bluuebunny/crossref_metadata_2025 huggingface dataset:
RELATIONSHIP BETWEEN CHOLINE AVAILABILITY AND ACETYLCHOLINE SYNTHESIS IN DISCRETE REGIONS OF RAT BRAIN
Authors: Wecker Lynn , Dettbarn Wolf‐D.
Published: October 2006

Abstract— The relationship between choline availability and the synthesis of acetylcholine in discrete brain regions was studied in animals treated with the organophosphorus cholinesterase inhibitor paraoxon. Administration of paraoxon (0.23 mg/kg) inhibited acetylcholinesterase activity by approx 90% in the striatum, hippocampus and cerebral cortex and increased acetylcholine levels to 149%, 124% and 152% of control values, respectively. Free choline levels were unaltered by paraoxon in the hippocampus and cerebral cortex, but were significantly decreased in the striatum to 74% of control. When animals were injected with choline chloride (60 mg/kg), 60 min prior to the administration of paraoxon, the paraoxon‐induced choline depletion in the striatum was prevented and the paraoxon‐induced acetylcholine increase was potentiated from 149% to 177% of control values. Choline pretreatment had no significant effect in either the hippocampus or cerebral cortex, brain regions that did not exhibit a decrease in free choline levels after paraoxon administration. Results indicate that choline administration, which had no significant effect on acetylcholine levels by itself, increased acetylcholine synthesis in the striatum in the presence of acetylcholinesterase inhibition. However, this effect was not apparent in either the hippocampus or the cerebral cortex at similar levels of enzyme inhibition. It appears that choline generated from the hydrolysis of acetylcholine may play a significant role in the regulation of neurotransmitter synthesis in the striatum, but not in the other brain areas studied. The evidence supports the concept that the regulatory mechanisms controlling the synthesis of acetylcholine in striatal interneurons may differ from those in other brain regions.
N‐SUBSTITUTED CHOLINE ANALOGUES AS SUBSTRATES FOR CHOLINE ACETYLTRANSFERASE
Authors: Sollenberg J. , Stensiö K.‐E. , Sörbo B.
Published: October 2006

Abstract— Alkyl, phenyl, phenylalkyl and pyridiniumalkyl derivatives of choline were studied as substrates for choline acetyltransferase from bovine brain. When one methyl group of choline was replaced by an ethyl group, pKm (negative logarithm of apparent Km) decreased, whereas Vmax was not significantly changed in comparison with choline. The n‐propyl derivative showed an even lower pKm with unchanged Vmax Further elongation of the n‐alkyl chain had little effect on the substrate parameters until the n‐decyl derivative was reached, when a pronounced decrease of Vmax occurred. The highest n‐alkyl homologue studied, n‐pentadecyl choline, was a very poor substrate. Phenylcholine was also a poor substrate, but introduction of an alkyl chain between the phenyl group and the quaternary nitrogen resulted in compounds with better substrate properties, although they were still inferior to choline. The lowest homologue of the pyridiniumalkyl cholines studied, pyridiniumpropylcholine, had a very low pKm and a lower Vm in comparison with choline. Increasing the chain length of the alkyl residue resulted in an increase of pKm, whereas Vmax was little affected. The results demonstrate that replacement of one methyl group of choline with a more bulky substituent resulted in impaired substrate properties, presumably due to steric effects. No evidence was obtained for hydrophobic interactions between the enzyme and non‐polar substituents in the choline analogues studied.
γ‐AMINOBUTYRIC ACID RECEPTOR BINDING and UPTAKE IN MEMBRANE FRACTIONS OF CRAYFISH MUSCLE
Authors: Meiners B. M. , Kehoe P. , Shaner D. M. , Olsen R. W.
Published: October 2006

Abstract— The uptake and binding of [3H]GABA and the binding of [3H]muscimol were measured in cell‐free fractions of crayfish muscle. The uptake of GABA was saturable, of high affinity (Km= 0.5μm), and inhibited by low concentrations of compounds believed to block GABA uptake specifically, such as nipecotic acid and 2,4,diaminobutyric acid. The GABA uptake activity was localized to sucrose gradient fractions enriched in sarcolemma as demonstrated by marker enzymes and electron microscopy. The binding of the potent GABAergic agonist muscimol was also localized to the sarcolemma. The binding was saturable, of high affinity (KD= 9 nm), and inhibited by GABA (K1= 125 nm) and by low concentrations of receptor‐specific GABA analogues, such as isoguvacine, imidazole acetic acid, and 3‐aminopropane sulfonic acid. The rank order for inhibition by GABA analogues of [3H]muscimol binding sites correlated very well with activity on GABA synapses in invertebrates, consistent with specific postsynaptic receptor labeling.
l‐[35S]CYSTINE UPTAKE BY RAT BRAIN SYNAPTOSOMES
Authors: Segal Stanton , Hwang Shing Mei
Published: October 2006

Abstract— The uptake of [35S]cystine at 37°C by synaptosomal fractions isolated from adult rat cerebrum can be divided into two components. About 60% of the uptake is due to binding to synaptosomal proteins while the remainder exists as a free amino acid pool. Chemical analysis of this soluble component indicates that considerable reduction of cystine to cysteine occurs with 75% or more of the labeled molecular species being cysteine. The process involved in the uptake into the soluble pool was composed of two saturable systems with apparent Km values of 0.14 and 1.4 mm. The low Km system was sodium and oxygen independent but inhibited by dinitrophenol. Dibasic amino acids, lysine, arginine and ornithine, did not inhibit cystine uptake. The characteristics of cystine uptake by synaptosomes from newborn brain are very similar to those of adult brain.
AXONAL TRANSPORT OF PROTEIN IN THE OPTIC NERVE OF OCTOPUS VULGARIS, LAM.
Authors: Cimarra Patrizia , Giuditta Antonio
Published: October 2006

Abstract— The presence of an axonal flow of proteins has been investigated in the optic nerve and lobe of Octopus vulgaris up to 5 days after the intraocular injection of [3H]leucine. In each of these regions and in the posterior half of the eye the content of radioactivity has been determined in the TCA‐soluble fraction and in the saline‐soluble and insoluble protein fractions.

After subtraction of the values of the control side, the concentration of radioactive proteins in the optic nerve and lobe of the injected side was found to increase according to a triphasic pattern. An initial phase of fast increase was followed by a period of essentially steady values and, eventually, by a second phase of less rapid but more prolonged increment. In both regions the per cent of radioactive soluble proteins increased after the completion of the first phase.
CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES
Authors: Mahony James B. , Brown Ian R.
Published: October 2006

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein‐RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [35S]methionine in a wheat embryo cell‐free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain‐specific S100 protein is present in both RNP particles and polysomes).

Addition of total brain RNP particles to a cell‐free wheat embryo system inhibits the endogenous incorporation of [35S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a‘light’and a‘heavy’fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell‐free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain.
THE SITE OF SYNTHESIS OF GANGLIOSIDES IN THE CHICK OPTIC SYSTEM
Authors: Landa C. A. , Maccioni H. J. F. , Caputto R.
Published: October 2006

Abstract– In the retinas of 1‐day‐old chickens that received an intraocular injection of N‐[3H]acetylmannosamine the labelling of N‐acetylneuraminic acid and CMP‐N‐acetylneuraminic acid increased for at least 8 h and that of gangliosides for at least 24 h after injection. In the optic tectum contralateral to the injected eye at 8 h after the intraocular injection, the labelling of gangliosides exceeded the labelling of gangliosides in the ipsilateral tectum by approx 20‐fold. In the contralateral tectum the highest concentration of labelled gangliosides was in subfractions enriched in synaptosomes and synaptic plasma membranes. No significant contralateral ipsilateral differences were found in the acid soluble substances of the tectum. In the optic tectum, labelled gangliosides appeared earlier in the neuronal perikarya than in synaptosomes when the injection was intracranial. Conversely, when the injection was intraocular the labelling appeared earlier in the synaptosomes than in the neuronal perikarya. The radioactivity pattern of the optic tectum gangliosides resembled the pattern of retina gangliosides when N‐[3H]acetylmannosamine was injected intraocularly, but when N‐[3H]acetylmannosamine was given intracerebrally the radioactivity pattern resembled that of optic tectum gangliosides. Intraocular injection of colchicine or vinblastine did not affect the labelling of retinal gangliosides from N‐[3H]acetylmannosamine injected into the same eye but prevented the appearance of labelled gangliosides in the optic tectum. In vitro the ganglioside glycosylating activity of optic tectum synaptosomes and synaptic plasma membranes was between 6 and 10‐fold lower than that found in the optic tectum neuronal perikarya. These findings support the notion that the main subcellular site of synthesis of neuronal gangliosides is in the neuronal perikarya, from which they are translocated to the nerve endings.
THE EFFECT OF dl‐ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN
Authors: Pajunen A. E. I. , Hietala Oili A. , Baruch‐Virransalo Eeva‐Lhsa , Piha R. S.
Published: October 2006

Abstract— dl‐Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l‐ornithine decarboxylase activity and a simultaneous decrease in S‐adenosyl‐l‐methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl‐allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl‐allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.
SELECTIVE LABELLING OF TWO PHASES OF AXONAL TRANSPORT OF CHOLESTEROL IN THE CHICK OPTIC SYSTEM
Authors: Rostas John A. P. , Austin I. , Jeffrey Peter L.
Published: October 2006

Abstract— In the chick optic system cholesterol is axonally transported in two phases which appear to take their cholesterol from different cellular pools. The intraocular injection of radioactire cholesterol results in the specific labelling of the slow phase which carries cholesterol in the unesterificd form and appears to move at the same rate as the slow phase of protein transport (Rostaset al., 1975). The intraocular injection of radioactive mevalonic acid, a metabolic precursor of cholesterol, results in the preferential labelling of a more rapid phase of axonal transport which also carries cholesterol in the unesterified form and is first detected at the optic tectum 10 h after the injection. It is likely that this rapid phase travels at the same rate as the rapid phase of protein transport and that the delayed arrival at the tectum is due to a lag time in the retina caused by the synthesis of cholesterol and its packaging for transport. Because the individual pools for the two transport phases can be selectively labelled, the retina and optic nerve provide a unique model system in which the metabolic turnover, intracellular compartmentalization and intracellular transport of cholesterol can be studied.
UPTAKE OF [14C]NIPECOTIC ACID INTO RAT DORSAL ROOT GANGLIA
Authors: Minchin M. C. W.
Published: October 2006

Abstract— [14C]Nipecotic acid was accumulated in isolated desheathed rat dorsal root ganglia by a saturable process with Km= 48.8 μm and Vmax= 2.2 nmol/g/min. The concentration of l‐2.4‐diamino‐butyric acid required to inhibit the uptake of nipecotic acid by 50% was three times the concentration of β‐alanine required to do the same. Light microscopic autoradiography indicated that the sites of uptake of [14C]nipecotic acid were principally confined to satellite glial cells. It is concluded that nipecotic acid is transported by the GABA uptake system in glia but that it has less affinity for this system than GABA.
STABILITY OF SYNAPTOSOMAL GABA LEVELS AND THEIR USE IN DETERMINING THE IN VIVO EFFECTS OF DRUGS: CONVULSANT AGENTS
Authors: Wood J. D. , Russell M. P. , Kurylo E. , Newstead J. D.
Published: October 2006

—The stability of the GABA content of synaptosomal‐enriched fractions was evaluated by two approaches. Firstly, the addition of 10−3m‐aminooxyacetic acid to the homogenizing medium totally inhibited the GABA‐degrading enzyme in the fractions but did not affect the GABA levels. This indicated that GABA was not being metabolized during the normal preparation of the synaptosomal‐enriched fraction. Secondly, when synaptosomal‐enriched fractions were re‐fractionated by discontinuous density gradient centrifugation, the GABA contents of the fractions before and after the second fractionation were very similar provided they were expressed on a per mg protein basis. It was therefore concluded that the GABA content of the organelles was not subject to change during the fractionation procedures. On the basis of these findings and others it was suggested that the synaptosomal‐enriched fraction could be used as a model to evaluate drug‐induced changes in GABA levels in nerve endings. In vivo experimentation indicated that the convulsant agents hydrazine, isonicotinic acid hydrazide and aminooxyacetic acid brought about similar decreases in the GABA content of the synaptosomal‐enriched fractions prepared from tissue at the onset of seizures despite the fact that no correlation was observed between seizure activity and whole brain GABA levels.
THE BINDING INTERACTION BETWEEN α‐LATROTOXIN FROM BLACK WIDOW SPIDER VENOM AND A DOG CEREBRAL CORTEX SYNAPTOSOMAL MEMBRANE PREPARATION
Authors: Tzeng Mu‐Chin , Siekevitz Philip
Published: October 2006

—The major toxin of black widow spider venom, α‐latrotoxin, can be iodinated with 125I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non‐specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre‐incubation with 2%β‐mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre‐treatment with 10m M‐EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non‐cooperative, with an apparent equilibrium dissociation constant, K1, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 105 M−1 s−1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10−3 and 5.2 × 10−5s−1 corresponding to half‐lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α‐latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma.
ELEVATED CONCENTRATIONS OF HISTAMINE IN THE RAT BRAIN
Authors: Orr Edward L. , Eichelman Burr
Published: October 2006

—Whole brain concentrations of histamine (Hm) in the rat were measured with a sensitive and specific single‐isotope enzymatic microassay and found to be elevated about 2‐fold in rats killed by focused microwave irradiation compared with rats killed by decapitation. However, regardless of the method of killing, whole brain Hm concentrations were elevated about 4‐fold when the brains were homogenized in 12.5 vol vs 2.5 vol of water or phosphate buffer. Homogenization of brain regions in 12.5 vol of water resulted in Hm concentrations that were about 10 times greater than previously reported values (except in the hypothalamus). Since (1) the‘extra’Hm could be extracted from pellets of low volume homogenates, and (2) synthesis of Hm is not likely to occur through an increase in the volume of water or buffer used for homogenization, we suggest that the elevated concentrations of Hm seen after high volume homogenization of brain tissue are due to a more complete extraction of Hm from brain tissue, and not to an artifactual production of this amine from its precursor (histidine) or some other compound(s).
UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS
Authors: WHELER G. H. T. , BRADFORD H. F. , DAVISON A. N. , THOMPSON E. J.
Published: October 2006

—Cortex slices, synaptosomes and C‐6 glioma cells were used to study [35S]taurine uptake and its electrically‐stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle‐bound taurine and 16% of the total in the tissue. The uptake of [14C]GABA by C‐6 glioma cells was inhibited 3‐fold more by β‐alanine than by l‐DABA, whilst synaptosome preparations showed the opposite pattern, l‐DABA being 2 or 3 times more effective than β‐alanine. [35S]Taurine uptake inhibition by l‐DABA was low for synaptosomes and C‐6 glioma, whereas β‐alanine showed considerable effect on C‐6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β‐alanine. When 30 min rather than 5 min incubations were employed, β‐alanine depressed [35S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium‐dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.
Changes in Prolyl Endopeptidase During Maturation of Rat Brain and Hydrolysis of Substance P by the Purified Enzyme
Authors: Kato Takeshi , Nakano Tamotsu , Kojima Kohichi , Nagatsu Toshiharu , Sakakibara Shumpei
Published: October 2006

: We determined changes in prolyl endopeptidase activity in developing rat brain. A new and highly sensitive fluorogenic substrate, 7‐(succinyl‐Gly‐Pro)‐4‐methylcoumarinamide, was used for determination of the enzyme activity. The enzyme activity per brain increased until 2 weeks of age, and then decreased during maturation. The enzyme was purified about 7800‐fold from the brain of the rat at 2 or 3 weeks of age. The enzyme has a pH optimum of 5.8 to 6.5, and an approximate molecular weight of 70,000. The enzyme activity was completely inhibited by low concentrations of diisopropylfluorophosphate and partially inhibited by high concentrations of phenylmethanesul‐phonylfluoride, which are potent serine protease inhibitors. Moreover, thiolblocking agents and some heavy metals also have a strong effect on the activity. Bacitracin was found to be a potent inhibitor, with an IC50 value of 2.5 × 10−6m at 0.5 mm of the substrate. The enzyme was proved to hydrolyze the NH2‐terminal tetrapeptide, Arg1‐Pro2‐Lys3‐Pro4, from substance P to produce the heptapeptide, Gln5‐Gln6‐Phe7‐Phe8‐Gly9‐Leu10‐Met11‐CONH2. The Km value of the hydrolysis of substance P was 1.0 mm. This enzyme may be related to the regulation of substance P in the brain, and to the development of neurones by forming the tetrapeptide because the tetrapeptide has almost the same effect as substance P on the neurite extension of neuroblastoma.
Heterogeneity of Benzodiazepine Receptors in the Central Nervous System Demonstrated with Kenazepine, an Alkylating Benzodiazepine
Authors: Williams Evan F. , Rice Kenner C. , Paul Steven M. , Skolnick Phil
Published: October 2006

: Binding studies using the alkylating benzodiazepine kenazepine strongly suggest the existence of several populations of benzodiazepine receptors in the CNS. Kenazepine reacts noncompetitively and irreversibly with some receptors and competitively (reversibly) with others. Cerebellum contains the largest proportion (approx. 80%) of the noncompetitive type, while hippocampus and cortex contain a preponderance of competitive‐type receptors (approx. 80 and 50%, respectively). The Hill coefficients for kenazepine are approx. 0.7 in cortex and cerebellum, and near unity in dorsal hippocampus. Different populations of benzodiazepine receptors may mediate different physiologic and pharmacologic effects in vivo.
Cyclic Guanosine Monophosphate in Primary Cultures of Glial Cells
Authors: Bloch‐Tardy M. , Fages C. , Gonnard P.
Published: October 2006

: Cyclic GMP was found in primary cultures of glial cells obtained by dissociation of newborn mouse brain hemispheres. Its basal level (0.52 pmoles/mg cell protein) was as high as that found in adult mouse brain cortex but 10 times lower than in cerebellum. When glia were grown in the presence of dBcAMP, astrocytes changed their morphology; cGMP level increased and reached about 8 to 10 times the basal value. This increase was dose dependant with cAMP and was enhanced by the presence of 5mM Theophylline. Two hypothesis are discussed, either a direct action of cAMP on glial cGMP metabolism or an indirect one on the protein activator of cGMP phosphodiesterase.
Laminar Distribution of Putative Neurotransmitter Amino Acids and Ligand Binding Sites in the Dog Olfactory Bulb
Authors: Nadi N. S. , Hirsch J. D. , Margolis F. L.
Published: October 2006

Abstract: Coronal sections of frozen dog olfactory bulb have been dissected into four anatomically distinct layers. The laminar distribution of ten amino acids, the dipeptide carnosine and nine [3H]ligand binding sites in these layers was determined. GABA and tyrosine levels were highest in the mitral cell‐granule cell layer and glutamate levels were slightly elevated in the glomerular layer. The distributions of all other amino acids did not show significant differences across the layers. Carnosine was predominantly localized in the fiber and glomerular layers. With the exception of quinuclidinyl benzilate, the [3H]ligand binding sites showed more discrete distributions. Muscimol, diazepam, kainic acid and spiroperidol binding were predominantly localized in the mitral cell‐granule cell layer, where clonidine binding was at a minimum. Dihy‐dromorphine binding was high in both the fiber and the mitral cell‐granule cell layers. Carnosine binding was maximal in the glomerular layer. The implications of these observations with regard to biochemical and neurophysiological data are discussed.
Regulation of Phospholipid Metabolism in Differentiating Cells from Rat Brain Cerebral Hemispheres in Culture: Ontogenesis of Carrier‐specific Transport of Choline and N‐Methyl‐substituted Choline Analogs
Authors: Yavin E.
Published: October 2006

Abstract: Dimethylaminoethanol was studied both as a substrate and as an inhibitor of choline uptake in long‐term cultures of foetal rat cerebral hemispheres. A saturable component with an apparent Km of 28 μM and Vmax of 11 pmol/min/μg DNA for dimethylaminoethanol, was observed. Like choline, dimethylaminoethanol was also taken up by a second, low‐affinity component, the apparent Vmax of which was about 102 pmol/min/μg DNA. Dimethylaminoethanol inhibited the high‐affinity but not the low‐affinity choline uptake in a competitive manner with an apparent inhibition constant of 6.0 μM. Monomethylaminoethanol (K1# 60 μM) competitively inhibited high‐affinity choline transport. At low concentrations hemicholinium‐3, but not ethanolamine, effectively inhibited high‐affinity uptake of choline and to a lesser degree the uptake of the dimethylaminoethanol. While the high‐affinity uptake of both substrates was inhibited by high concentrations of hemicholinium‐3 or ethanolamine, the low‐affinity system was not affected by hemicholinium‐3. From the kinetics of uptake and inhibition patterns of choline and its related analogs, the methyl group seems to play a major role in determining the affinity rate constants for these substrates. The maximum rate of choline uptake via the high‐affinity component increases about sixfold during a period of 2 weeks. In the absence of serum the maximum velocity of the high‐affinity component is greatly reduced. These observations suggest that the high‐affinity choline uptake component is an integral property and a useful marker, of the developing cerebral cells.
Concentration and In Vivo Synthesis of Cholecystokinin in Subcortical Regions of the Rat Brain
Authors: Goltermann Nils R. , Rehfeld Jens F. , Petersen Hans Røigaard
Published: October 2006

The in vivo biosynthesis of cholecystokinin (CCK) in subcortical regions of the rat brain was studied by intracisternal pulse injections of [35S]methionine. The rats were killed 1.0 or 1.5 h after the injection. Subcortical CCK extracted in boiling water and acetic acid was immunoabsorbed by an antiserum specific for the COOH‐terminal sequence of CCK. Gel chromatography of the absorbed CCK separated four molecular forms with elution constants (Kav) of 0.08, 0.50 (corresponding to the tritriacontapeptide amide, CCK‐33), 1.10 (corresponding to the COOH‐terminal octapeptide, CCK‐8), and 1.40 (a component which may correspond to the COOH‐terminal tetrapeptide amide). Significant incorporation of [35S]methionine occurred in the largest (Kav∼ 0.08) and octapeptide‐like form only. Chasing with methionine indicated a precursor relationship between the largest form and CCK‐8. The results demonstrate a substantial synthesis of CCK also in subcortical regions of the brain.
Endogenous Amino Acid Release from Rat Cerebellum In Vitro
Authors: Foster Alan C. , Roberts Peter J.
Published: October 2006

The release of six endogenous amino acids from superfused rat cerebellar slices was investigated. Only l‐glutamate, and GABA exhibited a calcium‐dependent, potassium‐stimulated release. Glutamine release was significantly inhibited during the stimulation period. Experiments were also performed with slices from cerebella which had received previously, injections of 2 μg kainic acid. This procedure failed to modify the release of any amino acid. At this time point (24 h), cell bodies of inhibitory neurons utilising GABA as their transmitter, have largely been destroyed; thus, the lack of effect on GABA release in particular, may indicate that the mechanisms for transmitter release from terminals are still operative under these conditions.
Brain Poly(A)RNA During Aging: Stability of Yield and Sequence Complexity in Two Rat Strains
Authors: Colman P. D. , Kaplan B. B. , Osterburg H. H. , Finch C. E.
Published: October 2006

To evaluate whether major age‐related changes occur during aging in the brain, we determined the sequence complexity of total poly(A)RNA and polysomal poly(A)RNA of brains from Sprague‐Dawley and Fischer 344 male rats, aged 2‐32 months. RNA complexity, determined by RNA‐driven hybridization reactions with nonrepetitive DNA, was 6‐7 × 108 nucleotides for total poly(A)RNA, whereas polysomal poly(A)RNA had a sequence complexity of 2.3‐2.8 × 108 nucleotides. Sprague‐Dawley rats had polysomal poly(A)RNA with a yield per gram of brain and a complexity that was 20% greater than Fischer 344 rats.

No age differences in total or polysomal poly(A)RNA complexity were detected in either strain. Mixtures of polysomal poly(A)RNA from different age groups gave complexity values that were indistinguishable from the complexity of each component, thereby indicating that most (>80%) of the RNA sequences are common to all age groups studied. Analysis of total poly(A)RNA hybridization kinetics indicated that the major kinetic component had similar pseudo‐first‐order rate constants and complexity in all age groups. Consideration of interassay variation suggests that the limits of age changes in poly(A)RNA sequence complexity or content are 10%. Thus, age changes may be smaller than interstrain differences in the rat. We conclude that if genomic function is grossly impaired during aging, as presumed in many contemporary theories, the impairments are not expressed in most brain cells.
Purification and Some Properties of Choline Acetyltransferase from Catfish Brain
Authors: Su Y. Y. Thomas , Wu J.‐Y. , Lam Dominic M. K.
Published: October 2006

Choline acetyltransferase (CAT) from catfish brain has been purified to electrophoretic homogeneity by a combination of ammonium sulfate fractionation, gel filtration, and preparative polyacrylamide gel electrophoresis. The purified enzyme solutions showed only one protein band on both 7.5% polyacrylamide gel and gradient (3.5% to 15%) polyacrylamide gel with the enzyme activity coincident with the protein band. Furthermore, when the gel slice from which the enzyme was extracted was applied to a gradient polyacrylamide gel, a single protein band was also obtained. The purified enzyme preparations represented a 1510‐fold purification over the crude homogenate. The moiecular weight of the enzyme was estimated as 128.000 and 140,000 from gel filtration and gradient (3.5% to 15%) polyacrylamide gel, respectively. On sodium dodecyl sulfate‐gel, the purified enzyme preparations showed two protein bands, with molecular weights of 34,000 and 68,000. Since the native enzyme may be a tetramer, the 68.000 subunit may be a dimer of the smallest subunit. The maximum activity of the enzyme appears at pH 7.2; the values of Km for choline and for acetyl CoA are 400 and 92 μM, respectively, and Vmax was obtained as 2.2 × 10‐3μmol/min/mg protein. CAT antibodies were obtained from rabbit that had received six biweekly injections with a total of 120 μg of the purified protein. Double immunodiffusion test with CAT antibodies and a crude enzyme extract from catfish brains showed only a single, sharp precipitin band, suggesting that the antibodies are specific only to CAT and antigen and that the purified CAT preparation is monodisperse.
Regulation of Adenosine‐Sensitive Adenylate Cyclase from Rat Brain Striatum
Authors: Anand‐Srivastava Madhu B. , Johnson Roger A.
Published: October 2006

An adenosine‐sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N6‐phenylisopropyl adenosine (PIA), 2‐chloroadenosine, and adenosine N′‐oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA‐stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg2‐. The degree of stimulation by PIA was greater at a low concentration of Mg2+, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg2+. Activation of adenylate cyclase by PIA was blocked by theophylline or 3‐isobutyl‐ 1‐methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)‐stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A2 treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA‐stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.
Sodium‐Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization
Authors: Sandoval Maria Elena
Published: October 2006

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca2+levels. Treatments known to inhibit Ca2+retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca2+, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca2+efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na+ levels on [3H]γ‐aminobutyric acid ([3H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [3H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K+‐free medium, which increase the internal sodium concentration. This effect was not observed when Na+ was omitted from the incubation medium and it was independent of external Ca2+, the experiments having been performed in a Ca2+‐free, EGTA‐containing medium. Since preincubation of synaptosomes with 2,4‐diaminobutyric acid did not prevent the stimulatory effect of increased internal Na+ levels on [3H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier‐mediated GABA transport. These results suggest that the augmented release of [3H]GABA may be due to an increased Ca2+efflux from mitochondria eiicited by the accumulation of Na+ at the nerve endings. Sandoval M. E. Sodium‐dependent efflux of [3H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35, 915–921 (1980).
Studies on the Wolfgram High Molecular Weight CNS Myelin Proteins: Relationship to 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase
Authors: Sprinkle Terry Joe , Wells Michael R. , Garver Fred A. , Smith Dennis B.
Published: October 2006

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500–3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform‐methanol‐insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNPI protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform‐methanol‐insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.
Activites of Enzymes Synthesizing Diacyl, Alkylacyl, and Alkenylacyl Glycerophosphoethanolamine and Glycerophosphocholine During Development of Chicken Brain
Authors: Freysz L. , Horrocks L. A. , Mandel P.
Published: October 2006

Abstract: Ethanolamine and choline glycerophospholipids are the major phospholipids of brain membranes. During brain development, the accumulation of these phospholipids is most intense when myelination occurs. In order to gain knowledge about the regulatory mechanisms for synthesis of these lipids in relation to membrane synthesis, we investigated the activities of the 1,2‐diradyl‐sn‐gIycerol: CDPethanolamine phosphoethanolarnine transferase and 1,2‐diradyl‐sri‐glycerol:CDPcholine phosphocholine transferase during chicken brain development. Diacyl, alkenylacyl, and alkylacylglycerols are substrates for both enzymes. The specific activities of microsomal phospho‐ethanolamine and phosphocholine transferases are constant between the 8th and 18th day of embryonic life. The specific activities of both enzymes double around hatching, which is the period of intense myelination and marked ac‐cumulation of ethanolamine and choline glycerophospholipids in brain. At the same time, the amount of microsomes increases by 50%; thus the total activities increase threefold. Four days after hatching the specific activities of both enzymes are at adult values. Similar results were obtained in the presence of exogenous diacyl or alkylacylglycerols. During brain development the apparent Km, value of rnicrosomal phosphoethanolamine transferase for CDP ethanolamine increases when assayed with diaclyglycerols or alkylacyl‐glycerols a s lipid substrates. The apparent Km, value of phosphocholine trans‐ferase for CDP choline does not change during brain development in the presence of exogenous diacylglycerols, but increases in the presence of exogenous alkylacylglycerols. These changes in Km, values may be due to the appearance of glial isoenzyme at the beginning of myelination. The apparent Km, values of diacylglycerol phosphocholine, alklyacylglycerol phosphocholine, and diacyl‐glycerol phosphoethanolamine transferases for their CDP bases are similar in adult brain microsomes and are threefold higher than the apparent Km, value of alkylacylglycerolphosphoethanolamine transferase. The high affinity of alkylacylglycerolphosphoethanolamine transferase for CDPethanolamine may be responsible for the preferential synthesis of ethanolamine plasmalogens in brain.
Changes in the Uptake of GABA and Taurine During Neuronal and Glial Maturation
Authors: Borg J. , Ramaharobandro N. , Mark J. , Mandel P.
Published: October 2006

Abstract: The influence of the time of culture on GABA and taurine uptake was investigated in spontaneously matured cultures of glial and neuronal origins and in cultures treated with cyclic nucleotides. In the spontaneously matured cultures the capacity of the high‐affinity neuronal GABA transport system increased with time in culture. Essentially opposite results were found for the uptake of GABA by glial cultures. In contrast with the neuronal uptake of GABA, the capacity of the taurine transport system was significantly decreased. Uptake of taurine into glia, however, exhibited a progressive increase with the period of culture. The values of Km, for the high‐affinity systems were always found to range around 10 μM. It is suggested that, in mature cells, neuronal uptake sites are of prime importance for GABA transport, while taurine uptake may be more specifically directed towards glial cells. When cultures were treated with cyclic nucleotide derivatives, a morphological differentiation was induced, which could not be linked to a stimulation of GABA or taurine uptake systems as compared with the non‐treated cultures.
In Vitro Synthesis of the Myelin Basic Proteins: Subcellular Site of Synthesis
Authors: Campagnoni Anthony T. , Carey Gregory D. , Yu Yie‐Teh
Published: October 2006

Abstract: Brains of 3‐week‐old C57BL/6J mice were homogenized and fractionated into several subcellular components, each of which was examined for ability to synthesize the myelin basic proteins (MBPs) in vitro. Myelin basic proteins were purified from incubation mixtures by conventional means. That the products of synthesis were the myelin basic proteins was established by solubility at pH 3, co‐chromatography with authentic proteins on carboxymethylcellulose and co‐migration with standards in two different polyacrylamide gel electrophoretic systems. The fractions examined for their ability to synthesize MBPs were the whole homogenate, postnuclear supernatant, postmitochondrial supernatant, crude mitochondrial pellet, free ribosomes and bound ribosomes. Although there was no requirement for exogenous energy sources for protein synthesis in the whole homogenate, as the homogenate was fractionated an increasing requirement emerged. Most of the label in the MBP preparations from whole homogenate and postnuclear supernatant incubations migrated with the large (L) and small (S) MBPs on gel electrophoresis; however, as the homogenate was subfractionated and incubated, a greater percentage of the label migrated more slowly than L and S on acetic acid‐urea gels. To show synthesis of the MBPs the L and S bands were cut out of these gels and rerun on sodium dodecylsulfate gels. Alternatively, MBP preparations were subjected directly to two‐dimensional gel electrophoresis and the bands corresponding to L and S were excised and counted. With this method only the whole homogenate, postnuclear supernatant, postmitochondrial supernatant and free ribosomes were observed to synthesize the MBPs in vitro. The “bound” ribosomes were not observed to synthesize significant amounts of the MBPs, incubated either intact or released from the membrane. It was concluded that the free ribosomes are the principal site of synthesis of the myelin basic proteins in the brain.
Kinetic Data on the Inhibition of High‐affinity Choline Transport into Rat Forebrain Synaptosomes by Choline‐like Compounds and Nitrogen Mustard Analogues
Authors: Rylett B. Jane , Colhoun E. Howard
Published: October 2006

Abstract: A series of choline analogues and nitrogen mustard derivatives were evaluated as inhibitors of high‐affinity transport of choline in rat forebrain synaptosomes. When synaptosomes were preincubated for 10 min with choline mustard aziridinium ion, monoethylcholine and monoethylcholine mustard aziridinium ion, the agents appeared to be equipotent as inhibitors of high‐affinity uptake (Ki=2.63, 3.15 and 2.72 μm, respectively). Acetylcholine mustard aziridinium ion was less potent than these compounds (Ki= 27.8 μm), but it was more potent than ethoxycholine and ethoxycholine mustard aziridinium ion (Ki= 500 and 403 μm) as a blocker of choline transport. From study with these compounds it was concluded that the high‐affinity choline transport mechanism shows specificity for hydroxylated compounds over those in which the same hydroxyl has been acetylated (10‐fold) and that the carbonyl oxygen of the acetylated analogues is important, as its removal (to form the ethylether derivative) decreased affinity another 20‐fold. The presence of an aziridinium ring on the quaternary nitrogen in place of two methyl groups did not affect the blocking of transport at 10 min of inhibitor preincubation and replacement of a methyl group on the nitrogen by an ethyl group did not alter affinity for the high‐affinity carrier. The aziridinium ring on the nitrogen of the mustard analogues was important, however, in determining the extent of reversibility of the binding of these agents to the carrier protein. Choline transport was not restored by washing synaptosomes that were incubated with choline mustard aziridinium ion or monoethylcholine mustard aziridinium ion, but was readily obtained in washed synaptosomes preincubated with monoethylcholine, hemicholinium‐3, or pyrrolcholine. The results indicate that the mustard analogues may be potent alkylators of the high‐affinity choline carrier and thus, useful agents in monitoring acetylcholine turnover in systems where the carrier is blocked.
Ionic and Metabolic Requirements for High‐Affinity Choline Uptake and Acetylcholine Synthesis in Nerve Terminals at a Neuromuscular Junction
Authors: Beach Robert L. , Vaca Ken , Pilar Guillermo
Published: October 2006

Abstract: We have shown previously that in the chick ciliary nerve‐iris muscle preparation Na+‐dependent high‐affinity choline uptake was confined to the nerve terminals. In this paper the sodium‐dependent high‐affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [3H]choline and its conversion to [3hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na+‐dependent choline (Ch+) uptake was shown to be linear for 16 min and to reach an equilibrium before Na+‐independent Ch+ uptake, which continued to increase for 60 min. Elevated [K+]0 concentrations inhibited Ch+ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K+]0. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K+]0. Reduction of SDHACU and sodium‐dependent ACh synthesis by depolarization with high [K+]0 or by inhibition of Na, K‐ATPase implies that the electrochemical gradients for Ch+ and Na+ are important in providing a driving force for high‐affinity Ch+ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K‐ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch+ uptake and ACh synthesis, in some cases Ch+ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K+]0 and ouabain. Na+‐dependent Ch+ uptake and ACh synthesis were found to be temperature‐dependent with a Q10 (20–30°) of 3.6 and 6.6, respectively and a Q10 (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch+ taken up which is converted to ACh. ACh did not reduce Ch+ transport when present at 100 μM.
Protein Synthesis in a Cell‐free System from Rat Brain Sensitive to ACTH‐like Peptides
Authors: Schotman P. , Heuven‐Nolsen D. v. , Gispen W. H.
Published: October 2006

Abstract: Protein synthesis was measured using a cell‐free system obtained from subcortical rat brain tissue. The concentrations of Mg2+ and K+ and the amount of tissue, during both the preparation and the final assay, were critical to the incorporation of amino acids as expressed per milligram protein. Even under optimal conditions mainly elongation of growing peptide chains was measured. Behaviorally active fragments of ACTH modulated the activity of the system in a biphasic manner; i.e., at a low concentration (10−8 M) of ACTH a stimulation of between 10 and 70% was found; a high concentration (10−4 M) was inhibitory (50 to 70%). Structure‐activity studies revealed that the stimulatory effect was confined to the N‐terminus of the peptide (1–24), whereas the C‐terminal sequence was responsible for the inhibition. The stimulation by ACTH1–24 was dependent on Ca2+ and Mg2+. Cyclic AMP (10−5 M) stimulated the amino acid incorporation too. When a similar cell‐free extract was prepared from brain tissue of hypophysectomized rats, the lower in vivo protein synthesis in these animals was preserved in the present cell‐free system. The data are discussed in terms of a possible direct intracellular effect of ACTH on brain protein synthesis.