bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in the Kinetics of Axonal Transport for Individual Lipid Classes in Rat Sciatic Nerve
Authors: Toews Arrel D. , Saunders Barry F. , Blaker William D. , Morell Pierre
Published: October 2006

Abstract: Lipid precursors ([2‐3H]gIycerol for phospholipids and [3H]acetate for cholesterol) were injected into the L‐5 dorsal root ganglion of adult rats. At various times, animals were killed, the ganglion and consecutive 5‐mm segments of sciatic nerve were dissected, and lipids were extracted and analyzed by TLC. Individual lipid classes exhibited markedly different transport patterns. The crest of radioactive phosphatidylcholine moved as a sharply defined front at about 300 mm/day, with a relatively flat plateau behind the moving crest. Although some radioactive phosphatidylethanolamine also moved at the same rate, the crest was continually attenuated as it moved so that a gradient of radioactive phosphatidylethanolamine along the axon was maintained for several days. Transported diphosphatidylglycerol exhibited a defined crest, as did phosphatidylcholine, but moved at about half the rate. Labeled cholesterol was transported at a rapid rate similar to that for phosphatidylcholine and phosphatidylethanolamine, but like phosphatidylethanolamine, the initial moving crest of radioactivity was continually attenuated. Relative to the phospholipids, cholesterol showed a more prolonged period of accumulation in the axons and was more metabolically stable. We propose that most labeled phosphatidylcholine, phosphatidylethanolamine, and cholesterol is transported in similar (or the same) rapidly moving membranous particles. Once incorporated into these particles, molecules of phosphatidylcholine tend to remain associated with them during transport. In contrast, molecules of phosphatidylethanolamine and cholesterol in these transported particles exchange extensively with unlabeled molecules in stationary axonal structures. Diphosphatidylglycerol, localized in a specialized organelle, the mitochondrion, is transported at a slower rate than other phospholipids, and does not exchange with other structures.
The Delipidation of Brain Proteolipid Protein by Ultrafiltration
Authors: Aguilar J. S. , de Cözar M. , Criado M. , Monreal J.
Published: October 2006

Abstract: It has been very difficult to prepare the apo‐protein moiety of brain white matter proteolipid so that it is completely devoid of complex lipids, without suffering aggregation and protein denaturation. The reason is that complex lipids are tightly bound to the proteolipid apo‐protein. Using a new ultrafiltration method, we obtained, in a gradual way and in a relatively short time, more than 99% delipidation in water‐saturated n‐butanol, with and without 0.1 M acetic acid and recovered up to 86% of the protein with no detectable reducing sugars remaining. The delipidated protein remained in solution and in a relatively nondenatured state for several days. In 1% sodium dodecyl sulfate (SDS)‐aqueous media, 90% of the lipids were removed and the yield of recovered protein in solution was near 90%; nearly 6% of the reducing sugars remained in the apoprotein. A higher delipidation was obtained by washing with 0.1 M NaOH. The content of reducing sugars was greater but the protein was less stable. When 10% SDS was employed to dissociate lipid‐protein interaction, an almost complete delipidation was obtained and reducing sugars disappeared.
Localization of Hyaluronectin in Oligodendroglial Cells
Authors: Asou Hiroaki , Brunngraber Eric G. , Delpech Bertrand
Published: October 2006

Abstract: Astroglia and oligodendroglia in primary cell cultures were identified by immunohistochemical staining with antiglial fibrillary acidic protein and anticerebroside antisera, respectively. The antiserum to hyaluronectin (HN) was utilized to show that this hyaluronic acid‐binding glycoprotein was localized in the Oligodendroglial cell. This is consistent with the recent finding that HN is localized in the node of Ranvier. Astroglia did not react with antihyaluronectin.
In Vitro Studies on the Effect of β‐Carbolines on the Activities of Acetylcholinesterase and Choline Acetyltransferase and on the Muscarinic Receptor Binding of the Rat Brain
Authors: Skup Małgorzata , Oderfeld‐Nowak Barbara , Rommelspacher Hans
Published: October 2006

Abstract: Acetylcholinesterase (acetylcholine acetylhy‐drolase, EC 3.1.1.7) activity and muscarinic receptor binding of homogenates from several brain structures were inhibited by β‐carbolines. The inhibition was of the noncompetitive type in the case of the enzyme and of the mixed type in the case of the receptor binding. This effect was most strongly manifested by pyridoindoles(harmane, norharmane), i.e., carbolines containing an aromatic C ring than by the corresponding piperidoindoles (tetrahy‐droharmane, tetrahydronorharmane), i.e., those with a reduced C ring. The activity of choline acetyltransferase (acetyl‐CoAxholine O‐acetyltransferase, EC 2.3.1.6) was not altered. These data are further evidence of the interactions between indoleamine derivatives and the cholinergic system. The results are discussed in terms of their possible biological significance.
Phase‐Shifting of Cycles in Level of Serotonin N‐Acetyltransferase Activity and Cyclic GMP Content of Cultured Chick Pineal Glands by Methotrexate
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: October 2006

Abstract: Methotrexate at 1 μM stimulated increase of serotonin N‐acetyltransferase (NAT) activity in chick pineal glands cultured under each of three conditions of illumination. The peak of the circadian rhythm in NAT activity and the “spike” in content of cyclic GMP were both advanced in pineal glands cultured in the dark from midphotoperiod. In contrast, the time of peak NAT activity in glands cultured in the dark from late photoperiod was unaffected. In addition, methotrexate did not affect times of reaching maximum NAT activities in glands cultured from midphotoperiod in the light or under diurnal illumination. Doubling the concentration of methotrexate also eliminated the lag phase in increase of NAT activity in glands cultured in the dark. However, at a concentration of 5 μM methotrexate the curve depicting increase of NAT activity was biphasic, and neither time nor level of peak NAT activity differed from those of control glands. Results of attempts to demonstrate persistent effects of exposure to methotrexate were inconclusive.
MDL 72145, an Enzyme‐Activated Irreversible Inhibitor with Selectivity for Monoamine Oxidase Type B
Authors: Zreika Monique , McDonald Ian A. , Bey Philippe , Palfreyman Michael G.
Published: October 2006

Abstract: MDL 72145, (E)‐2‐(3′,4′‐dimethoxyphenyl)‐3‐fluoroallylamine hydrochloride, was designed and synthesised as a potential enzyme‐activated irreversible inhibitor of monoamine oxidase (MAO). In vitro, the compound displayed time‐dependent pseudo‐first‐order irreversible inhibitory characteristics with high selectivity for the B form of rat brain mitochondrial MAO At 10°C the Kt and T50 values for the B enzyme were 40 μM and 1.7 min, respectively, while these same kinetic constants for the A enzyme were 131 μM and 14.5 mm, respectively. Selective protection against inactivation of the two forms of MAO by MDL 72145 was obtained by preincu‐bating the enzyme with suitable concentrations of the selective A and B substrates, 5‐hydroxytryptamine and benzylamine.
Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding
Authors: Oliveira C. R. , Duarte E. P. , Carvalho A. P.
Published: October 2006

Abstract: [3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 μg/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 ± 9.2 fmol/mg protein before phospholipase treatment to a value of 52 ± 7.8 fmol/mg protein after treatment, but no change in affinity (KD= 0.24 ± 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidyl‐choline produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 μg/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase‐mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.
Accumulation of Pantothenic Acid by the Isolated Choroid Plexus and Brain Slices In Vitro
Authors: Spector Reynold , Boose Barbara
Published: October 2006

Abstract: In vitro, the transport of [14C]pantothenic acid into and from the isolated rabbit choroid plexus, an anatomical locus of the blood‐CSF barrier, and brain slices was studied. The choroid plexus accumulated [14C]pantothenic acid from the medium against a concentration gradient, although at low concentrations (< 1 μM) there was substantial intracellular phosphorylation and binding of the [14C]pantothenic acid. The saturable accumulation process in choroid plexus was inhibited by probenecid and caproic acid but not by nicotinic acid or by weak bases. The accumulation process was markedly inhibited by N‐ethylmaleimide, poly‐l‐lysine (which blocks sodium transport), and low temperatures. [14C]Pantothenic acid was readily released from choroid plexus by a temperature‐dependent process. Brain slices also accumulated and, at low concentrations, phosphorylated [14C]pantothenic acid from the medium by a temperature‐, probenecid‐, and N‐ethylmaleimide‐sensitive saturable process. However, unlike choroid plexus, brain slices did not concentrate free pantothenic acid and [14C]pantothenic acid accumulation was not sensitive to poly‐l‐lysine. [14C]Pantothenic acid was readily released from brain slices by a temperature‐sensitive process. These results are consistent with the view that [14C]pantothenic acid enters the isolated choroid plexus and brain slices by active transport and facilitated diffusion, respectively.
Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists
Authors: Horwitz Joel , Perlman Robert L.
Published: October 2006

Abstract: Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4‐dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a twofold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8‐bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8‐bromo cyclic AMP are not dependent on extracellular Ca2+Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.
Bulk Separation and Long‐Term Culture of Oligodendrocytes from Adult Pig Brain. I. Morphological Studies
Authors: Gebicke‐Härter P. J. , Althaus H.‐H. , Rittner I. , Neuhoff V.
Published: October 2006

Abstract: A method is described by which oligodendrocytes from adult pig brains can be isolated. It results in a cellular preparation suitable for long‐term culture. The entire procedure can be accomplished within 2–3 h. The purity of oligodendrocytes ranges between 80 and 95% depending on the Percoll gradient used and on the time in vitro. Yields between 2.5 and 4 × 107 cells per brain and plating efficiencies on the order of 60% make the system very useful for biochemical investigations. It was shown by immunocytochemical studies that oligodendrocytes produce extensive networks of processes, some of them having elaborate membranous expansions. Anti‐galactocerebroside (GC) antibodies as well as anti‐myelin basic protein (MBP), anti‐Wolfgram protein (WP), antiglial fibrillary acidic protein (GFAP), and monoclonal antibodies O1 and O4 are used to identify the cell types and to characterize the cellular composition of the cultures. Anti‐GC and O1 are suitable markers for these oligodendrocytes. Both antibodies label similar cells, and the staining intensities are equally strong. In the case of O4, variable staining intensities are observed, and a few additional cells are labeled that are anti‐GC−. After 31/2 weeks in culture, about 60% of the cells can be labeled by anti‐MBP. Here too differences in staining intensities are observed. The anti‐WP stain is too weak to be defined as positive. The percentage of GFAP+ cells lies in the range 15–20% at maximum. Cells were also mixed into collagen gels. This method appears to be more useful for outgrowth and branching of fibers than are monolayer systems. Drawbacks, however, include limited access for the antibodies and poor recovery of undamaged cells with their fibers.
Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide
Authors: Wood J. D. , Kurylo E.
Published: October 2006

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ‐aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long‐acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine‐induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of “aspartate + glutamate + glutamine + GABA” was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.
Increase of Junctional and Background 16S (Tailed, Asymmetric) Acetylcholinesterase During Postnatal Maturation of Rat and Mouse Sternocleidomastoid Muscle
Authors: Rieger François , Goudou Danièle , Tran Lam H.
Published: October 2006

Abstract: Acetylcholinesterase (AChE) is found both in motor end‐plate (MEP)‐free and MEP‐rich regions of rat or mouse muscle. We studied the developmental aspects of the localization of asymmetric 16S AChE in both regions of the sternocleidomastoid muscle, which has a well‐defined zone of motor innervation. In the rat, the proportion of 16S AChE to total AChE increases in the MEP‐rich region, and becomes significantly higher than in the MEP‐free regions between the first and the second weeks after birth. In the mouse, at birth, the MEP‐rich region already has a higher relative content in 16S AChE than the MEP‐free regions. Total 16S AChE amounts increase during postnatal development, not only in the MEP‐rich region but also in the MEP‐free regions. Thus, 16S AChE is not eliminated from MEP‐free regions during muscle maturation and growth. Two distinct pools of 16S AChE are distinguished in the muscles, both of which increase during postnatal development: junctional and background 16S AChE.
Effect of Cerebral Puncture on Brain Protein Synthesis in Adult and Young Rodents
Authors: Dunlop David S. , McHale Denise M. , Lajtha Abel
Published: October 2006

Abstract: In adult mice cerebral puncture results in an inhibition of brain protein synthesis, as suggested previously by Dunn (1975). The inhibition is apparent within a few minutes but subsides by 15 min after puncture. The percent inhibition therefore depends on the length of time between the puncture and the measurement. Mice receiving a puncture were less active than controls, and a decrease in brain temperature was observed in these animals. The decrement is, however, too small to account for the inhibition of synthesis. Diphenylhydantoin had no effect on the inhibition. Cerebral puncture of young mouse (7‐day‐oId) or rat (8‐day‐old) brain induced no inhibition of brain protein synthesis.
Effect of Immobilization Stress on Rat Pineal β‐Adrenergic Receptor‐Mediated Function
Authors: Yocca Frank D. , Friedman Eitan
Published: October 2006

Abstract: The results of this investigation indicate that stress produced by immobilization alters rat pineal function. Chronic stress reduced the density of pineal β‐adrenergic receptors and the activities of the intracellular enzyme serotonin N‐acetyltransferase (NAT), its product N‐acetylserotonin (NAS), and the pineal hormone melatonin, which was measured during the dark phase of the diurnal lighting cycle. Removal of the adrenal medulla did not prevent the reduction of pineal β‐adrenergic receptor binding sites that is observed after chronic stress. Acute immobilization stress suppressed the dark‐induced elevations of pineal NAT activity and NAS levels 10 h after the stress session without altering pineal β‐adrenergic receptor binding. Although the precise mechanisms responsible for these effects are not completely clear, the results indicate that they are related to changes in sympathetic neuronal activity and not mediated by stress‐induced elevations in plasma catecholamines.
Aerobic Glycolysis by the Pituitary Gland In Vivo
Authors: Viña Juan R. , Page Robert B. , Davis Donald W. , Hawkins Richard A.
Published: October 2006

Abstract: Arteriovenous differences of glucose, lactate, and pyruvate were measured across the pituitary glands of overnight‐fasted female pigs to determine whether net uptake of glucose occurred, and if so, whether it was oxidized or converted to lactate. Arteriovenous differences were also measured across a portion of the cerebral cortex as a control. Cerebral cortex oxidized 84% of the glucose taken up, but this was not true in the pituitary gland, where glucose was almost completely converted to lactate. The arteriovenous difference across the pituitary gland was 0.61 μmol/ml for glucose while the veno‐arterial differences of lactate and pyruvate were 1.0 and 0.07 μmol/ml, respectively. The results indicated that little net oxidation of glucose either by the Krebs cycle or the pentose phosphate pathway occurs in the pituitary gland in vivo. Estimates of the amount of energy released from aerobic glycolysis indicate that, unless the requirements of the neurohypophysis are very different from those in the rest of brain, very little energy could be derived from the metabolism of glucose.
Concurrent Determination of Brain Dopamine and 5‐Hydroxytryptamine Turnovers in Individual Freely Moving Rats Using Repeated Sampling of Cerebrospinal Fluid
Authors: Hutson P. H. , Sarna G. S. , Kantamaneni B. D. , Curzon G.
Published: October 2006

Abstract: 5‐Hydroxytryptamine (5‐HT) turnover and dopamine (DA) turnover values were obtained in individual conscious rats by measuring the rates of accumulation of 5‐hydroxyindoleacetic acid (5‐HIAA), 3,4‐dihydroxy‐phenylacetic acid (DOPAC), and homovanillic acid (HVA) in cisternal CSF samples taken from each rat at 0, 30, and 60 min after probenecid (200 mg/kg i.p.) administration. In a separate experiment, 5‐HT and DA turnover values were determined in CSF, striatum, and rest of brain of groups of rats killed 0, 30, or 60 min after probenecid. Whole brain turnover values were calculated from striatal and rest of brain values. Mean turnover values using CSF were comparable with both procedures. DA turnover values were greater when based on total (i.e., free + conjugated) DA metabolites than when based on free metabolites. After partial inhibition of monoamine synthesis with the decarboxylase inhibitor DL‐α‐monofluoromethyl‐DOPA (MFMD, 100 mg/kg p.o.) DA and 5HT turnover values were comparably reduced in whole brain, rest of brain, and CSF but more markedly reduced in the striatum. Mean DA and 5‐HT turnover values obtained using CSF were similar with probenecid doses over the range 150‐250 mg/kg i.p. but were variable when repeatedly determined in the same rats after administration of 200 mg/kg probenecid. Results in general show that the CSF procedure may be used to determine concurrently both 5‐HT and DA turnover (when estimated from the sum of total but not free metabolites) and that it provides a good index of whole brain turnover of these transmitters in the conscious individual rat.
Opposite Effects of Extracellular Sodium Removal on the Uptake of Tryptophan into Rat Cortical Slices and Synaptosomes
Authors: Wilkinson Lawrence S. , Collard Keith J.
Published: October 2006

Abstract: The removal of extracellular sodium decreased L‐tryptophan (L‐TRP) uptake into brain slices but in‐creased uptake into synaptosomes. The uptake of 5‐hy‐droxytryptamine (5‐HT) was inhibited in both preparations under these conditions. It is suggested that the dif‐ferent effects on L‐TRP uptake may be due to differencesin cellular composition between the two preparationsrather than to any differences in sample handling.
Pyruvate Dehydrogenase Activity in Regions of the Rat Brain During Postnatal Development
Authors: Butterworth Roger F. , Giguère Jean‐François
Published: October 2006

Abstract: Activity of the pyruvate dehydrogenase complex (PDHC) was measured in seven brain regions of themale rat at various times during the postnatal period usingan arylamine acetyltransferase coupled assay. Three daysafter birth, PDHC activity was found to be < 15% ofadult values in all brain regions with the exception of hypothalamus and medulla‐pons (30% of adult values ineach case). Activity of the enzyme complex in these latterregions attained adult levels by 21 days postnatally, some 5‐15 days ahead of that found in cerebral cortex, striatum, hippocampus, and cerebellum. Such differences in PDHC maturation reflect the greater degree of earlymaturity of the phylogenetically older brain structures. Cerebellar PDHC developed more slowly than in otherbrain regions to attain only 40% of adult levels by thetime of weaning. The pattern of maturation of cerebellarPDHC is paralleled by increased incorporation of glucoseinto cerebral amino acids and by the pattern of develop‐ment of parallel fiber synaptogenesis. These findings sug‐gest that PDHC may play a key role in the regional de‐velopment of metabolic compartmentation and the asso‐ciated maturation of cerebral function in the rat.
Norepinephrine Metabolism in Man Using Deuterium Labelling: Origin of 4‐Hydroxy‐3‐Methoxymandelic Acid
Authors: Mårdh Göran , Änggård Erik
Published: October 2006

Abstract: A double isotope labelling technique was used to simultaneously determine the in vivo turnover rates of 4‐hydroxy‐3‐methoxyphenylglycol (HMPG) and 4‐hydroxy‐3‐methoxymandelic acid (HMMA, VMA) and the rate of HMPG oxidation to HMMA. Six healthy men were given intravenous injections of [2H3]HMPG and [2H6]HMMA and their plasma and urine samples analysed by gas chromatography‐mass spectrometry (GC/MS) for the protium and deuterium species. HMPG and HMMA production rates were calculated by isotope dilution. The rate of HMPG oxidation to HMMA was obtained from the fraction of [2H3]HMPG recovered as [2H3]HMMA. The results showed that the entire production of HMMA, 1.11 ± 0.21 μmol/h (mean ± SE), could be accounted for by oxidation of HMPG, 1.49 ± 0.31 μmol/h. In another experiment designed to avoid expansion of the HMPG body pool, a tracer dose of [14C]HMPG was given to the same subjects. The levels of [14C]HMPG and [14C]HMMA were measured in urine after extraction and separation by thin layer chromatography. Urinary excretion of endogenous HMPG and HMMA was determined by GC/MS. The results showed that the endogenous HMMA fraction of the total HMPG and HMMA urinary excretion rate, 0.57 ± 0. 04, was the same as the fraction of [14C]HMPG oxidized to [14C]HMMA, 0.62 ± 0.01. Thus, HMPG is the main intermediate in the metabolic conversion of norepinephrine and epinephrine to HMMA in man.
Octopamine Release from Centrifugal Fibers of the Limulus Peripheral Visual System
Authors: Battelle B‐A. , Evans J. A.
Published: October 2006

Abstract: Octopamine, a biogenic amine, is synthesized and stored within centrifugal (efferent) fibers that project from the brain to the lateral and ventral eyes of the horseshoe crab, Limulus polyphemus. The experiments described here show that depolarization of Limulus lateral and ventral eyes, produced by elevating the concentration of extracellular K+, causes the selective release of newly synthesized octopamine from centrifugal fibers in a manner that requires the influx of extracellular Ca2+. Conjugates of octopamine and tyramine that are also stored within centrifugal fibers are not released in response to K+‐induced depolarization. These findings add further support to the hypothesis that octopamine is a neurotransmitter synthesized by and released from centrifugal fibers in Limulus eyes. This amine may be responsible for many of the alterations in lateral eye structure and function that are mediated by centrifugal inner‐vation.
Biochemical and Immunological Properties of a Membrane‐Bound Brain Metalloendopeptidase: Comparison with Thermolysin‐Like Kidney Neutral Metalloendopeptidase
Authors: Almenoff June , Orlowski Marian
Published: October 2006

Abstract: Membrane‐bound neutral metalloendopeptidase (“enkephalinase”) was purified from rabbit brain and compared with a homogeneous preparation of a similar enzyme (EC 3.4.24.11) isolated from rabbit kidney. The two enzymes had the same pH optimum and the same apparent molecular weight. They showed identical specificity toward several synthetic substrates and cleaved both Met‐ and Leu‐enkephalin at the Gly‐Phe bond. Minor, but significant, differences were found between the two enzymes in the inhibitory constants determined for phosphoramidon and the N‐[1(R,S)‐carboxy‐2‐phenylethyl] derivatives of phenylalanyl and alanyl‐p‐aminobenzoate. A guinea pig antiserum obtained against the rabbit kidney enzyme showed strong crossreactivity with the rabbit brain enzyme when tested in an anticatalytic immunoinhibition assay. Ouchterlony immunodiffusion experiments gave a pattern of precipitation consistent with partial identity of the two enzymes. The kidney enzyme, however, seemed to contain antigenic determinants not present on the brain enzyme. The data indicate that the two enzymes are identical with respect to specificity, pH optimum, and molecular weight, but show minor, although significant, differences in interaction with active‐site‐directed inhibitors and specific antisera.
Ethanolamine Accumulation by Photoreceptor Cells of the Rabbit Retina
Authors: Pu Glen A‐Wen , Anderson Robert E.
Published: October 2006

Abstract: The rabbit retina accumulates ethanolamine by an overall process that has a high affinity for ethanolamine. This process is different from the choline uptake, since ethanolamine accumulation was unaffected by high choline concentrations. Autoradiography identified the major site of high‐affinity uptake as the perinuclear region of the photoreceptor cells. Ethanolamine accumulated by the high‐affinity uptake was not used for neurotransmission by photoreceptor cells but was used to synthesize phosphatidylethanolamine. However, only a small percentage of the accumulated ethanolamine was converted into phospholipid. The rate of phosphorylation may contribute to control of phospholipid synthesis, since choline kinase activity is much greater than ethanolamine kinase activity in the rabbit retina.
Size and Surface Charge Properties of Myelin Vesicles from Normal and Diseased (Multiple Sclerosis) Brain
Authors: Moscarello M. A. , Chia L.‐S. , Leighton D. , Absolom D.
Published: October 2006

Abstract: Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross‐sectional area of the vesicles was 5.69 ± 0.17 μm2 from normal myelin and 3.71 ± 0.28 μm2 for diseased myelin. Vesicle size was reduced to 4.08 ± 0.21 μm2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2 during the preparation of the vesicles had no effect on the mean cross‐sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 ± 0.28 to 5.40 ± 0.31 μm2 Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was ‐5.169 ± 0.193 × 10−8 compared with ‐6.093 ± 0.202 × 10−8 m2 s−1 V−1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to ‐6.483 ± 0.151 × 10−8 m2 s−1 V−1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be ‐2.93 and ‐5.39 mV m−1 respectively. The phase transition temperature determined by wide angle x‐ray diffraction studies was 63°C for normal myelin vesicles and 43°C for MS myelin vesicles. The presence of Ca2 did not affect the phase transition temperature, whereas EDTA decreased it to 40°C in the case of normal myelin vesicles. In the case of MS vesicles, Ca2 increased the phase transition temperature from 43 to 53°C, whereas EDTA had no effect. From these data, we conclude that both the surface properties (surface charge) and the physical structure of the bilayer are affected in MS. The Ca2 concentration of the vesicles from normal myelin was slightly elevated over that of vesicles from MS myelin. The Mg2 concentrations were not significantly different.
Species Differences in the Binding of [3H]Nitrobenzylthioinosine to the Nucleoside Transport System in Mammalian Central Nervous System Membranes: Evidence for Interconvertible Conformations of the Binding Site/Transporter Complex
Authors: Hammond James R. , Clanachan Alexander S.
Published: October 2006

Abstract: The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site‐specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high‐affinity (KD 0.11–4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 < 20 nM and IC50 > 6 μM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitorinduced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.
Does Phospholipid Methylation Play a Role in the Primary Mechanism of Action of Nerve Growth Factor?
Authors: Ferrari Giovanna , Greene Lloyd A.
Published: October 2006

Abstract: Nerve growth factor (NGF)‐untreated (naive) and neurite‐bearing NGF‐treated (“primed”) PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite‐bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl‐3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono‐, di‐, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite‐bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.
Soluble and Particulate Forms of Rat Catechol‐O‐Methyltransferase Distinguished by Gel Electrophoresis and Immune Fixation
Authors: Grossman Mark H. , Creveling C. R. , Rybczynski Robert , Braverman Muriel , Isersky Chaviva , Breakefield Xandra O.
Published: October 2006

Abstract: Catechol‐O‐methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDSPAGE) of all tissue homogenates examined revealed three major immune‐specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 × g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an “aggregate” of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two‐dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pl of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1–5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is “soluble” with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.
Brain Gangliosides of Quaking and Shiverer Mutants: Qualitative and Quantitative Changes of Monosialogangliosides in the Quaking Brain
Authors: Iwamori Masao , Harpin Marie‐Luce , Lachapelle Francois , Baumann Nicole
Published: October 2006

Abstract: Ganglioside compositions in the brains of the mutant mice quaking and shiverer were compared with those of their littermate controls, C57BL/6 and C3HSWV. Neither ganglioside content nor composition of shiverer brains differed from those of the control brains. Change in the ganglioside composition of the mutant brain from that of the control was observed only in the quaking mutant brain, in which monosialoganglioside GM1 was significantly reduced and GM4 was completely absent. The structures of the gangliosides were determined by negative ion fast atom bombardment mass spectrometry, and the GM3 and GM4 gangliosides in the quaking brain were found to be altered in regard to their long‐chain base and fatty acid compositions when compared to the normal C57BL/6 brain.
Radiation Inactivation Studies of the Benzodiazepine/γ‐Aminobutyric Acid/Chloride Ionophore Receptor Complex
Authors: Schwartz Rochelle D. , Thomas John W. , Kempner Ellis S. , Skolnick Phil , Paul Steven M.
Published: October 2006

Abstract: Radiation inactivation was used to estimate the molecular weight of the benzodiazepine (BZ), γ‐amino‐butyric acid (GABA), and associated chloride ionophore (picrotoxinin/barbiturate) binding sites in frozen membranes prepared from rat forebrain. The target size of the BZ recognition site (as defined by the binding of the agonists [3H]diazepam and [3H]flunitrazepam, the antagonists [3H]Ro 15–1788 and [3H]CGS 8216, and the inverse agonist [3H]ethyl‐β‐carboline‐3‐carboxylate) averaged 51,000 × 2,000 daltons. The presence or absence of GABA during irradiation had no effect on the target size of the BZ recognition site. The apparent molecular weight of the GABA binding site labelled with [3H]muscimol was identical to the BZ receptor when determined under identical assay conditions. However the target size of the picrotoxinin/barbiturate binding site labelled with the cage convulsant [35S]t‐butylbicyclophosphorothionate was about threefold larger (138,000 daltons). The effects of lyophilization on BZ receptor binding activity and target size analysis were also determined. A decrease in the number of BZ binding sites (Bmax) was observed in the nonirradiated, lyophilized membranes compared with frozen membranes. Lyophilization of membranes prior to irradiation at – 135°C or 30°C resulted in a 53 and 151% increase, respectively, in the molecular weight (target size) estimates of the BZ recognition site when compared with frozen membrane preparations. Two enzymes were also added to the membrane preparations for subsequent target size analysis. In lyophilized preparations irradiated at 30°C, the target size for (i‐galactosidase was also increased 71% when compared with frozen membrane preparations. In contrast, the target size for glucose‐6‐phosphate dehydrogenase was not altered by lyophilization. Thus, in some cases lyophilization may lead to an increase in protein target size, possibly due to aggregation of protein subunits. We conclude that in frozen membrane preparations the functional target size of the BZ and GABA binding sites reflects the subunit molecular weights for the BZ/GABA/chloride ionophore receptor complex.
Characteristics of Tyrosine Hydroxylase Activation by K+‐Induced Depolarization and/or Forskolin in Rat Striatal Slices
Authors: El Mestikawy S. , Gozlan H. , Glowinski J. , Hamon M.
Published: October 2006

Abstract: The mechanisms of tyrosine hydroxylase (TH) activation by depolarization or exposure of dopaminergic terminals to cyclic AMP have been compared using rat striatal slices. Tissues were incubated with veratridine or 60 mM K+ (depolarizing conditions), on the one hand, and forskolin or dibutyryl cyclic AMP, on the other. K+ ‐(or veratridine‐)induced depolarization triggered an activation of TH (+ 75%) that persisted in soluble extracts of incubated tissues. This effect disappeared when drugs (EGTA, N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulfon‐amide, Gallopamil) preventing Ca2+‐ and calmodulin‐de‐pendent processes were included in the incubating medium. In contrast, prior in vivo reserpine treatment or in vitro addition of benztropine did not affect the depolarization‐induced activation of TH. In vitro studies of soluble TH extracted from depolarized tissues indicated that activation was associated with a marked increase in the enzyme Vmax but with no change in its apparent affinity for the pteridin cofactor 6‐methyl‐5,6,7,8‐tetrahydrop‐terin (6‐MPH4) or tyrosine. Furthermore, the activated enzyme from depolarized tissues exhibited the same optimal pH (5.8) as native TH extracted from control striatalslices. In contrast, TH activation resulting from tissue incubation in the presence of forskolin or dibutyryl cyclic AMP was associated with a selective increase in the apparent affinity for 6‐MPH4 and a shift in the optimal pH from 5.8 to 7.0–7.2. Clear distinction between the two activating processes was further confirmed by the facts that heparin‐ and cyclic AMP‐dependent phosphorylation stimulated TH activity from K+‐exposed (and control) tissues but not that from striatal slices incubated with forskolin (or dibutyryl cyclic AMP). In contrast, the latter enzyme but not that from depolarized tissues could be activated by Ca2+ ‐dependent phosphorylation. These data strongly support the concept that Ca2+– but not cyclic AMP‐dependent phosphorylation is responsible for TH activation in depolarized dopaminergic terminals.
Gastrocnemius Muscle Lipids in Relation to Diet in Two Mouse Mutants, 129Re‐dy and A2G‐adr, with Abnormal Muscle Function
Authors: Rebello Tessio , Watts David C.
Published: October 2006

Abstract: The lipids of gastrocnemius muscle from normal and dystrophic (dy) mice of the Bar Harbor, 129Re strain were studied. Animals were fed diets containing either 3.1% or 1.1% of total calories as linoleic acid. Lipid analyses were also done on muscle from a new mouse mutant, A2G‐adr, which has abnormal muscle function, characterised by an arrested development of the righting response. These animals were fed the “high” linoleic acid diet only. Total lipid, triacylglycerol, and cholesterol were elevated in the 129Re‐dy irrespective of the diet, whereas A2G‐adr possessed significantly higher levels of cholesterol. Total phosphorus (μg P/g muscle) and cholesterol/phospholipid ratios were elevated in the dy strains only. Cardiolipin was raised in the dy (“low” linoleic diet) and adr muscle, whereas phosphatidylcholine was lower in the adr strain only. Linoleic acid esterified to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine was elevated whereas arachidonic acid in phosphatidylserine was decreased in both mutants. Docosahexanoic acid (22:6) in all three dy phospholipids was decreased, independent of dietary treatment. The adr strain possessed normal levels of this fatty acid. The results specifically point to an abnormality in long‐chain polyunsaturated fatty acid metabolism in gastrocnemius muscle in the 129Re‐dy mutant; in the adr mutant they could reflect an abnormal increase in the number of muscle mitochondria.
Biochemical Studies on 5′‐Nucleotidase of Schwann Cells in Degenerated Nerve
Authors: Ross Stephen M. , Sabri Mohammad I. , Spencer Peter S.
Published: October 2006

Abstract: This report describes the partial characterization of 5′‐nucleotidase (5′‐AMPase) in Schwann‐cell plasmalemmae (PM) prepared from degenerated cat sciatic nerve. 5′‐AMPase was enriched 3.7‐fold in the PM fraction over that of the crude homogenate preparation. The plant lectin concanavalin‐A (Con‐A) reduced Schwann cell PM 5′‐AMPase activity in a concentration‐dependent manner (30‐600 μg/ml). Plasma membrane 5′‐AMPase activity was maximally inhibited to 20% of control values by Con‐A (400–600 μg/ml), and activity returned to control levels by pretreatment with the hapten sugar α‐methyl‐d‐mannoside (50 mM). Equimolar concentrations of UDP and ADP (100 μM) reduced the rate of hydrolysis of labeled AMP to labeled adenosine in PM to 45% and 35% of controls, respectively. This is the first study to characterize a Schwann‐cell PM enzyme and demonstrates that 5′‐AMPase may be used as a Schwann‐cell PM marker enzyme.
Neurochemical Abnormalities in Brains of Renal Failure Patients Treated by Repeated Hemodialysis
Authors: Perry Thomas L. , Yong Voon Wee , Kish Stephen J. , Ito Masatoshi , Foulks James G. , Godolphin William J. , Sweeney Vincent P.
Published: October 2006

Abstract: We examined autopsied brain from 10 patients with end‐stage renal failure who had undergone repeated hemodialysis. Eight had classic symptoms, and two had suggestive symptoms of dialysis encephalopathy. Findings were compared with those in autopsied brain from control adults who had never been hemodialyzed. Mean γ‐aminobutyric acid (GABA) contents were significantly reduced in frontal and occipital cortex, cerebellar cortex, dentate nucleus, caudate nucleus, and medial‐dorsal thalamus of the hemodialyzed patients, the reduction being >40% in cerebral cortex and thalamus. Choline acetyltransferase activity was reduced by 25–35% in three cortical regions in the hemodialyzed patients. These two abnormalities were observed in the brain of each hemodialyzed patient, regardless of whether or not the patient died with unequivocal dialysis encephalopathy. Pyridoxal phosphate contents were substantially reduced in brains of the hemodialyzed patients, but metabolites of noradrenaline, 3,4‐dihydroxyphenylethylamine (dopamine), and 5‐hydroxytryptamine (serotonin) were present in normal amounts. Aluminum levels were abnormally high in frontal cortical gray matter in the hemodialyzed patients. Although this study does not clarify the role played by aluminum toxicity in the pathogenesis of dialysis encephalopathy, the abnormalities we found suggest the need for further neurochemical investigations in this disorder.