoport Stanley I.
Published: October 2006

Abstract: 36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed‐nephrectomized rats with plasma Cl concentrations between 14 and 120 μmol × ml−1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood‐to‐brain transfer constants for Cl, kCl, ranged from 1.8 × 10−5 s−1 at the parietal cortex to 3.8 × 10−5 s−1 at the thalamus‐hypothalamus. kCl fell by 42–62% when mean plasma [Cl] was elevated from 16 to 114 μmol × ml−1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis‐Menten saturation kinetics, where, for the parietal cortex, Km= 43 μmol × ml−1 and Vmax= 2.5 × 10−3μmol × s−1× g−1, and for CSF Km= 68 μmol × ml−1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration‐dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.
Ergopeptine‐Sensitive Calcium‐Dependent Protein Phosphorylation System in the Brain
Authors: Stratford Carol A. , Fisher Stephen K. , Ueda Tetsufumi
Published: October 2006

Abstract: We studied a protein phosphorylation system that is regulated by the dopamine‐mimetic ergot bromocriptine. Bromocriptine was found to inhibit selectively the endogenous phosphorylation of a threonine residue(s) in 50,000‐ and 60,000‐dalton proteins in a synaptosome fraction. The bromocriptine‐sensitive phosphorylation is stimulated by calcium and by calmodulin, and occurs predominantly in the brain. The inhibitory effect of bromocriptine was not mimicked by 3,4‐dihydroxyphenylethylamine or by any of the neurotransmitters and related agents tested, but was mimicked, although less effectively, by other ergots that contain peptide moieties. In the hippocampus, the brain region with the highest content of the 50,000‐ and 60,000‐dalton proteins, the ergopeptine‐sensitive protein phosphorylation appears to be localized to interneurons or cell bodies whose axons synapse outside the hippocampus. The results raise the possibility that some of the bromocriptine‐ and ergopeptine‐induced pharmacological effects in the CNS may be mediated by the inhibition of the calcium/calmodulin‐dependent phosphorylation of these specific proteins.
Developmental and Biochemical Analysis of Chick Brain Tubulin Heterogeneity
Authors: Sullivan Kevin F. , Wilson Leslie
Published: October 2006

Abstract: Tubulin, isolated from brain tissue of chicks at different stages during late embryonic and early posthatched development by ion‐exchange chromatography and by in vitro microtubule reassembly, was analyzed by high‐resolution isoelectric focusing and by two‐dimensional polyacrylamide gel electrophoresis. Similar results were obtained with tubulins purified by the two methods. Sixteen isoelectric species of tubulin that differ in apparent net charge under denaturing conditions were detected by isoelectric focusing. By two‐dimensional polyacrylamide gel electrophoresis, the chick brain tubulins were resolved into at least seven forms of α and 10 forms of β tubulin. The number and relative proportions of the maltiple brain tubulins were modulated during development. Since there are only four α tubulin and four β tubulin genes in chickens, posttranslational modification of the tubulins must play a prominent role in the heterogeneity. Analysis of isotubulin distributions through cycles of microtubule assembly and disassembly indicated that the tubulins differ very little, if at all, in their capacity to assemble into microtubules. Therefore, the chemical differences that distinguish the multiple tubulins have very little structural impact on the protein surface areas involved in microtubule formation. Partial fractionation of the multiple tubulins during ion‐exchange chromatography was observed, suggesting that it may be possible to isolate individual native tubulin variants for biochemical studies.
Interaction of N‐(2‐Nitro‐4‐Azidophenyl)‐Serotonin with Two Types of Monoamine Oxidase in Rat Brain
Authors: Chen Shiuan , Shih Jean C. , Xu Qin‐Ping
Published: October 2006

The effects of N‐(2‐nitro‐4‐azidophenyl) serotonin (NAP‐5‐HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (K1 values of 0.19 μM and 0.21 μM for types A and B MAO, respectively). Upon photolysis, NAP‐5‐HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP‐5‐HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP‐5‐HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP‐5‐HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.
Effect of Age on Human Brain Serotonin (S‐1) Binding Sites
Authors: Marcusson Jan , Oreland Lars , Winblad Bengt
Published: October 2006

The effect of age on the binding of [3H]5‐hydroxytryptamine ([3H]5‐HT, serotonin) to postmortem human frontal cortex, hippocampus, and putamen from individuals between the ages of 19 and 100 years was studied. One high‐affinity binding site was observed in adult brains, with a mean KD of 3.7 nM and 3.2 nM for frontal cortex and hippocampus, respectively, and 9.2 nM for putamen. Decreased binding capacities (Bmax) with age were detected in frontal cortex and hippocampus. In putamen a decrease in affinity was noted. Postmortem storage did not significantly contribute to the age‐related changes. No significant sex differences were detected. [3H]5‐HT binding was also studied in brains from human neonates. The specific binding was 1.5–3 times lower than in adult frontal cortex and putamen, and Scatchard analysis suggested more than one binding site. In infant hippocampus a single binding site was observed and except for a premature individual, the binding capacity approximated adult values.
Bradykinin Receptor‐Mediated Cyclic GMP Formation in a Nerve Cell Population (Murine Neuroblastoma Clone N1E‐115)
Authors: Snider R. Michael , Richelson Elliott
Published: October 2006

A clone of murine neuroblastoma (N1E‐115) was shown to have functional receptors for the nonapeptide bradykinin. These receptors mediated a large, rapid (about 1 min to peak) and calcium‐dependent increase in cyclic GMP. The median effective concentration (EC50) averaged 1.4 nM. In addition, this event was inhibited by quinacrine, 5,8,11,14‐eicosatetraynoic acid, and nordi‐hydroguaiaretic acid, suggesting involvement of phospholipase A2 with subsequent formation of lipoxygenase metabolites of arachidonic acid. [3H]Bradykinin binding to intact cells, investigated under conditions nearly identical to those used in the cyclic GMP assay, yielded binding sites with KDs of 0.83 pM, 1.0 nM, and 4.9 nM with respective Bmax values of 12, 160, and 250 fmol/106 cells. Apparently, the cyclic GMP response was associated with the binding site in which the KD= 1.0 nM. Peptide analogs of bradykinin stimulated cyclic GMP with EC50s nearly identical to their respective KDs determined in binding assays with [3H]bradykinin, thus providing evidence for receptor specificity of this response. This finding of a biochemical response of bradykinin promises to make N1E‐115 cells a convenient model system for study of neuronal bradykinin receptors.
Increased Phosphorylation of a Membrane Protein Consequent to Amygdaloid Kindling
Authors: Patel Jitendra , Marangos Paul J. , Contel Nancy , Gardner Glen , Post Robert M.
Published: October 2006

Abstract: The phosphorylation of both particulate and soluble proteins in the amygdala was examined in electrically kindled rats. In animals receiving electrical stimulation in the left amygdala for 5–6 days that displayed electrical after‐discharges but no motor seizures, no changes were observed in the phosphorylation of either particulate or soluble proteins. In animals stimulated for 20–21 days where major motor seizures were produced, the phosphorylation of a protein having a molecular weight of 45,000 (45K) was markedly increased. The phosphorylation of this protein was increased in both the right (unstimulated) and left (stimulated) amygdala. Major motor seizures induced by electroconvulsive shocks, however, did not alter phosphorylation of this protein. Phosphorylation of the 45K protein was stimulated by calcium and calmodulin. The 45K protein is a major phosphoprotein of amygdala, representing 3.2% of the total particulate phosphoproteins in control animals and 7.4% in the kindled animals. In the presence of calcium‐calmodulin, 16.2% of net protein phosphorylation was accounted for by the 45K protein.
Muscarinic Receptors in the Cochlear Nucleus and Auditory Nerve of the Guinea Pig
Authors: Whipple Michael R. , Drescher Dennis G.
Published: October 2006

Abstract: The specific‐binding properties of l‐[3H]quinuclidinyl benzilate, a muscarinic acetylcholine‐receptor antagonist, were investigated in synaptic and other membrane preparations of the guinea pig cochlear nucleus and auditory nerve. Binding parameters for all experiments were consistent with a single binding site with a Hill coefficient of 1.0. The binding of the ligand was specific and of high affinity, with values of KD in the range of 30–80 pM. Bmax was 0.352 ± 0.023 pmol/mg protein for the dorsal cochlear nucleus and 0.215 ± 0.011 pmol/mg protein for the ventral cochlear nucleus. The dorsal cochlear nucleus/ventral cochlear nucleus ratio for density of muscarinic receptors (1.6/1.0) was maintained across two different buffer systems, which varied with respect to the inclusion of proteolysis inhibitors. The results for auditory nerve indicated a level of binding much below that of the cochlear nucleus, with Bmax= 0.052 ± 0.011 pmol/mg protein. The results of specific‐binding experiments for l‐[3H]quinuclidinyl benzilate support a role for acetylcholine as a neurotransmitter in the cochlear nucleus. The greater density of muscarinic receptors in the dorsal cochlear nucleus may indicate greater cholinergic activity in the dorsal relative to the ventral cochlear nucleus.
Early Inhibition of Acetylcholinesterase and Choline Acetyltransferase Activity in Herpes simplex Virus Type 1 Infection of PC12 Cells
Authors: Rubenstein Richard , Price Richard W.
Published: October 2006

Abstract: Early in the course of productive Herpes simplex virus type 1 (HSV‐1) infection of PC12 cells, activities of both acetylcholinesterase (AChE) and choline acetyltransferase (CAT) fell. Studies using metabolic inhibitors and a temperature‐sensitive mutant of the virus suggested that the decline in activities of both enzymes was associated with events occurring early in the replicative cycle related to expression of the immediate‐early (α) group of viral polypeptides. HSV‐1 gene products thus may alter specialized cell functions well before the production of viral progeny and initiation of cell lysis. The early clinical manifestations of nervous system viral infection may reflect focal metabolic disturbance rather than, or in addition to, simple cell death.
Prolyl Endopeptidase: Inhibition In Vivo by N‐Benzyloxycarbonyl‐Prolyl‐Prolinal
Authors: Friedman Theodore C. , Orlowski Marian , Wilk Sherwin
Published: October 2006

Abstract: The activity of prolyl endopeptidase in homogenates of mouse tissues was determined 30 min after intraperitoneal injection of N‐benzyloxycarbonyl‐prolylprolinal (1.25 mg/kg), a potent transition state analog inhibitor (K1= 14 nM) of prolyl endopeptidase (EC 3.4.21.26). A more than 85% decrease of enzyme activity was obtained in all tissues. The in vivo degradation of potential prolyl endopeptidase substrates was studied by following the release of sulfamethoxazole from N‐benzyloxycarbonylglycyl‐prolyl‐sulfamethoxazole, a model synthetic substrate of the enzyme. When this substrate was given intraperitoneally, its enzymatic degradation was blocked after administration of the inhibitor in a dose‐ and time‐dependent manner, indicating inhibition of the enzyme in vivo. Of interest is the long duration of the inhibition. After a relatively low inhibitor dose (5 mg/kg) significant inhibition was seen in most tissues even after 6 h. The brain was particularly sensitive to the effect of the inhibitor. Since prolyl endopeptidase readily degrades many proline‐containing neuropeptides, the inhibitor should be of value in studies on the role of the enzyme in neuropeptide metabolism.
Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination
Authors: Schwob Valerie S. , Clark H. Brent , Agrawal Daya , Agrawal Harish C.
Published: October 2006

Abstract: Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti‐large basic protein serum was shown by the immunoblot technique to cross‐react with all five forms of basic protein present in the myelin of 8‐day‐old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.
Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: I. Interaction with δ and μ Sites
Authors: Castanas E. , Bourhim N. , Giraud P. , Boudouresque F. , Cantau P. , Oliver C.
Published: October 2006

Abstract: In the present study we examined the interaction of opiates with the δ and μ opioid binding sites in the bovine adrenal medulla. [3H][D‐Ala2, D‐Leu5]‐enkephalin ([3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze δ site interactions, whereas either [3H]DADLE in the presence of saturating concentrations of [D‐Ser2, Leu5]‐enkephalinThr6 (DSLET) or [3H][D‐Ala2, Me‐Phe4, Gly5‐ol]‐enkephalin ([3H]DAGO) was used for the determination of μ sites. Both binding sites were found to interact stereo selectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, α‐chymotrypsin, pepsin), N‐ethylmaleimide, and A2‐phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second‐order bi molecular kinetics. Competition for binding by opiates and opioid peptides confirmed the δ and μ selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.
Lipoxygenase Metabolism of Arachidonic Acid in Brain
Authors: Adesuyi Sunday A. , Cockrell Carolyn S. , Gamache Daniel A. , Ellis Earl F.
Published: October 2006

Abstract: When blood‐free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12‐hydroxy‐5,8,10,14‐eicosatetraenoic acid (12‐HETE), with lesser amounts of 5‐hydroxy‐6,8,11,14‐eicosatetraenoic acid and 15‐hydroxy‐5,8,11,13‐eicosatetraenoic acid. When 12‐[2H]HETE was used to measure endogenous 12‐HETE in brain tissue frozen with liquid nitrogen, the level of 12‐HETE was 41 ± 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12‐HETE level increased to 964 ± 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12‐HETE concentration by one‐half.
Rat Brain Phosphatidyl‐N,N‐Dimethylethanolamine Is Rich in Polyunsaturated Fatty Acids
Authors: Tacconi Mariateresa , Wurtman Richard J.
Published: October 2006

Abstract: Phosphatidyl –N,N ‐ dimethylethanolamine (PDME), an intermediate in the formation of phosphatidylcholine (PC) by the sequential methylation of phosphatidylethanolamine (PE), was purified from rat brain and its fatty acid (FA) composition compared with those of brain PC and PE. The proportion of polyunsaturated fatty acids (PUFAs) in the PDME (29.8%) was similar to that of PE (27.7%) and much greater than in PC (2.8%). Like the PUFAs of PE, the major PUFAs found in PDME were arachidonic acid (20:4) and docosahexaenoic acid (22:6). An isotopic method was developed to quantify the PDME purified from brain; a tritiated methyl group from CH3I was transferred to the PDME in the presence of cyclohexylamine to form [3H]PC, and the radioactivity of the PC was then counted. The concentration of rat brain PDME obtained using this method (33.0 ± 1.8 μg/g brain) was very similar to that obtained using quantitative GLC analysis of its FAs (36.9 ± 1.8 μg/g). The FAs in the PE and PC of rat brain synaptosomes were also analyzed; too little PDME was present in synaptosomes to permit similar analysis. The percentage of unsaturated FAs insynaptosomal PE was even higher (43.4 vs. 27.7) than that in PE prepared from whole brain. Since synaptosomes have a very high activity of phosphatidyl‐N‐methyltransferase, the enzyme complex that methylates PE to form PC, this enzyme may serve, in nerve endings, to produce a particular pool of PC, rich in PUFAs, which may have a distinct physiological function.
Decreased Cerebrospinal Fluid Glutamine Levels in Patients with Benign Brain Tumors
Authors: El‐Yazigi Adnan , Al‐Mefty Ossama
Published: October 2006

Abstract: CSF glutamine concentrations were studied in 12 patients with benign brain tumors (meningioma, craniopharyngioma, or osteofibroma), 12 patients with malignant brain tumors (astrocytoma, medulloblastoma, pinealoblastoma, or chondrosarcoma), 9 patients with non‐cerebral tumors, and a reference group of 24 patients. The mean ± SD levels in the benign tumor group (424 ± 124 μM) were significantly lower (p < 0.0004) than those in the reference group (642 ± 195 μM). There was no significant difference between the CSF glutamine concentrations in the malignant cerebral tumor group (643 ± 210 μM) or noncerebral tumor group (599 ± 127 μM) and those in the reference group. In patients with benign brain tumors there was indication of an inverse linear relationship between the logarithm of CSF glutamine concentration and tumor diameter.
Dissociation of [35S]t‐Butylbicyclophosphorothionate Binding Differentiates Convulsant and Depressant Drugs that Modulate GABAergic Transmission
Authors: Maksay G. , Ticku M. K.
Published: October 2006

Abstract: The dissociation of [35S]t‐butylbicyclophosphorothionate ([35S]TBPT) from binding sites on membranes from rat cerebral cortex, after addition of saturating concentrations of convulsant and depressant drugs, was studied. The addition of unlabeled TBPT, picrotoxinin, or pentamethylenetetrazol resulted in dissociation patterns that were monophasic and not distinguishable, suggesting that these convulsants bind competitively to the same (convulsant) sites. In contrast, γ‐aminobutyric acid (GABA) greatly facilitated [35S]TBPT dissociation by binding allosterically to the GABA recognition site of the receptor‐ionophore complex. TBPT dissociation was similarly accelerated by the depressants etazolate, (+)‐etomidate, and barbiturates. The convulsant and depressant S(+) and R(‐) stereoisomers of N‐methyl‐5‐phenyl‐5‐propyl‐barbituric acid displayed large stereoselectivity in the acceleration of TBPT dissociation. These results suggest that depressants bind to sites different from the convulsant sites of the allosteric GABA receptor complex, or the binding of depressants to the same population of sites elicits negative cooperativity and dissociates the convulsants.
Influence on 5‐[3H]Hydroxytryptamine Binding Site Development in Chick Embryo by Serotonergic Compounds
Authors: Soblosky Joseph S. , Jeng Ingming
Published: October 2006

Abstract: Saturable and specific binding sites for 5‐[3H]hydroxytryptamine (5‐HT, serotonin) characterized by a KD of 3.5–4.5 nM were detected in the chick embryo brain and were shown to develop linearly as a function of age, weight, and protein content. Saturation and displacement studies using unlabeled 5‐HT as the displacing ligand suggested a single population of binding sites. However, displacement studies using 5‐methoxytryptamine, lysergic acid diethylamide (LSD), 2‐bromo‐lysergic acid diethylamide (BOL), methysergide, and spiperone as competing ligands suggested the existence of subclasses of [3H]5‐HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5‐HT binding sites (5‐HT1) in the rat forebrain, the IC50 values of the competing ligands were similar. However, the Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5‐HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5‐HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l‐5‐hydroxytryptophan, 5‐HT, or BOL were found to have no significant effects on either the affinity (KD) or number (Bmax) of specific [3H]5‐HT binding sites. Multiple pretreatments with p‐chlorophenylalanine were found to increase the Bmax of specific [3H]5‐HT binding by 23% (p < 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (p < 0.01). Neither of these pretreatments affected KD values which indicated that only the number of specific [3H]5‐HT binding sites was altered. Evidence was presented suggesting that these effects were probably not due to excess endogenous 5‐HT or LSD remaining in the tissue preparation. The overall evidence indicated that the chick embryo brain may have a functioning serotonergic system and that the chick embryo may be an ideal system for the study of [3H]5‐HT binding site regulation and development.
Comparative Analysis of Nicotine‐Like Receptor‐Ligand Interactions in Rodent Brain Homogenate
Authors: Larsson Christer , Nordberg Agneta
Published: October 2006

Abstract: The effects of different variables such as incubation time, temperature, tissue protein content, and pH on the interactions of various labelled nicotinic ligands with nicotine‐like binding sites in vitro were studied in rodent brain preparations. The ligands tested were α‐[3H]bungarotoxin (α‐[3H]BTX), [3H]tubocurarine ([3H]TC), and [3H]nicotine ([3H]NIC). The regional distribution of the labelled nicotinic ligand binding was also studied and affinity constants and maximal binding (Bmax) values for the equilibrium [3H]NIC binding are given. Association kinetics for [3H]NIC and [3H]TC binding to brain homogenate were similar, with maximal binding within 5–10 min of incubation, followed by a continuous decrease. In contrast, the binding of α‐[3H]BTX to brain homogenate was much slower, reaching equilibrium after 30–60 min of incubation. Scatchard analysis of equilibrium binding data for [3H]NIC in the hippocampus indicated two binding sites: a high‐affinity site (Bmax 60 pmol/ g protein; KD, 6 nM) and a low‐affinity site (Bmax, 230 pmol/g protein; KD, 125 nM). The data for the high‐affinity [3H]NIC binding site are very similar to previously found data for the high‐affinity binding site of [3H]TC and the binding site of α‐[3H]BTX. Each ligand showed regional differences in binding, and the binding pattern also differed between the ligands.
Calcium Enhances In Vitro Free Radical‐Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons
Authors: Braughler J. Mark , Duncan Lois A. , Goodman Tracey
Published: October 2006

Abstract: Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+‐free Krebs buffer resulted in a 27% inhibition of synaptosomal γ‐aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 μM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+‐ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical‐induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.
Kinetics of the Mechanism of Action of Monoamine Oxidase in the Regulation of Na+, K+‐ATPase Activity in Rat Brain
Authors: Mayanil C. S. K. , Baquer N. Z.
Published: October 2006

Kinetic studies on the action of monoamine oxidase (MAO) in the regulation of Na+,K+‐ATPase were performed using 3‐methoxy‐4‐hydroxybenzaldehyde (MHB), which is an analogue of 3‐methoxy‐4‐hydroxyphenylacetylaldehyde (product of MAO‐catalysed reaction with dopamine as substrate). It was observed that at 2.6 μM MHB, the activation of Na+,K+‐ATPase may be the result of the removal of the inhibitory Ca2+, thereby increasing the Vmax. Double‐reciprocal plots of Pi versus MHB showed that Ca2+ counteracted the effect of the aldehyde not by changing the Km, but be decreasing the Vmax of the Na+,K+‐ATPase stimulation. The removal of 3′,5′‐cyclic AMP‐dependent protein kinase from the microsomes by sodium dodecyl sulphate treatment abolished the activation and/or inhibition of the Na+,K+‐ATPase by aldehyde; it can therefore be inferred that 3′,5′‐cyclic AMP‐dependent protein kinase is involved in the regulation of Na+,K+‐ATPase.
Characterization of Glutamate Uptake into Synaptic Vesicles
Authors: Naito Shigetaka , Ueda Tetsufumi
Published: October 2006

Recent evidence indicates that L‐glutamate is taken up into synaptic vesicles in an ATP‐dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP‐dependent vesicular uptake of glutamate. Evidence is provided that a Mg‐ATPase, not Ca‐ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATP‐dependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg‐ATPase. Of particular interest is the finding that the ATP‐dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L‐glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter.
Simultaneous Determination of 5‐Hydroxytryptophan and 3,4‐Dihydroxyphenylalanine in Rat Brain by HPLC with Electrochemical Detection Following Electrical Stimulation of the Dorsal Raphe Nucleus
Authors: Duda N. J. , Moore K. E.
Published: October 2006

HPLC coupled with electrochemical detection was used to make concurrent measurements of the rate of accumulation of 5‐hydroxytryptophan and 3,4‐dihydroxyphenylalanine in selected brain regions (striatum, nucleus accumbens, septum, medial periventricular hypothalamus) and thoracic spinal cords of rats treated with NSD 1015, an inhibitor of aromatic‐L‐amino‐acid decarboxylase. 5‐Hydroxytryptophan and 3,4‐dihydroxyphenylalanine accumulated in all brain regions 30 min after the intravenous infusion of various doses of NSD 1015; there were no significant differences in the responses to 12.5, 25, 50, and 100 mg/kg. After the intravenous administration of 25 mg/kg NSD 1015 the concentrations of 5‐hydroxytryptophan and 3,4‐dihydroxyphenylalanine increased linearly with time in all brain regions for at least 30 min. Electrical stimulation of 5‐hydroxytryptamine neurons in the dorsal raphe nucleus for 30 min at 5 or 10 Hz increased 5‐hydroxytryptophan accumulation in all brain regions but not in the spinal cord. Unexpectedly, this stimulation also increased the accumulation of 3,4‐dihydroxyphenylalanine in the hypothalamus and spinal cord. These results suggest that 5‐hydroxytryptophan accumulation following the administration of NSD 1015 is a valid index of 5‐hydroxytryptamine neuronal activity in the brain.
Hydrophobic Regions in Myelin Proteins Characterized Through Analysis of “Hydropathic”Profiles
Authors: Weise Michael J.
Published: October 2006

Computer‐generated “hydropathic’ profiles were constructed for graphic comparison of the amino acid sequences for P2 protein, 18.5 kilodalton (kDa) myelin basic protein (BP), and myelin proteolipid protein (PLP). Profiles were also obtained for cytochrome b5, a membrane protein known to be capable of reversible association with lipid bilayers and of a size comparable to that of the myelin BPs. Analysis of the PLP sequence produced profiles generally compatible with the suggestions that PLP has three transbilayer and two bilayer intercalating segments. Profiles for P2 and 18.5 kDa BP were found to contain hydrophilic segments separated by relatively short hydrophobic regions. Whereas hydropathic indices in hydrophobic regions of P2, 18.5 kDa BP, and PLP fall in the value ranges recently reported for cores of globular proteins and intrabilayer domains of membrane proteins, hydrophobic sections of P2 and 18.5 kDa BP have hydropathic indices similar to those in the hydrophobic core (transprotein) regions of globular proteins. None of them are comparable to the region of cytochrome b5 known to anchor that protein in its membrane or to the segments of PLP sequence proposed as intrabilayer domains. This comparison suggests that neither BP has structural characteristics compatible with insertion into the hydrocarbon core of the myelin lipid bilayer, a conclusion that is consistent with a recently published study that identified the bilayer penetrating proteins of myelin with a hydrophobic probe. The above findings suggest an enhancement for some details of myelin architecture and a cautious approach to interpreting data for BP intercalation into bilayers.
Free and Conjugated Amines in Human Lumbar Cerebrospinal Fluid
Authors: Tyce Gertrude M. , Rorie Duane K. , Byer David E. , Danielson David R.
Published: October 2006

Significant amounts of acid‐hydrolyzable conjugates of 3,4‐dihydroxyphenylethylamine, norepinephrine, and 5‐hydroxytryptamine were detected in lumbar CSF from 22 awake unpremedicated healthy individuals. In the CSF samples, the amounts of conjugated amines almost always exceeded the amounts of free amines, but were less than the amounts of the acid metabolites 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid.
Taurine Stimulation of Isolated Hamster Brain Na+, K+‐ATPase: Activation Kinetics and Chemical Specificity
Authors: Hastings David F. , Welty Joseph D. , Rohani Farrokh
Published: October 2006

Epileptic foci are associated with locally reduced taurine (2‐aminoethanesulfonic acid) concentration and Na+, K+‐ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na+, K+‐ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na+, K+‐ATPase by taurine. Normal whole brain homogenate Na+, K+‐ATPase activity is 5.1 ± 0.4 (4) μmol Pi± h−1± mg−1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane‐bound Na+, K+‐ATPase activity of 204.6 ± 5.8 (4) mol Pi± h−1± mg−1 Lowry protein. Taurine activates the Na+, K+‐ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β‐alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na+, K+‐ATPase. Its action is mediated by a membrane‐bound protein.
Cerebrospinal Fluid Glucose: Turnover and Metabolism
Authors: Hochwald Gerald M. , Magee Jill , Ferguson Vicki
Published: October 2006

The turnover of cerebrospinal fluid (CSF) glucose was studied in cats during steady‐state perfusion. In all experiments, the perfusion fluid contained either tracer [14C]glucose alone or tracer glucose along with 4.45 mM unlabeled glucose. In some studies, serum glucose was lowered with insulin. The concentration of glucose and [14C]glucose in the effluent fluid was measured, and the distribution of 14C between glucose and lactate was determined by chromatography. From these values, the extraction of glucose and the metabolism of glucose to lactate were calculated. From the decrease in the specific activity of glucose in the perfusion fluid, the influx of glucose from serum was also determined. During steadystate perfusion, 71% of the radioactivity was recovered in the effluent fluid: 50% in the form of glucose, 6% in the form of lactate, and 15% in forms that were not identified. Thus, 50% of the perfusion fluid glucose was cleared, of which 29% was extracted and 21% metabolized. The influx of glucose was proportional to the serum glucose when the latter was about 2.5 mM or 10.0 mM. During perfusion with tracer glucose only, the concentration of glucose in the effluent fluid was 25% that of serum. The transport of glucose from serum was independent of the glucose concentration gradient between serum and perfusion fluid. However, when perfusion fluid glucose concentration was greater than that of serum, transport was inhibited. These studies suggest that in maintaining CSF glucose at a lower concentration than serum glucose, with equal amounts of glucose entering and leaving the CSF, 50% of CSF glucose concentration cleared is replaced by 25% of serum glucose concentration.
Potassium‐Induced Release of Endogenous Amino Acids in the Guinea Pig Cochlea
Authors: Jenison G. L. , Bobbin R. P. , Thalmann R.
Published: October 2006

Guinea pig cochleae were perfused with highpotassium solutions to depolarize hair cells artificially and induce the release of afferent neurotransmitter. Sequential injections of artificial perilymph containing 5 mM KCl, then 50 mM KCl, and finally 5 mM KCl were made into the scala tympani. This injection sequence was conducted under either normal divalent‐cation conditions (2.0 mM CaCl2, 1.0 mM MgCl2) or calcium‐deficient conditions intended to antagonize evoked transmitter release (0.1 mM CaCl2, 20.0 mM MgCl2). The levels of 21 endogenous primary amines in effluent collected from the scala vestibuli were determined by gradient‐elution, reverse‐phase HPLC using o‐phthaldialdehyde‐thiol adducts with fluorescence detection. Analyses indicated effluent concentrations of glutamate, taurine, and a coeluting taurine‐γ‐aminobutyrate (GABA) fraction (but not GABA alone) increased significantly after exposure to 50 mM KCl and returned to baseline levels after reintroduction of 5 mM KCl under normal divalent‐cation conditions. Correspondent changes in the release of these constituents were significantly attenuated under calcium‐deficient conditions. This was not the case for potassium‐induced changes in the release of arginine, aspartate, and isoleucine. These data are consistent with the hypothesis that the receptoneuronal transmitter is glutamate and further suggest a calcium‐dependent mechanism involving taurine.