bluuebunny/crossref_metadata_2025 huggingface dataset:
Deoxycytidine Transport and Metabolism in the Central Nervous System
Authors: Spector Reynold , Huntoon Sheryl
Published: October 2006

Abstract: The mechanisms by which deoxycytidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [3H]deoxycytidine intraarterially, intravenously, and intraventricularly. After intracarotid injection of deoxycytidine (1.0 μM) into rats, deoxycytidine did not pass through the blood‐brain barrier at a faster rate than sucrose. [3H]Deoxycytidine, either alone or together with unlabeled deoxycytidine, was infused at a constant rate into conscious adult rabbits. At 130 min, [3H]deoxycytidine readily entered CSF, choroid plexus, and brain. In brain, approx. 60% of the nonvolatile radioactivity was attributable to [3H]deoxycytidine phosphates. The addition of 0.22 mmol/kg unlabeled deoxycytidine to the infusion syringe decreased the phosphorylation of [3H]deoxycytidine in brain by approx. 50%; the addition of 2.2 mmol/kg of unlabeled deoxycytidine to the infusion syringe decreased the relative entry of [3H]deoxycytidine into CSF and brain by approx. 50 and 75%, respectively. Two hours after the intraventricular injection of [3H]deoxycytidine, [3H]deoxycytidine was rapidly cleared from CSF, in part, to brain, where approx. 65% of the [3H]deoxycytidine was converted to [3H]deoxycytidine phosphates. The intraventricular injection of unlabeled deoxycytidine with the [3H]deoxycytidine decreased the phosphorylation of [3H]deoxycytidine in the brain significantly and also decreased the clearance of [3H]deoxycytidine from the CSF. These results were interpreted as showing that the entry of deoxycytidine from blood into CSF occurs by a saturable transport system within the choroid plexus. Once within the CSF, the deoxycytidine can enter brain, undergo phosphorylation to deoxycytidine phosphates, and subsequently be incorporated into DNA.
High‐Affinity, Sodium‐Dependent Γ‐Aminobutyric Acid Uptake by Slices of Rat Ovary
Authors: Erdó Sándor L.
Published: October 2006

Abstract: The high concentration of γ‐aminobutyric acid (GABA) recently demonstrated in rat ovary prompted us to examine the capacity of ovarian slices to take up [3H]‐GABA. Active uptake, dependent on temperature and sodium concentration, was observed and a kinetic constant (Km) of 1.0 μM found for the uptake process. Ouabain (100 μM) reduced the rate of accumulation of [3H]GABA. Uptake was inhibited only partially by 100 μM d, l‐nipecotic acid, but more strongly by 100 μMβ‐alanine. These results suggest that the uptake system in ovary possesses properties similar to those of high‐affinity GABA transport systems in the brain.
Analytical Subcellular Fractionation of Rat Cortex: Resolution of Serotonergic Nerve Endings and Receptors
Authors: Laduron P. M. , Janssen P. F. M. , Ilien B.
Published: October 2006

Abstract: An analytical procedure for the subcellular fractionation of rat brain cortex is presented; it consists of a two‐step procedure involving a differential centrifuga‐tion using the five‐fraction scheme and an isopycnic cen‐trifugation in continuous sucrose gradients. All fractions obtained were analyzed for their content of various constituents, such as receptor binding, uptake, and several marker enzymes. Special attention was paid to the subcellular distribution of the serotonin S2 receptors; they were mainly recovered in the microsomal P fraction, but a significant amount was also associated with the mito‐chondrial (M and L) fractions. After equilibration in density gradients, serotonin S2 receptors revealed two peaks, which were similarly affected after treatment with ami‐triptyline and/or yohimbine. There is no evidence to suggest that serotonin S2 receptors are associated with nerve endings containing the neurotransmitter serotonin. Although three main profiles, a microsomal, a mitochondrial, and a mixed one, clearly appear from the differential centrifugation, subgroups of these main profiles were also found. For instance, the microsomal distribution patterns of serotonin S2 receptors and 5′‐nucleoti‐dase are very similar, but differ from that of UDP‐galactosyltransferase. Similarly, the mitochondrial profiles of cytochrome oxidase and 5‐HT (serotonin) uptake are different. An analytical approach for brain fractionation, when performed with appropriate measurements (cytochrome oxidase, amine uptake, 5′‐nucleotidase, and receptor binding), is rapid and clearly differentiates pre‐and postsynaptic constituents.
Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin
Authors: Miller Sheldon L. , Pleasure David , Herlyn Meenhard , Atkinson Barbara , Ernst Carolyn , Tachovsky Thomas G. , Baird Lynn
Published: October 2006

Abstract: Monoclonal antibodies against P0, myelin basic protein, or myelin‐associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso‐type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin‐associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin‐associated glycoprotein. Antibodies were characterized in solid‐phase radioimmunoassay for their species cross‐reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin‐producing cells, since in both cases the concentration of myelin proteins is low.
Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons
Authors: Hayashi Yokichi , Higashida Haruhiro , Kuo Che‐Hui , Miki Naomasa
Published: October 2006

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8‐day‐old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine‐coated well exposed to NOF (NOF‐bound POR well), marked neurite outgrowth was observed. When NOF‐bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48‐h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.
Distribution of Acid Protease Activity in the Squid Nervous System
Authors: Gallant Paul E. , Pant Harish C. , Gainer Harold
Published: October 2006

Abstract: Acid protease activity was measured in homogenized stellate ganglion, axoplasm extruded from the squid giant axon, homogenized fin nerves, and in lysed synaptosomes prepared from the optic lobe of the squid. At least two different acid protease classes were distinguished on the bases of their inhibitor profiles. Acid protease activity was present in each of the above tissues except extruded axoplasm. This result suggests that the acid protease activity found in our homogenized finnerves might be located not within the axons but rather in glial cells or extracellular tissue. The absence of acid protease activity in extruded axoplasm indicates that acid proteases are unlikely to play a significant role in the catabolism of intracellular proteins along the length of the axon.
Tryptophan Uptake and Hydroxylation in Rat Forebrain Synaptosomes
Authors: Knowles Richard G. , Pogson Christopher I.
Published: October 2006

Abstract: Tryptophan uptake, hydroxylation, and decarboxylation in isolated synaptosomes were studied to assess how their properties may determine the rate of serotonin synthesis in the presynaptic nerve terminals of the brain. Simultaneous measurements of the rates of uptake, hydroxylation, and decarboxylation in the presence and absence of various inhibitors showed that tryptophan hydroxylase is rate‐limiting for serotonin synthesis in this model system. There was significant direct decarboxylation of tryptophan to tryptamine. Measurement of tryptophan hydroxylase flux with varying internal concentrations of tryptophan allowed the determination of the Km of tryptophan hydroxylase in synaptosomes for tryptophan of 120 15 μM. Depolarisation of synaptosomes with veratridine caused both a reduction in the internal tryptophan concentration and an apparent activation of tryptophan hydroxylase. This activation did not occur in the absence of Ca2+ or in the presence of trifluoperazine. Synaptosomal serotonin synthesis and brain stem‐soluble tryptophan hydroxylase were inhibited by low concentrations of noradrenaline or dopamine. Dibutyryl cyclic AMP, glucagon, insulin, and vasopressin were observed to have no effect on tryptophan uptake or hydroxylation in synaptosomes.
Positional Distribution of Acyl and Alk‐1‐enyl Groups in Grey and White Matter Ethanolamine and Choline Phosphoglycerides of a Marsupial, the Koala (Phascolarctos cinereus)
Authors: Naughton Joan M. , Trewhella Maurice A.
Published: October 2006

Abstract: The major phosphoglycerides in grey and white matter from the brain of the koala have been separated and examined. The major polyunsaturated fatty acids present in both the diacyl‐ and alk‐1‐enyl acylglycerophosphorylethanolamines from grey matter were 22:6ω3, 20:4ω6, and 22:4ω6. In both grey and white matter, 22:6ω3 and 20:4ω6 were concentrated in the 2‐position of diacylglycerophosphorylethanolamines and 22:4ω6 in the 2‐position of alk‐1‐enylacylglycerophos‐phorylethanolamines; polyunsaturated fatty acid levels were higher in diacylglycerophosphorylethanolamines. Ethanolamine phosphoglyceride fractions from grey matter were enriched in polyunsaturated fatty acids compared with those from white matter. The acyl groups 18:0, 18:1, and 16:0 and their alk‐1‐enyl analogues were prominent in grey and white matter ethanolamine phosphoglycerides; 18:1 was dominant in white matter alk‐1‐enylacylglycerophosphorylethanolamines. The plasmalogen composition of ethanolamine phosphoglycerides was 55% in grey matter and 76% in white matter.

Choline phosphoglycerides contained negligible plasmalogen and low polyunsaturated fatty acid levels. Diacylglycerophosphorylcholine was characterized by high levels of 16:0 and 18:1. Similar acyl group distributions were estimated in the 1‐position in both grey and white matter, 16:0 being present at >50%.

The presence of the molecular species 18:0/22:6ω3 was indicated in grey matter diacylglycerophosphorylethanolamine, 18:1/18:1 in white matter alk‐1‐enylacylglycerophosphorylethanolamine, and 16:0/18:1 in white matter diacylglycerophosphorylcholine.
Axoplasmic Transport of Transfer RNA in the Chick Optic System
Authors: Scheffer J. W. , Howe N. , Gunning P. W. , Austin L.
Published: October 2006

Abstract: It has previously been shown that 4S RNA is transported in the optic nerve of the chick, but that no movement of rRNA can be detected. The 4S component behaved as though it were composed mainly of transfer RNA (tRNA), but the possibility remained that it could contain significant amounts of material resulting from RNA degradation. The transport of this 4S component has been examined in more detail to determine its nature. In addition, the transported material was examined to establish whether the transport of tRNA is a general phenomenon or that there are only a limited number of species involved. This was done using the same principles applied in the previous study; i.e., the specific activities of separated 4S RNA species appearing in the optic tectum 4 days after intraocular injection of [3H]uridine were compared with that of 5S RNA, a nontransported species. The separation was accomplished using 2.8‐5‐10‐17% slab polyacrylamide gels, and 18 separate regions of 4S species could be identified. The results show that at least most, if not all 4S RNA species are transported. In a separate series of experiments the 4S RNA was aminoacylated and again separated on slab gels. In this instance, the RNA was labelled with [3H]uridine and the aminoacyl component with [14C]amino acids. Gel profiles of these dual‐labelled components showed excellent correspondence between the two labels, demonstrating that 4S RNA species could be aminoacylated and were therefore tRNA species. In contrast to tRNA, the small‐molecular‐weight RNA (smwRNA), species L (7S RNA) is not transported. This smwRNA is largely cytoplasmic in location, as is tRNA. If the transport system were nonspecific it would be expected that smw‐L would also be transported. A number of potential roles for axonal tRNA are considered.
PO Protein and 2′,3′‐Cyclic‐Nucleotide 3′‐Phosphodiesterase Activity in the Peripheral Nerve and Subcellular Fractions of the Trembler Mouse
Authors: Lewis Lorraine N. , Nunn David J. , Mezei Catherine
Published: October 2006

Abstract: To investigate the biochemical abnormalities of the Trembler mouse, the level of the PO protein (as % of total protein) and the activity of CNP was compared in the sciatic nerve and subcellular fractions of normal and mutant littermates. There was a significant decrease in both of these myelin markers in total nerve homgenates of the neurological mutant compared with the control animals. Immunoassay of the PO protein and polyacrylamide gel analysis of proteins indicated an accumulation of a protein with an apparent molecular weight of 67K in mutant nerve extracts. The mutant nerve also had relatively decreased levels of a protein of molecular weight about 41K that cross‐reacted with antibody to PO protein. The Trembler mouse exhibited a larger percentage recovery of PO protein and CNP activity in subcellular fractions denser than the myelin sheath. Together these results are consistent with the theories that these denser components represent immature forms of myelin and that the Trembler mutant is characterized by hypomyelination.
Tyrosine Increases Tissue Dopamine Concentration in the Rat
Authors: Oishi Tomonari , Szabo Sandor
Published: October 2006

Abstract: Dopamine and norepinephrine concentrations in the stomach, duodenum, and brain were measured after subcutaneous injection of tyrosine methyl ester in the rat. Tyrosine significantly increased dopamine concentrations in each tissue without altering norepinephrine levels. The increase in dopamine concentration in the stomach or duodenum was observed 1 h after tyrosine methyl ester injection, but it returned to normal 4 h after the administration of the chemical.
Isolation of Protoplasmic Astrocytes: A Procedure Based on Controlled Trypsin Digestion
Authors: Chatterjee D. , Sarkar P. K.
Published: October 2006

Abstract: Trypsinization of rat brain tissue for shorter (10 min) and longer (60–90 min) periods is shown to yield two distinctly different types of dissociated cell populations. The over‐all yield of intact dissociated cells declines when the period of trypsinization exceeds 10 min. Comparison of the types of dissociated cells obtained after different lengths of trypsinization indicates that the protoplasmic astrocytes, which represent the bulk of the neuroglial cells in brain, are highly susceptible to degradation during tissue trypsinization. Based on this observation, a new procedure involving controlled trypsin digestion for tissue disruption and Percoll gradient cen‐trifugation has been developed for the isolation of virtually pure populations of intact protoplasmic astrocytes (97–98% particle purity). Identification of the purified cells is based on morphological, histochemical staining, and biochemical (marker enzyme) characteristics.
Microsomal Flavin‐Containing Monooxygenase Activity in Rat Corpus Striatum
Authors: Duffel Michael W. , Gillespie Stephen G.
Published: October 2006

Abstract: Microsomal fractions isolated from rat corpus striatum catalyze the oxidation of thiobenzamide to the sulfoxide. The rate of thiobenzamide sulfoxidation is 6.9 ± 4.8 nmol (min)−1 (mg microsomal protein)−1. The reaction is inhibited by an excess of sulfur‐and nitrogen‐containing substrates for the microsomal flavin‐containing monooxygenase. These inhibitors of thiobenzamide sulfoxidation include methimazole, cysteamine, and trimethylamine. Enzyme activity is also destroyed by treatment of the microsomal preparation at 60± for 1 min. In parallel experiments, rat liver microsomes exhibit similar inhibition characteristics. The data indicate the presence in corpus striatum of a microsomal monooxygenase with catalytic properties of the hepatic microsomal flavin‐containing monooxygenase.
Measurement of Serotonin Turnover Rate in Rat Dorsal Raphe Nucleus by In Vivo Electrochemistry
Authors: Echizen Hirotoshi , Freed Curt R.
Published: October 2006

Abstract: 5‐Hydroxytryptamine (5‐HT; serotonin) turnover rate in dorsal raphe nucleus of the urethane‐anes‐thetized rat was estimated by using the in vivo electrochemical detector to measure the decay of extraneuronal 5‐hydroxyindole acetic acid (5‐HIAA) after monoamine oxidase inhibition. Carbon paste electrodes were scanned by semiderivative voltammetry and revealed two peaks: one at +0.15 V and the other at +0.25 V. The higher potential peak is composed primarily of the 5‐HT metabolite 5‐HIAA. After administration of pargyline, 75 mg/ kg i.p., this peak declined exponentially. Regression analysis of these data by an exponential decay model yielded the fractional rate constant 0.82 ± 0.06 h−1 (mean ± SEM). This rate constant of 5‐HIAA disappearance measured by in vivo electrochemistry is identical to the rate constant found by others measuring 5‐HIAA disappearance by direct tissue assay methods. In animals not treated with pargyline, tissue 5‐HIAA concentrations in the dorsal raphe nucleus were measured by HPLC with electrochemical detection. The average 5‐HT turnover rate calculated as the product of the fractional rate constant and steady‐state tissue 5‐HIAA concentration was 12.6 nmol/g/h. These results demonstrate that electrochemical detection of extraneuronal 5‐HIAA combined with monoamine oxidase inhibition can be used to measure neurotransmitter turnover in vivo in a discrete brain region.
Glycoproteins of the Chromaffin Granule Membrane: Separation by Two‐Dimensional Electrophoresis and Identification by Lectin Binding
Authors: Gavine Fiona S. , Pryde James G. , Deane David L. , Apps David K.
Published: October 2006

Abstract: The proteins of highly purified chromaffin granule membranes were separated by one or two‐dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organdies. Two dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin‐binding bands are discussed: neither cytochrome b561 nor the F1‐like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin‐A binding proteins, as well as dopamine β‐hydroxylase, are present in the chromaffin granule lysate.
Effect of α‐Amino‐4‐Phosphonobutyrate on the Release of Endogenous Glutamate and Aspartate from Cortical Synaptosomes of Epileptic Rats
Authors: Abdul‐Ghani Abdul‐Salam
Published: October 2006

Abstract: The effect of the glutamate antagonist α‐amino‐4‐phosphonobutyrate (APBA) on the release of endogenous amino acids from sensorimotor cortical synaptosomes of rats with a cortical cobalt focus and from nonepileptic rats was studied: (1) The release of endogenous glutamate, aspartate, and γ‐aminobutyric acid (GABA) from synaptosomal preparations of cobalt‐induced epileptogenic tissues was increased compared with the release from the contralateral (sensorimotor) region or the sensorimotor cortex of normal animals. The intrasynaptosomal content of these amino acids was reduced in proportion to the amount released. The levels of other amino acids were unaffected or showed much smaller changes. (2) APBA (0.5‐1 mM) decreased significantly the spontaneous release of aspartate and glutamate from the epileptic foci without affecting GABA or any other amino acid. (3) APBA produced no effect whatsoever on the release of any amino acid from synaptosomal preparations of nonepileptic focus.
Renin and Angiotensin‐Converting Enzyme in Human Neuroblastoma Tissue
Authors: Mizuno Kenji , Ojima Motoko , Hashimoto Shigeatsu , Fukuchi Soitsu
Published: October 2006

Abstract: High activity of renin was demonstrated in human neuroblastoma tissue. This activity was inhibited by specific antibody raised against human renal renin, indicating that it was not due to the nonspecific action of proteases. The specific activity of renin was 122.8 ng of angiotensin I generated mg of protein −1h −1. It shared some biochemical features with well‐known kidney renin, such as molecular weight, optimum pH, the presence of trypsin‐activatable inactive renin, and glycoprotein nature. Furthermore, angiotensin‐converting enzyme (ACE) activity (2.64 nmol mg of protein−1 min−1) was found in the tissue. This activity was inhibited by captopril, a specific ACE inhibitor, or by omission of chloride ion. These results suggest that true renin in addition to ACE exists in human neuroblastoma tissue.
Medium Isotope Effect in [3H]Diazepam Binding to Benzodiazepine Receptors of Synaptic Membranes
Authors: Kardos J. , Kovács I. , Simonyi M.
Published: October 2006

Abstract: Replacement of H2O by D2O resulted in significantly higher amount of [3H]diazepam specifically bound to synaptic membranes. The isotope effect arises from increased number of binding sites in D2O and is associated with a stronger solvation of membrane receptors by heavy water.
Free Mitochondria and Synaptosomes from Single Rat Forebrain. A Comparison Between Two Known Subfractionation Techniques
Authors: Dagani F. , Zanada F. , Marzatico F. , Benzi G.
Published: October 2006

Abstract: Two published subcellular subfractionation techniques employing Ficoll‐sucrose or sucrose‐density gradient centrifugation, respectively, are evaluated for their capacity to yield fractions containing free mitochondria and synaptosomes from a single rat forebrain. The enzymes lactate dehydrogenase, acetylcholinesterase, NAD(P)H‐cytochrome c reductase, and citrate synthase, markers of different subcellular components, were used to assess the purity and integrity of the fractions. Judged by the distribution of these specific enzymatic markers, the free mitochondria obtained by the Ficoll‐sucrose gradient technique were less contaminated by synaptosomes and had greater biochemical integrity than those obtained by the sucrose‐gradient technique. By contrast, the synaptosomes obtained by the Ficollsucrose gradient technique resulted in more contamination by microsomes than those prepared in a sucrose gradient.
Alteration in the Postnatal Ontogeny of Cerebellar Norepinephrine Content Following Chronic Prenatal Carbon Monoxide
Authors: Storm Jan E. , Fechter Laurence D.
Published: October 2006

Abstract: The postnatal ontogeny of norepinephrine content in the cortex and cerebellum was determined in rats exposed prenatally to a chronic low level of carbon monoxide (150 parts per million). In the cerebellum, norepinephrine concentration and total norepinephrine content among carbon monoxide‐exposed rats were consistently elevated over that of control rats from the second through the sixth postnatal weeks. In the cortex, norepinephrine concentration and total norepinephrine content among carbon monoxide‐exposed rats did not differ from that of control rats over the same period. These results identify the cerebellum as a region whose postnatal development is altered by prenatal exposure to low levels of carbon monoxide‐induced hypoxia.
Effects of Short‐ and Long‐Term Protriptyline Treatment on Phospholipid Metabolism in Rat Brain
Authors: Trzeciak Henryk I.
Published: October 2006

Abstract: Wistar rats were injected intraperitoneally with 10 mg/kg of protriptyline according to one of the following schedules: a single dose or daily for 4 days (short‐term), or daily for 2 or 13 weeks (long‐term). Total lipid, total phospholipid, and individual phospholipid contents in the brain were determined. Further, the incorporation of 32P into individual phospholipids in vivo and the fatty acid composition of phosphatidylethanolamine in the brains of rats treated with protriptyline for 13 weeks were studied. Three alternative phases of changes of total and individual phospholipid contents in the brain during 13 weeks of experimentation were distinguished. An increase of phospholipid contents after 4 days, a decrease after 2 weeks, and a further increase after 13 weeks of protriptyline administration were found. However, phosphatidylinositol and phosphatidic acid levels after 13 weeks of protriptyline administration were diminished. The decrease of specific radioactivity of phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine and the increase of phosphatidylinositol, phosphatidic acid, and sphingomyelin in rats treated with the drug for a longer period of time were noted. No greater differences in fatty acid composition of phosphatidylethanolamine in the brains of the same group of rats were observed as compared to control. These results indicate that during long‐term treatment with protriptyline the contents of lipids and phospholipids in rat brain are altered. The modification of the biological function of phospholipids in brain cell membranes is suggested.
Comparison of Protein Synthesis in Mitochondria, Synaptosomes, and Intact Brain Cells
Authors: Irwin Carol C.
Published: October 2006

Abstract: Qualitative aspects of protein synthesis in organelles and intact cultured cells of brain origin were compared to clarify the distinction between synaptosomal and mitochondrial protein synthesis. Brain mitochondria and synaptosomes were isolated either on a traditional Ficoll‐sucrose gradient or by a new Percoll gradient procedure, and were incubated in an amino acid incorporation system containing [35S]methionine, then electrophoresed on gradient slab gels. Autoradiography of the gels revealed that in the presence of cycloheximide both mitochondria and synaptosomes synthesized at least 17 proteins in the 6,000‐50,000 MW range, and that incubation with chloramphenicol reduced or eliminated these bands. With minor variation these patterns in the low‐molecularweight region also resembled patterns obtained from cycloheximide‐inhibited rat liver mitochondria and intact brain cells (cultured glia, glioma, and neuroblastoma). In the higher molecular weight region of the gels (>50,000) banding patterns were more complex and tended to differ between organelles and intact cells. These polypeptides probably reflect nonmitochondrial protein synthesis, and their variable response to inhibitors may account for confusion in the literature with regard to the effects of inhibitors of protein synthesis in brain mitochondria and synaptosomes.
Calcium‐Dependent Calmodulin Binding to Chromaffin Granule Membranes: Presence of a 65‐Kilodalton Calmodulin‐Binding Protein
Authors: Bader M. F. , Hikita T. , Trifaró J. M.
Published: October 2006

Abstract: The presence of calmodulin‐binding sites on chromaffin granule membranes has been investigated. Saturable, high‐affinity 125I‐calmodulin‐binding sites (KD= 9.8 nM; Bmax= 25 pmol/mg protein) were observed in the presence of 10−4M free calcium. A second, nonsaturable, calmodulin‐binding activity could also be detected at 10−7M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10−4M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10−7M promotes the binding of calmodulin to some solvent‐soluble components of the chromaffin granule membrane. Calmodulin‐binding proteins associated with the granule membrane were identified by photoaffinity cross‐linking. A calmodulin‐binding protein complex, of molecular weight 82K, was formed in the presence of 10−4M free calcium. This cross‐linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross‐linker activation. When solvent‐treated membranes were used, a second, specific, calmodulin‐binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin‐binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin‐affinity chromatography. When detergent‐solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin‐binding protein, it is concluded that only the 65K calmodulin‐binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.
Evidence that [3H]Dopamine Is Taken Up and Released from Nondopaminergic Nerve Terminals in the Rat Substantia Nigra In Vitro
Authors: Kelly E. , Jenner P. , Marsden C.D.
Published: October 2006

Abstract: Potassium chloride (25 mM) and (+)‐amphet‐amine (100 μM) both stimulated the release of radioactivity from slices of substantia nigra preincubated with [3H]3,4‐dihydroxyphenylethylamine ([3H]dopamine). Potassium chloride (25 mM) released radioactivity from slices of both zona compacta and zona reticulata. Prior 6‐hydroxydopamine (6‐OHDA) lesions of one nigro‐striatal pathway did not reduce the spontaneous release of radioactivity, or the potassium chloride‐ or amphetamine‐induced release of radioactivity from slices of nigra ipsilateral to the lesion after preincubation with [3H]dopamine. The accumulation of radioactivity following incubation of nigral slices from 6‐OHDA‐lesioned animals with [3H]dopamine was increased when compared to uptake into slices from intact tissue. In synap‐tosomal preparations of striatum, nomifensine but not de‐sipramine or fluoxetine inhibited [3H]dopamine uptake. In contrast, nomifensine, desipramine, and fluoxetine all inhibited [3H]dopamine uptake in nigral synaptosomal preparations. Following 6‐OHDA lesions of one nigro‐striatal pathway the uptake of [3H]dopamine into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was substantially decreased. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]dopamine uptake into nigral preparations but not into striatal synaptosomes. The uptake of [3H]5‐hydroxytryptamine ([3H]5‐HT) into synaptosomal preparations of substantia nigra was abolished by fluoxetine and reduced by desipramine, but was unaffected by nomifensine. In contrast, fluoxetine, desipramine, and nomifensine all inhibited [3H]5‐HT uptake into striatal synaptosomal reparations. Following 6‐OHDA lesions of one nigro‐striatal pathway the uptake of [3H]5‐HT into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was reduced. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]5‐HT uptake into both nigral and striatal synaptosomal preparations. These results suggest that in tissue preparations from substantia nigra, the in vitro uptake and release of [3H]dopamine may not reflect dendritic dopamine function alone, but may involve other neuronal processes, particularly 5‐HT neurones.
In Vitro Phosphorylation of Microtubule‐Associated Protein 2: Differential Effects of Cyclic AMP Analogues
Authors: Richter‐Landsberg Christiane , Jastorff Bernd
Published: October 2006

Abstract: Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)‐dependent protein kinase activity, and microtubule‐associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2′, 3′, or 5’ position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3′,5′‐monophosphorothioate (cAMPS). The (Sp)‐cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)‐cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP‐stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP‐dependent phosphorylation on functional properties in microtubules in general.
Isolation of Eukaryotic Initiation Factor 2 from Rat Brain
Authors: Calés Carmela , Salinas Matilde , Fando Juan L.
Published: October 2006

Abstract: Eukaryotic initiation factor 2 (eIF‐2) was isolated from salt‐washed microsomes of 4‐day‐old rat brain which show a high rate of protein synthesis. A three‐step purification scheme was employed, including heparin‐Sepharose, phosphocellulose, and DEAE‐cellulose column chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF‐2 forms ternary complexes with [3H]methionyl‐tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF‐2 and explains the high rate of activity of the crude fraction.
β‐Glucosidase and β‐Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes
Authors: Hof Liselotte , Kimelberg Harold K.
Published: October 2006

In primary astrocyte cultures β‐glucosidase (EC 3.2.1.21) and β‐galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco‐and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3–4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12–20 days after birth. The specific activities were two‐ to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of β‐galactosidase, but not β‐glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.
Effect of Intraventricular Putrescine on Adenosylmethionine Decarboxylase in Rat Hypothalamus and Caudate Nucleus
Authors: Ientile Riccardo , Ruggeri Pietro , Russo Pietro , Macaione Salvatore
Published: October 2006

Abstract: Effects of intracerebroventricularly injected putrescine on adenosylmethionine decarboxylase activity in rat hypothalamus and caudate nucleus were studied. Doses that lacked the capacity to produce behavioral and electrocortical epileptogenic disorder caused a significant activation of enzyme occurring between 10 and 15 min after injection. The changes in hypothalamic and caudate nucleus enzyme activity after putrescine administration were dose‐dependent, and they supported the assumption that limiting putrescine concentration in vivo may play a role in adenosylmethionine decarboxylase regulation.
Characterization of Sodium‐Dependent [3H]GBR‐12935 Binding in Brain: A Radioligand for Selective Labelling of the Dopamine Transport Complex
Authors: Janowsky Aaron , Berger Paul , Vocci Frank , Labarca Rodrigo , Skolnick Phil , Paul Steven M.
Published: October 2006

Abstract: High‐affinity and saturable binding sites for the diphenyl‐substituted piperazine derivative [3H]GBR‐12935 have been characterized in crude synaptosomal membranes prepared from rat brain. The specific binding of [3H]GBR‐12935 is sodium‐dependent and is unevenly distributed among various brain regions, with the highest concentration of binding sites being found in the corpus striatum and nucleus accumbens. Sodium‐dependent [3H]GBR‐12935 binding in all other brain areas was 10% or less of the binding found in the striatum. The affinity of [3H]GBR‐12935 for binding sites in the striatum is increased in the presence of Na+ but other cations, including K+, Ca2+, or Mg2+, inhibit specific binding. There is an excellent correlation (r= 0.96, p < 0.01) between the potencies of a series of drugs in inhibiting [3H]GBR‐12935 binding to striatal membranes and their potencies in inhibiting [3H]3,4‐dihydroxyphenylethylamine ([3H]dopamine) uptake in synaptosomes. Agonists and antagonists of other neurotransmitter receptor or drug recognition sites have little or no effect on specific [3H]GBR‐12935 binding to striatal membranes. In addition, prior intracerebroventricular administration of 6‐hydroxydopamine results in a decrease in the number of specific [3H]GBR‐12935 binding sites in the striatum. These data indicate that [3H]GBR‐12935 is a selective radioligand of the presynaptic dopamine transport complex in brain.
Specific Inhibition of Dopamine D‐1‐Mediated Cyclic AMP Formation by Dopamine D‐2, Muscarinic Cholinergic, and Opiate Receptor Stimulation in Rat Striatal Slices
Authors: Kelly Eamonn , Nahorski Stefan R.
Published: October 2006

Abstract The ability of different receptors to mediate inhibition of cyclic AMP accumulation due to a variety of agonists was examined in rat striatal slices. In the presence of 1 mM 3‐isobutyl‐1‐metbylxanthine, dopamine D‐2, muscarinic cholinergic, and opiate receptor stimulation by RU 24926, carbachol, and morphine (all at 10−‐8‐−1(10−‐5M), respectively, inhibited the increase in cyclic AMP accumulation in slices of rat striatum due to dopamine D‐1 receptor stimulation by 1 μM SKF 38393. In contrast, these inhibitory agents were unable to reduce the ability of a number of other agonists, including isoprenaline, prostaglandin E1, 2‐chloroadenosine, vasoactive intestinal polypeptide, and cholera toxin, to increase cyclic AMP levels in striatal slices.

These results suggest that in rat striatum either (a) dopamine D‐2, muscarinic cholinergic, and opiate receptors are only functionally linked to dopamine D‐l receptors or that (b) the D‐1 and D‐2 receptors linked to adenylate cyclase lie on the cells, distinct from other receptors capable of elevating striatal cyclic AMP levels.
Acute Effect of Ammonia on Branched‐Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures
Authors: Murthy Ch. R. K. , Hertz Leif
Published: October 2006

Abstract: 14CO2 production and incorporation of label into proteins from the labeled branched‐chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mMammonium chloride. Production of 14CO2 from [I‐14C]leucine and [I‐14C]valine was larger than 14CO3 production from [U‐14C]leucine and [U‐14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched‐chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mMammonium chloride greatly suppressed 14CO2 production from [I‐14C]‐labeIed branched chain amino acids but had little effect on 14CO2 production from [U‐14C]‐labeled branched‐chain amino acids in astrocytes. Ammoniumion, at this concentration, suppressed the incorporation of label from all three branched‐chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.
Peptide E and Its Products, BAM 18 and Leu‐Enkephalin, in Bovine Adrenal Medulla and Cultured Chromaffin Cells: Release in Response to Stimulation
Authors: Boarder Michael R. , Evans Christopher , Adams Maria , Erdelyi, Elizabeth , D. Barchas Jack
Published: October 2006

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys‐Arg) typical processing site, would generate two opioid fragments, the amino‐terminal fragment BAM 18 and the carboxy‐terminal fragment Leu‐enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu‐enkephalin). Here we present evidence that BAM 18 and Leu‐enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys‐Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu‐enkephalin was found to be present in much greater amounts than was BAM 18‐like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu‐enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu‐enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu‐enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.
Effects of Lead Ions on Events Associated with Exocytosis in Isolated Bovine Adrenal Medullary Cells
Authors: Pocock G. , Simons T. J. B.
Published: October 2006

Abstract: Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1–6 μM) of Pb2+ on stimulus‐secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 μM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol‐evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor‐mediated channels, measured in the presence of tetrodotoxin (2 μM) and ouabain (50 μM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K‐ and carbachol‐evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine‐induced channels as well as through voltage‐sensitive calcium channels.
Effects of α2‐Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents
Authors: Sakakibara Manabu , Collin Carlos , Kuzirian Alan , Alkon Daniel L. , Heldman Eliahu , Naito Shigetaka , Lederhendler Izja
Published: October 2006

Abstract: Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presyn‐aptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (≥ 100 μM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage‐dependent outward K+ currents (IA, an early current, Ica2+‐K+, a late Ca2+‐dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the α2‐receptor antagonist, yohimbine (0.5 μM), but not the α1‐blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde‐induced fluorescence or Falck‐Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine‐like synaptic effect on type B cells, therefore, could amplify conditioning‐specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.