bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Triethyl Tin on Myelination in the Developing Rat
Authors: Blaker William D. , Krigman Martin R. , Thomas David J. , Mushak Paul , Morell Pierre
Published: October 2006

Myelinogenesis in developing rats was studied following chronic dosing with triethyl tin (TET), at a level of 1.0 mg TET/kg body wt/day. Experiments included starved controls with body weights depressed by 17 to 40% to equal those of the TET‐treated groups. Rats at ages of 16, 21, and 30 days showed decreases relative to well‐nourished controls in body weight, forebrain weight, myelin yield, cerebroside level, and specific activity of brain 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase when dosed with TET. At 30 days, myelin and cerebroside yields were reduced by approximately 55%, while CNP activity was reduced by less than 20%. No differences in the forebrain myelin protein composition between control, starved, and TET animals were noted. The rate of myelin protein synthesis relative to brain total protein (assayed by incorporation of intracranially injected [3H]glycine into brain homogenate and myelin proteins) was decreased in the TET rats in proportion to the decreased yield of myelin, but no particular myelin protein was preferentially affected. Matching starved controls exhibited similar body weight decreases, less pronounced forebrain weight decreases, and little or no decrease in myelin concentration. There was a relative increase in the myelin protein synthesis rate in the starved rats, indicating preferential utilization of limited protein precursors for myelin protein synthesis. Spinal cord myelin was also decreased in the TET rats, but less severely than in the forebrain. At all ages optic, but not sciatic, nerves showed decreases in myelin concentration with TET treatment. We conclude that TET inhibits forebrain growth and CNS myelination more severely than can be accounted for by a general metabolic insult.
The Activity of Rat Pineal and Brain Tyrosine Hydroxylase During the Daily Cycle of Light and Darkness as Determined by the Modified 14CO2 Assay Method
Authors: Watanabe Shuzo , Tom Michio , Ichiyama Arata , Kataoka Tohru
Published: October 2006

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a two‐step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X‐100 were as follows. The apparent Km values of the brain enzyme for L‐tyrosine with 1 mM‐(6‐dl)‐5,6,7,8‐tetrahydro‐L‐erythro‐biopterin (BPH4) as cofactor and for BPH4 with 62 μM‐L‐tyrosine as substrate were approximately 25 μM and 85 μM, respectively. The Km for L‐tyrosine with 1 mM‐(6‐dl)‐5,6,7,8‐tetra‐hydro‐6‐methylpterin (6MPH4) as cofactor and for 6MPH, with 210 μM‐L‐tyrosine as substrate were 68 μM and 270 μM, respectively. The marked substrate inhibition by high concentrations of L‐tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver‐Burk plot of reciprocal velocity versus the reciprocal concentration of BPH4 with 62 μM‐L‐tyrosine as substrate deviated downward at a BPH4 concentration of about 100 μM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (61.‐and 6d‐isomers) of the tetrahydrobiopterin cofactor, with apparent Kmfor BPH4 of 23 μM and 1025 μM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.
Differential Uptake of Horseradish Peroxidase Isoenzymes by Cultured Neuroblastoma Cells
Authors: Chan Kwan Y. , Haschke Richard H.
Published: October 2006

The endocytotic uptake and intracellular decay of horseradish peroxidase isoenzymes C and A by cultured mouse neuroblastoma cells were analyzed quantitatively by a direct spectrophotometric assay. At concentrations below 1 mg/ml, the rate of uptake of the isoenzyme C was more than three times as much as the isoenzyme A. This differential uptake suggests that previous claims of horseradish peroxidase being endocytosed only in the nonselective fluid phase are oversimplified. The implication of this selectivity in the biological significance of retrograde axonal transport of proteins by neuronal systems is discussed.
Disposition of Gangliosides and Sialosylglycoproteins in Neuronal Membranes
Authors: Landa C. A. , De Filpo S. S. , Maccioni H. J. F. , Caputto R.
Published: October 2006

Abstract Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP‐[3H]N‐acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [3H] sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 m‐KCl, 125 mm‐EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of the neuraminidase‐labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase‐labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labeled in vivo by an intracerebral injection of N‐[3H]acetylmannos‐amine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with the behavior of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.
Developmental Changes of N‐Acetyl‐L‐Aspartic Acid, N‐Acetyl‐α‐Aspartylglutamic Acid and β‐Citryl‐L‐Glutamic Acid in Different Brain Regions and Spinal Cords of Rat and Guinea Pig
Authors: Miyake Masaharu , Kakimoto Yasuo
Published: October 2006

Abstract The developmental changes of N‐acetylaspartic acid (NA‐Asp), N‐acetyl‐α‐aspartylglutamic acid (NA‐Asp‐Glu), and β‐citryl‐L‐glutamic acid (β‐CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA‐Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA‐Asp‐Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA‐Asp‐Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β‐CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β‐CG content decreased to an adult level at birth.
Choline Acetyltransferase in Skeletal Muscle from Patients with Myasthenia Gravis
Authors: Molenaar P. C. , Newsom‐Davis J. , Polak R. L. , Vincent A.
Published: October 2006

Abstract— Acetylcholine synthesis in homogenates of human intercostal muscle was measured by a radiochemical method. Choline acetyltransferase activity in control muscle was about 20 nmol.g−1.h−1. The enzyme was found only in the endplate area of the muscle. At high substrate concentrations its activity was overshadowed by the acetylcholine synthesizing activity of a different enzyme not saturated by 10 mm‐choline. The nonspecific enzyme was present at and away from the endplate area. Choline acetyltransferase in parasternal samples of intercostal muscle from myasthenia gravis patients was about 2.5 times higher than in samples, taken from a more lateral location, of control patients, but the Km for choline was not altered (0.24 mm). It is suggested that in myasthenia gravis the shortage of acetylcholine receptors is partially compensated for by increased synthesis, storage, and release of the transmitter.
Ontogenetic and Imprinting‐Induced Changes in Chick Brain Protein Metabolism and Muscarinic Receptor Binding Activity
Authors: Longstaff Alan , Rose Steven P. R.
Published: October 2006

Abstract— Over the 20‐min period following exposure of young chicks to a flashing light as an imprinting stimulus there is an increased incorporation of [14C]leucine into an acidic (tubulin‐enriched) protein fraction of the anterior dorsal forebrain in birds which have learnt the characteristics of the stimulus as compared with, either birds which have been exposed to an imprinting stimulus but learn poorly, or chicks kept in the dark. This brain region has been implicated in several studies as the locus for a number of biochemical modulations that accompany learning. The amount of [14C]leucine incorporated does not seem to be determined by precursor pool availability; it does, however, correlate with a well‐validated measure of the extent to which birds have learnt to recognise the characteristics of the stimulus, as shown by a two‐choice discrimination test. There is no change in the total content of tubulin dimer as assayed by colchicine binding under these conditions. Additionally, in birds which show evidence of learning, the binding of quinuclidinyl benzilate, an irreversible muscarinic ligand, is altered in both the posterior dorsal forebrain and midbrain regions. None of these effects could be simply the result of visual stimulation. The meaning of these changes is discussed.
Compartments of Labeled and Endogenous γ‐Aminobutyric Acid Giving Rise to Release Evoked by Potassium or Veratridine in Rat Cortical Slices
Authors: Szerb John C. , Ross T. Elaine , Gurevich Lea
Published: October 2006

Abstract— To establish compartments involved in depolarization‐induced release of γ‐aminobutyric acid (GABA) in rat brain slices, the amount of exogenous labeled and endogenous GABA released and retained was followed during 48 min exposure to 50 mm‐K+ or to 50 μm‐veratridine. Endogenous GABA was measured with high performance liquid chromatography. The presence of 10 μm‐aminooxyacetic acid throughout prevented both the metabolism of GABA and the formation of endogenous GABA due to depolarization. During super‐fusion with 50 mm‐K+ and 2.6 mm‐Ca2+ the efflux of labeled and endogenous GABA after an initial large increase declined to 10% of the highest value with constant and identical rates. Kinetic analysis of efflux showed that 10% of endogenous and 25% of labeled GABA present is available for release by high K+ and Ca2+. In the absence of Ca2+, release by high K+ of both labeled and endogenous GABA was nearly suppressed. Veratridine, unlike high K+, caused an efflux which declined with an initial fast and late very slow phase. The slow efflux by veratridine was doubled in the absence of Ca2+. Exposure to veratridine in the absence of Ca2+ during 120 min released nearly 70% of labeled and endogenous GABA present. Results suggest that only about 0.25 μmol g−1 endogenous GABA is the source of physiological Ca2+‐dependent release, while much of the remaining GABA present is released only under unphysiological conditions.
Dual Action of Pentobarbitone on GABA Binding: Role of Binding Site Integrity
Authors: Willow Max , Johnston Graham A. R.
Published: October 2006

Abstract— The effects of pentobarbitone on the binding of γ‐aminobutyric acid (GABA) to crude synaptosomal rat brain membranes were studied. In extensively washed P2 membranes, pentobarbitone had a biphasic action: at concentrations ranging between 12.5 and 500 μm, pentobarbitone enhanced GABA binding in a concentration‐dependent manner; at concentrations greater than 500 μm, this enhancement was progressively reversed towards control levels of GABA binding. The effect of pentobarbitone seen at higher concentrations may reflect a GABA‐mimetic action, since similar concentrations enhanced diazepam binding to washed P2 membranes, an effect antagonized by bicuculline methochloride and picrotoxinin. When washed P2 membranes were incubated in 0.5% Triton X‐100 (30 min at 37°C), the enhancement of GABA binding by low concentrations of pentobarbitone was abolished, while at higher concentrations GABA binding was progressively inhibited, suggesting that the GABA‐mimetic action is retained. When washed P2 membranes were subjected to high‐frequency homogenization, the biphasic dose‐response relationship for pentobarbitone was markedly shifted to the right. The choice of membrane preparation appears to be a critical factor in examining drug‐receptor interactions in vitro, at least for those involving GABA and the barbiturates.
Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells
Authors: Toews Arrel D. , Morell Pierre
Published: October 2006

Abstract— We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2‐3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half‐lives of approximately 7 days. Apparent half‐lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half‐lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half‐lives for axonally transported phospholipids labeled with [32P]‐phosphate were only slightly longer than for [2‐3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.
Occurrence of Two Types of Ca2+‐Dependent Protein Kinases in the Cytosol Fraction of the Brain
Authors: Miyamoto E. , Fukunaga K. , Matsui K. , Iwasa Y.
Published: October 2006

Abstract— Two types of Ca2+‐dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE‐cellulose, Sephadex G‐200, and calmodulin‐affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a Ka value of 16.7 nm. The Ka values of peaks I and II for Ca2+ were 2.4 and 1.6 μm, respectively.
The Effect of Anti‐Synaptosomal Membrane Antibodies on Neurotransmitter Uptake
Authors: Hofstein Raphael , Barnett Barbara , Samuel David
Published: October 2006

Abstract: Antibodies raised against synaptosomal plasma membranes of rat hippocampus (anti‐HPC IgG) caused inhibition of [3H]noradrenaline, [3H]5‐hydroxytryptamine, [3H]GABA and [3H]aspartate uptake into S1 fractions and slices of hippocampus and cerebral cortex, but not those of caudate nucleus and hypothalamus. Similar inhibition was not observed on using antibodies against synaptosomal membranes of rat caudate nucleus. Anti‐HPC JgG raised against synaptosomal membranes of hippocampus failed to alter both spontaneous and K+‐evoked release of [3h]noradrenaline. They did not interfere with the binding of [3H]desipramine (the potent noradrenaline‐uptake inhibitor) and with the binding of [3H]dihydroalprenolol, thus excluding any interaction of the antibodies with drug receptors which are located on either the pre‐ or postsynaptic membrane. The anti‐HPC IgG inhibit the enzymatic activity of [Na+‐K+]ATPase by 30% upon incubation of the antibodies with crude membrane preparations. A comparison of their inhibitory effects with those of the neurotoxin 6‐hydroxydopamine suggests that the corresponding hippocampal specific antigens are located at a presynaptic site.
Purification of Sciatin Using Affinity Chromatography on Concanavalin A‐Agarose
Authors: Markelonis George J. , Oh Tae Hwan
Published: October 2006

Abstract: A glycoprotein from chicken sciatic nerves, sciatin, has been shown to have trophic effects on the maturation and maintenance of skeletal muscle cells in culture. This protein was purified 24‐fold from sciatic nerve extracts by affinity chromatography on concanavalin A‐agarose followed by ion‐exchange on diethylaminoethyl cellulose. The purity of sciatin obtained by this procedure was greater than 97% as estimated by densitometric integration of sodium dodecyl sulfate gels, and represented 33% of the sciatin present in sciatic nerve extracts as determined by rocket immunoelectrophoresis. Sciatin purified by this technique retained full biological activity since (1) addition of the protein to embryonic chicken skeletal muscle cells in culture enhanced the morphological development of the cells, and (2) the protein increased the number of acetylcholine receptors as measured by binding of 125I‐α‐bungarotoxin to 261% of the control value after 4 days in vitro. The purification procedure described in the present communication provides a more rapid and convenient method for the isolation of this trophic protein.
Uptake of 36Cl and 22Na by the Choroid Plexus‐Cerebrospinal Fluid System: Evidence for Active Chloride Transport by the Choroidal Epithelium
Authors: Smith Quentin R. , Woodbury Dixon M. , Johanson Conrad E.
Published: October 2006

Abstract: Cl and Na transport by the lateral ventricle (LVCP) and fourth ventricle (4VCP) choroid plexuses were examined by kinetic analysis of 36Cl and 22Na uptake into the choroid plexus‐CSF system of the adult rat. Both radioisotopes required more than 5 h to reach steady‐state distribution in the in vivo choroid plexuses and CSF after intraperitoneal injection. Whereas the LVCP and 4VCP 36Cl steady‐state spaces were comparable (55–56%), the 4VCP 22Na space (39%) tended to be greater than the LVCP 22Na space (36%). No evidence for inexchangeable Cl or Na was found for the choroid plexuses; the radioisotopic and chemical spaces were not significantly different. Choroid plexus 36Cl and 22Na uptake curves were resolved into two components, a fast component (t1/2 0.02–0.05 h) and a slow component (t1/2 0.85–1.93 h). By analysis of the distribution of [3H]inulin, [3H]mannitol, and 51Cr‐tagged erythrocytes within the choroid plexuses, the fast component of 36Cl and 22Na uptake was found to represent extracellular and erythrocyte contributions to the tissue radioactivity, whereas the slow component represented isotope movement into the epithelial cell compartment. The calculated cell [Cl] of LVCP and 4VCP, 67 mmol/kg cell water, was 3.9 times greater than that predicted by the membrane potential for passive distribution. It is postulated that Cl is actively transported into the choroid epithelial cell across the basolateral membrane; the energy source for active Cl transport may be the Na electrochemical potential gradient (˜90 mV), which is twice that of the Cl electrochemical potential gradient (˜45 mV).
Immunological, Physical, and Chemical Evidence for the Identity of Brain and Kidney Post‐Proline Cleaving Enzyme
Authors: Hersh Louis B.
Published: October 2006

Abstract: Recent studies from this laboratory have suggested a similarity, if not identity, of thyrotropin releasing factor (TRF) deamidase and post‐proline cleaving enzyme. Bovine brain TRF deamidase was purified to homogeneity and used to elicit antibodies to the enzyme. These antibodies were used to demonstrate identical immunological reactivity between rat brain TRF deamidase and rat kidney post‐proline cleaving enzyme. In addition, both proteins exhibit a molecular weight of 75,000, and have identical Km values for the synthetic substrate pGlu‐(N‐benzyl‐l‐His)‐Pro‐β‐naphthylamide and identical K1 values for TRF and luteinizing hormone releasing factor as inhibitors. Finally, the enzymes exhibit the same sensitivities to inhibition by mercury, iodoacetamide, N‐ethylmaleimide, and 5, 5′‐dithiobis‐(2‐nitrobenzoic acid). These results strongly suggest that brain TRF deamidase and kidney postproline cleaving enzyme are identical.
2‐Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice
Authors: Mikoshiba Katsuhiko , Kohsaka Shinichi , Takamatsu Ken , Tsukada Yasuzo
Published: October 2006

Abstract: The 2‐deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2‐deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2‐deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2‐deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.
Mutually Independent Cyclic AMP and Sodium Responses to Nerve Growth Factor in Embryonic Chick Dorsal Root Ganglia
Authors: Skaper Stephen D. , Varon Silvio
Published: October 2006

Abstract: Chick embryo dorsal root ganglia display a rapid and transient rise in their cyclic AMP content when presented with nerve growth factor. These ganglia also depend on nerve growth factor for control of their intracellular Na+ and K+ levels. A sequential relationship between the cyclic AMP and Na+ responses is not readily apparent. Incubation of chick sensory ganglia in a sodium‐free medium does not prevent the cyclic AMP response to nerve growth factor from occurring. When ganglia are first incubated with ouabain for 6 h, presentation of nerve growth factor elicits a cyclic AMP response, but no Na+ response. The cyclic AMP response therefore does not depend on the Na+ environment. An initial presentation of nerve growth factor to the ganglia for 30 min, followed by its withdrawal and subsequent re‐administration at different intervals over several hours failed to result in a second cyclic AMP response. Nevertheless, the expected Na+ behaviors were still observed. Dibutyryl cyclic AMP is capable of eliciting a cyclic AMP response in chick sensory ganglia after 6 h of nerve growth factor deprivation. When both agents were presented simultaneously to the ganglia, only a single cyclic AMP response was obtained, corresponding in time to the response elicited by dibutyryl cyclic AMP alone‐indicating that this drug acts on the NGF‐sensitive cells. At the same time dibutyryl cyclic AMP alone failed to result in a Na+ response, leading one to conclude that the cyclic AMP response to nerve growth factor is truly not mediating the Na+ response. Additional support for the mutual independence of these two short‐latency responses is provided by the apparent inability of nerve growth factor to cause a cyclic AMP response in chick embryo sympathetic ganglia, another traditional target for the factor, which is capable of displaying a Na+ response.
Impairment of Protein Synthesis in the Retinal Tissue in Diabetic Rabbits: Secondary Reduction of Fast Axonal Transport
Authors: Chihara Etsuo
Published: October 2006

Abstract: Protein biosynthesis in the retina and fast axonal transport along the optic pathway were studied in rabbits in which diabetes had been experimentally induced. Retinal protein biosynthesis and axonal transport were significantly reduced in the diabetic rabbits, and the reduction was correlated to the severity of the diabetes. The 'somal delay time’was only slightly elongated and the O/R ratio was fairly constant in the various levels of blood glucose; thus intrasomal protein movement seems to be less affected in diabetic rabbits. Velocity and the distribution pattern of axonally transported protein remained unaffected in the diabetic rabbits. These findings suggest that a disturbance in the metabolism in the cell body is the most important factor related to quantitative reduction of fast axonal transport in diabetic rabbits.
Quantitation of Cholinergic Synaptosomes from Guinea Pig Brain
Authors: Richardson Peter J.
Published: October 2006

Abstract: An antiserum raised to nerve terminal sacs derived from the electric organ of Torpedo marmorata was used to lyse guinea pig brain synaptosomes in the presence of complement. From the release of the cytoplasmic enzymes choline acetyltransferase, lactate dehydrogenase, tyrosine hydroxylase and glutamate decarboxylase it appears that the antiserum binds specifically to cholinergic terminals. The amount of lactate dehydrogenase released was used to estimate the proportion of cholinergic nerve terminals in different synaptosome preparations.
In Vitro Release of Endogenous Amino Acids from Granule Cell‐, Stellate Cell‐, and Climbing Fiber‐Deficient Cerebella
Authors: Flint R. S. , Rea M. A. , McBride W. J.
Published: October 2006

Abstract: The K+‐stimulated, Ca2+‐dependent release of glutamate, aspartate, ‐γ‐aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell‐, granule cell plus stellate cell‐, or climbing fiber‐deficient cerebella of the rat. The 55 mm‐K+‐stimulated release of glutamate and GABA was 10‐fold greater in the presence of Ca2+ than in its absence. The stimulated release of aspartate was 4‐fold higher when Ca2+ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca2+. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg2+ concentration to 16 HIM inhibited the K+‐stimulated, Ca2+‐dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K+‐stimulated, Ca2+ dependent release of glutamate, aspartate, and alanine from x‐irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x‐irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K+‐stimulated, Ca2+‐dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber‐deficient cerebella obtained from 3‐acetylpyridine‐treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co‐released along with glutamate from granule cells.
Conformations of P2 Protein of Peripheral Nerve Myelin by Nuclear Magnetic Resonance Spectroscopy
Authors: Chapman B. E. , James G. E. , Moore W. J.
Published: October 2006

Abstract: High‐resolution 1H NMR spectra of P2 protein from bovine peripheral nerve myelin indicate that the protein contains a high degree of tertiary structure in aqueous solution. Denaturation of the protein in urea solutions is a multi‐step process. Binding of lysophosphatidylcholine micelles to the protein causes a conformational change and a broadening of NMR peaks from side chains of aromatic amino acid and methionine residues, with much less effect on upfield methyl resonances.
Increased Incorporation of [3H]Lysine into Synaptic Membranes of the Visual Cortex Following First Exposure of Dark‐Reared Rats to Light Is Confined to Particular Polypeptides
Authors: Burgoyne Robert D. , Rose Steven P. R. , Harding Susan
Published: October 2006

Abstract: The incorporation of [3H]lysine into separated polypeptides of synaptic‐membrane fractions prepared from the visual cortices of dark‐reared rats and littermates exposed to light for 1 h was examined. Increased incorporation of [3H]lysine was found in synaptic membranes from light‐exposed compared to dark‐reared rats in polypeptides of four molecular weights: 100,000, 71,000, 44,000, and 38,000. The 44,000‐molecular‐weight peak has been suggested to be actin on the basis of its comigration with pure brain actin. These results indicate that increased incorporation of [3H]lysine into synaptic membranes of the visual cortex, following first exposure of dark‐reared rats to light, is confined to particular polypeptides.
Distribution of Catecholamines in the Ventral Mesencephalon of Human Brain, with Special Reference to Parkinson's Disease
Authors: Javoy‐Agid F. , Taquet H. , Ploska A. , Cherif‐Zahar C. , Ruberg M. , Agid Y.
Published: October 2006

Abstract: The high levels of dopamine (DA) detected in the ventral tegmental area (VTA), as well as in the substantia nigra (SN) of human brain, suggest the presence of DA cells in these areas. This favors the possible existence of a mesocortico‐limbic system besides the mesostriatal pathway. In Parkinson's disease both DA systems seem to be deficient.
Biochemical and Histochemical Studies on Catecholamines in Bullfrog Spinal Ganglia
Authors: Kojima H. , Fukushima S. , Hirono T. , Suetake K. , Toshima N. , Yoshida M. , Anraku S. , Inanaga K.
Published: October 2006

Abstract: Bullfrog spinal ganglia were subjected to two types of examination to determine whether catecholamines were present. A biochemical microassay developed by Kissinger and co‐workers and a histofluorescence technique for cellular demonstration of biogenic monoamines developed earlier by Falck and co‐workers were used. Bullfrog spinal ganglia and dorsal roots were found to contain catecholamines, primarily adrenaline.
Effects of Pentobarbitone on 45Ca2+ Transport by Rat Brain Mitochondria
Authors: Willow Max , Bygrave Fyfe L.
Published: October 2006

Abstract: The effects of pentobarbitone on the transport of 45Ca2+ by rat brain mitochondria were studied, using the Ruthenium Red‐EGTA quench technique. In the presence of succinate and inorganic phosphate, mitochondria rapidly accumulate 45Ca2+. Pentobarbitone (0.1–1.0 mM) stimulates the initial rate of Ca2+ transport. In contrast, pentobarbitone (1 mM) did not affect the NaCl (50 mM)‐induced efflux of 45Ca2+ from mitochondria. Dibucaine (60 μM), a clinically used local anaesthetic, inhibits both 45Ca2+ uptake and efflux. The results suggest that barbiturate stimulation of mitochondrial Ca2+ uptake may, in combination with effects on other Ca2+ sequestering processes, contribute to the inhibition of transmitter release observed at a number of synapses.
Effects of Drugs Interfering with Dopamine and Noradrenaline Biosynthesis on the Endogenous 3,4‐ Dihydroxyphenylalanine Levels in Rat Brain
Authors: Westerink B. H. C. , van Es T. P. , Spaan S. J.
Published: October 2006

Abstract: A chemical assay of 3,4‐dihydroxyphenylalanine (DOPA) in nervous tissue is described. The method is based on a rapidly performed isolation of DOPA on small Sephadex G‐10 columns, followed by reverse‐phase HPLC with a trichloroacetic acid‐containing eluent, in conjunction with a rotating disk electrochemical detector. The detection limit of the assay (about 100 pg/tissue sample) permits a detailed investigation of the regional distribution of endogenous DOPA levels in the rat brain. DOPA as well as dopamine (DA) could be quantified in the same chromatographic run. The assay was applied to a study of the effects of α‐methyl‐p‐tyrosine, apomorphine, chlorpromazine, clonidine, γ‐butyrolactone, haloperidol, morphine, oxotremorine, pargyline, reserpine, and tyrosine methylester on the concentration of DOPA in the striatum, hypothalamus, frontal cortex, and cerebellum of the rat brain. Drugs known to interact with DA biosynthesis all caused characteristic changes of the DOPA content in the striatum and not in nondopaminergic brain areas. A close correlation existed between drug‐induced changes in tyrosine hydroxylase activity and changes in the DOPA content in the striatum. Tyrosine methylester increased DOPA concentrations in all brain areas studied.
Monoamine Synthesis and Concentration in Neonatal Rat Brain During Hypercapnia and Recovery
Authors: Hedner T. , Lundborg P.
Published: October 2006

Abstract: One‐day‐old rats were exposed to a gas mixture of 15% CO2–21% O2–64% N2 for a 30‐min period. Monoamine synthesis in whole brain was measured during, and at various intervals after, hypercapnia by estimating the accumulation of dihydroxyphenylalanine (DOPA) and 5‐hydroxytryptophan (5‐HTP) after inhibition of aromatic l‐amino‐acid decarboxylase with NSD 1015. Endogenous concentrations of tyrosine, dopamine (DA), noradrenaline (NA), tryptophan, 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) were measured at the same intervals. Exposure to CO2 induced an increased synthesis of catecholamines and 5‐HT. Further, an increase in DA concentration was seen during hypercapnia, while NA and 5‐HT were unchanged. After the CO2 exposure the increased in vivo synthesis rates of catecholamines and 5‐HT were rapidly normalized, as was the endogenous DA concentration. A slight increase in 5‐HT and 5‐HIAA concentrations was seen immediately after CO2 exposure. These results indicate that in neonatal animals, hypercapnia induces changes in central monoamine neurons, primarily an increased synthesis. These alterations may be relevant to some physiological changes seen during CO2 exposure, such as the alteration in central respiratory performance.
Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes
Authors: Bauer Ulrike , Brand Karl
Published: October 2006

Abstract: Synaptosomes were isolated from rat cerebral cortex and incubated with [U‐14C]‐, [1‐14C]‐ or [6‐14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10−5M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.
Application to Mammalian Tissues of the Chemiluminescent Method for Detecting Acetylcholine
Authors: Israël Maurice , Lesbats Bernard
Published: October 2006

Abstract: It is now possible to extend to mammalian tissues the chemilumi‐nescent acetylcholine assay. Mammalian tissue extracts must be treated with oxidants (which is not necessary for electric organ extracts). The assay can then be performed as previously described (acetylcholinesterase hydrolyses acetylcholine; choline oxidase converts choline to betaine and H2O2, which gives off light in the presence of luminol and peroxidase). It is also shown that release experiments can be performed on mammalian tissue slices (mouse caudate nucleus) after the slice is washed in oxygenated saline solutions.
The Regional Distribution and Chromatographic Characterisation of Neurotensin‐Like Immunoreactivity in the Rat Central Nervous System
Authors: Emson P. C. , Goedert M. , Horsfield P. , Rioux F. , St. Pierre S.
Published: October 2006

Abstract: Carboxy‐ and amino‐terminal specific neurotensin antisera have been characterized and used to determine the nature of neurotensin‐like immunoreactivity in the rat central nervous system. Using these antisera, together with the separation of neurotensin‐like immunoreactivity on reversephase HPLC columns, it is clear that the majority of rat central nervous system neurotensin‐like immunoreactivity is indistinguishable from the synthetic tridecapeptide. However, smaller amounts of carboxy‐ and amino‐terminal neurotensin‐like immunoreactivity were detected, which may correspond to carboxy‐ and amino‐terminal fragments of neurotensin. In addition, using the amino‐terminal directed neurotensin antiserum, a detailed distribution of neurotensin‐like immunoreactivity in the rat central nervous system is described. Highest amounts were found in the hypothalamus, central amygdaloid nucleus, bed nucleus of the stria terminalis and the substantia gelatinosa of the spinal cord and of the trigeminal region.
Comparison of the Molecular Forms of the Cholinesterases in Tissues of Normal and Dystrophic Chickens
Authors: Lyles Joan M. , Silman Israel , Di Giamberardino Luigi , Couraud Jean‐Yves , Barnard Eric A.
Published: October 2006

Abstract: The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (ΦChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed‐fibre‐type twitch muscles varies in proportion to the percentage of glycolytic fast‐twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow‐tonic, oxidative‐glycolytic fast‐twitch, or oxidative slow‐twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and ΦChE are more severe in muscles richer in glycolytic fast‐twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or ΦChE in a slow‐tonic muscle. In the pectoral or PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the ΦChE pattern remains abnormal. Muscle ΦChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of ΦChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or ΦChE were found in cardiac or neural tissues from dystrophic chickens.
A Regional Study of 4‐Aminobutyrate Metabolism and Amino Acid Levels in Rat Brain Following Chronic Oral Administration of Ethanolamine O‐Sulphate
Authors: Fletcher Allan , Fowler Leslie J.
Published: October 2006

Abstract: Ethanolamine O‐sulphate (EOS) dissolved in the drinking water (5mg‐ml−1) was administered ad libitum to rats for 26 days. At the end of this period, glutamate decarboxylase (GAD) and GABA‐transaminase (GABA‐T) activities, 4‐aminobutyrate (GABA) concentration, and the levels of six other amino acids were measured in various brain regions. Significant inhibition of GABA‐T accompanied by significant increases in GABA content were observed throughout the brain, although the magnitudes of these effects varied according to region. GAD activity was significantly reduced in most brain regions, although this effect was apparently not related to cofactor availability or the direct actions of EOS or increased GABA concentration. Glutamine levels were significantly reduced to approximately 72% of control values in all brain regions. Aspartate levels were significantly reduced to approximately 84% of control values in all regions except the striatum and cerebellum. Minor changes in other amino acid levels were also detected. These neurochemical changes which accompanied the primary effect of EOS on GABA‐T are discussed in terms of indirect secondary metabolic changes rather than nonspecific enzyme inhibition by EOS.
Pyrrolines as Prodrugs of γ‐Aminobutyric Acid Analogues
Authors: Callery Patrick S. , Geelhaar Linda A. , Nayar M. S. Balachandran , Stogniew Martin , Rao K. Gurudath
Published: October 2006

Abstract: Δ′‐Pyrroline, S‐methyl‐Δ′‐pyrroline, and 5.5‐dimethyl‐Δ′‐pyrroline have been identified as substances metabolized to γ‐aminobutyric acid (GABA), 4‐aminopentanoic acid (raethyl GABA), and 4‐amino‐4‐methylpen‐tanoic acid (dimethyl GABA), respectively. An enzyme system residing in the soluble fraction of rabbit liver catalyzes the conversion of Δ′‐pyrroline to GABA and its lactam, 2‐pyrrolidinone. Acetaldehyde, allopurinol, and cyanide inhibited the reaction. Incubation of deuterium‐labeled Δ′‐pyrroline with mouse brain homogenates produced deuterated GABA. Mouse liver 10,000 g supernatant and mouse brain homogenates converted S‐methyl‐Δ′‐pyrroline to methyl GABA, and 5,5‐dimethyl‐Δ′‐pyrroline to dimethyl GABA. Four hours after intraperitoneal injection of 5‐methyl‐Δ′‐pyrroline (200 mg/kg), methyl GABA was detected in mouse brain (0.27 μ‐mol/g). Dimethyl GABA (1.21 μmol/g) was determined in mouse brain 30 min after intraperitoneal administration of 5.5‐dimethyl‐Δ′‐pyrroline (200 mg/kg). Neither methyl GABA nor dimethyl GABA penetrated into the central nervous system when administered in the periphery. The present studies suggest that pyrrolines may represent a chemical class of brain‐penetrating precursors of pharmacologically active analogues of GABA.
On the Identification of α‐ and β‐Tubulin Subunits
Authors: Perry G. W. , Wilson David L.
Published: October 2006

Abstract: Confusion appears to have arisen in the literature regarding the designation of α‐and β‐tubulin in polyacrylamide gels. The presence or absence of 8 M‐urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M‐urea‐SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post‐translational tyrosylation of protein appears exclusively restricted to α‐tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α2 subunit of tubulin is tyrosylated.
A High‐Affinity, Na+‐Dependent Uptake System for γ‐Hydroxybutyrate in Membrane Vesicles Prepared from Rat Brain
Authors: Benavides J. , Rumigny J. F. , Bourguignon J. J. , Wermuth C. G. , Mandel P. , Maitre M.
Published: October 2006

Abstract: γ‐Hydroxybutyrate (GHB) is a compound with numerous neuropharmacological properties. The discovery of its biosynthetic system, together with its endogenous repartition, have prompted its possible implication in neurotransmission. This role is also supported by the existence, reported here, of a high‐affinity uptake system for GHB (Km= 46.4 μM)in both purified brain plasma membrane vesicles and in the crude mitochondrial fraction. GHB uptake is dependent on a Na+ gradient but is independent of the membrane electrical potential. Cl− and K+ can also modulate the uptake. As an approach to determine the conformation required for GHB uptake, a series of related compounds, including aryl‐or alkyl‐derivatives, has been examined for ability to inhibit GHB uptake. The regional distribution of uptake is also indicative of its possible physiological role, since in striatum, an area where GHB has a known pharmacological effect on dopaminergic neurons, this uptake activity is the highest.
Phospholipid Methylation and Noradrenaline Exchanges in a Synaptosomal Preparation from the Rat Brain
Authors: Fonlupt P. , Rey C. , Pacheco H.
Published: October 2006

Abstract: When synaptosomes from rat brain were incubated with S‐adenosylmethionine (10−3M), noradrenaline uptake and KCI‐stimulated release were decreased. These effects were dependent on MgCl2, temperature, and incubation time. We have investigated the enzymatic methylation of phosphatidylethanolamine to give phosphatidylcholine.
Aggregation Properties of the Acetylcholinesterase from the Central Nervous System of Manduca sexta
Authors: Silver Lewis H. , Prescott David J.
Published: October 2006

Abstract: The CNS of the tobacco hornworm, Manduca sexta, provides a rich source of true acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7.). Optimal extraction of the enzyme was obtained with a nonionic detergent at high ionic strength (1% Triton X‐100, 0.5 M NaCl). Velocity sedimentation of the Triton + salt‐extracted enzyme demonstrated a single peak whose sedimentation coefficient was dependent upon the enzyme concentration layered on top of the gradient. When more than 20 units were applied to the gradient, a sedimentation coefficient of 8.6 S (205,000) was obtained, and extrapolation to zero units yielded a 5.7 S (110,500) species. Sedimentation in the absence of detergents (1.0 M NaCl or 10 mM phosphate buffer, pH 7.4) yielded pelleted enzyme and species with mean values of 18.6 S (650,000) and 17.5 S (600,000), respectively. The detergent‐extracted enzyme also demonstrated a concentration‐dependent size in gel filtration experiments. When less than 300 units were applied to the column, a single species was recovered, with a molecular radius of 40.15 ± 2.08 Å, (108,000) or 43.4 ± 2.38 Å, (117,000) calculated by different methods. If the sample contained 300 to 1300 units, two species were observed, with molecular radii of 40.15 ± 2.08 Å or 43.4 ± 2.38 Å and 78.4 ± 3.94 Å, (319,000) or 80.25 ± 3.01 Å (326,000). Velocity sedimentation and gel filtration of AChE have demonstrated that the enzyme has a minimum molecular weight of approximately 110,000 and also exists as higher‐molecular‐weight aggregates of this value.