bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Subunit Structure of DNA‐dependent RNA Polymerase BII from Rat Brain Nuclei1
Authors: Yamamoto H. , Takahashi Y.
Published: October 2006

DNA‐dependent RNA polymerase BII from rat brain nuclei was purified by ammonium sulphate fractionation, successive chromatography on ion‐exchange resins, and glycerol density gradient centrifugation. The purified enzyme was resolved into two components (BIIa and BIIb) on native polyacrylamide gel electrophoresis. The subunit compositions of these enzymes were analysed on SDS‐polyacrylamide gel. BIIa enzyme consisted of nine subunits: BII1 (215,000), BII3 (150,000), BII4 (35,000), BII5 (25,000), BII6 (21,500), BII7 (17,500), BII8 (15,500), BII9 (14,500), and BII10 (11,500); in BIIb enzyme, which also contained nine subunits, BII1 was replaced by a BII2 (180,000) subunit. Addition of both 30% glycerol and dithiothreitol to the preparation medium was essential for stabilizing the enzyme.
Lipid Labeling After Intracerebral Injection of Radioactive Malonic Acid
Authors: Koeppen Arnulf H. , Mitzen Edward J. , Papandrea John D.
Published: October 2006

Growing albino rats between the ages of birth and 50 days received single intracranial injections of either [2‐14C]malonate or [1‐14C]malonate. After 1 h, 90% of the nonmalonate radioactivity was recovered in the free amino acids. Fatty acids and sterols accounted for the remainder. Most of the fatty acid radioactivity resided in palmitate and stearate. The label of the polar lipid fraction was concentrated in choline phosphoglyceride (CPG) (65%).Over half of the CPG‐fatty acid radioactivity was present in the 2‐acyl group. Fatty acid biosynthesis occurred predominantly by a de novo mechanism although some elongation was also present. The incorporation of the precursor into amino acids increased rapidly after birth and reached stable levels at 14 days. Fatty acid labeling also increased with brain growth but the fatty acid‐amino acid ratio declined steadily from birth to 50 days. Synthesis of malonyl‐CoA is the rate‐limiting step in the biosynthesis of amino acids, fatty acids, and sterols from free malonic acid.
An Improved DEAE‐Cellulose Filter Assay for Colchicine Binding Protein (During Mouse Brain Development)
Authors: Tse C. , Doherty R. A.
Published: October 2006

A DEAE‐cellulose filter assay for colchicine binding protein (CBP) has been modified to include 1 m‐sucrose in the incubation medium (single‐point assay). Due to the much greater stability of colchicine binding capacity under the conditions described, multiple time‐point assays (time‐decay method) were not necessary for accurate determination of CBP in brain samples, as shown by the agreement of results obtained using the two methods to measure CBP in C129F1 hybrid mouse brains at different stages of development. In this study, the highest concentrations of CBP (millimoles of colchicine bound per milligram of total protein) were observed in the 160,000g supernatant and pellet of newborn‐brain homogenate. The CBP concentrations in these two fractions were approximately twice those observed in the earliest stage measured (13.5 days gestation) and in the 11‐month‐old adult. However, CBP concentration, in terms of millimoles of colchicine bound per gram of brain wet weight, reached a maximum at the end of the 1st week after birth. Between 17.5 days gestation and the end of the 1st postnatal week, more CBP accumulated in the pellet fraction than in the supernatant. Tse C. and Doherty R. A. An improved DEAE‐cellulose filter assay for colchicine binding protein (during mouse brain development). J. Neurochem.35, 767–774 (1980).
The Effects of Various Incubation Temperatures, Particulate Isolation, and Possible Role of Calmodulin on the Activity of the Base Exchange Enzymes of Rat Brain
Authors: Buchanan Alfred G. , Kanfer J. N.
Published: October 2006

The involvement of calmodulin in the choline, ethanolamine, and serine exchange activities of rat brain microsomes was investigated. Calmodulin stimulated choline exchange activity to a greater extent than ethanolamine and serine exchange activities. The three base exchange activities were inhibited by antipsychotic drugs believed to prevent calmodulin interaction, but not by calmodulin‐binding protein. The solutions employed for tissue homogenization and subsequent isolation of microsomes greatly influenced the base exchange activites. The process of resuspending isolated microsomes and recentrifugation, or “washing,” produced major losses of detectable activity. The base exchange enzyme activities were maximal at 45°, and Arrhenius plots revealed a common transition temperature of 31°. The activation energies for the base exchange reactions decreased at temperatures above the observed transition temperature. Kinetic data, Km and Vmax, for the base exchange activities at 27, 37, and 45° are presented. Buchanan A. G. and Kanfer J. N. The effects of various incubation temperatures, particulate isolation, and possible role of calmodulin on the activity of the base exchange enzymes of rat brain. J. Neurochem.35, 814–822 (1980).
Comparative Analysis of Rapidly Transported Axonal Proteins in Sensory Neurons of the Frog and Rat
Authors: Neale Joseph H. , Formant David S. , Shibla Deborah B. , Shortell Sheryl A.
Published: October 2006

35S‐labeled proteins carried by fast axonal transport in sciatic sensory axons of bullfrog and rat were separated electrophoretically on discontinuous polyacrylamide gradient slab gels. In contrast to the previously reported similarity in the electrophoretic profiles of rapidly transported proteins from functionally different neurons, we have found that there is very little correspondence in the profiles of these proteins in functionally similar neurons from two widely studied species. We also found very little correspondence between the two species in the profiles of locally synthesized sciatic nerve protein. The results demonstrate the difficulty inherent in comparing the electrophoretic profiles obtained using these two model systems for the study of rapidly transported axonal proteins. In particular, relationships between the major rapidly transported proteins in the two species could_not be analyzed with this technique.
The Response of Ornithine Decarboxylase During Neuronal Degeneration and Regeneration in Olfactory Epithelium
Authors: Rochel S. , Margolis F. L.
Published: October 2006

Mature olfactory neurons are continually replaced from a population of progenitor cells. Olfactory nerve section, bulbectomy, or treatment with certain chemicals induces degeneration of olfactory neurons followed in some cases by regeneration. Ornithine decarboxylase (ODC) activity was measured in mouse olfactory tissues as an indicator of cellular regeneration. ODC activity in olfactory tissue (0.2–0.4 nmol/mg protein/h) is 10‐30 times higher than in a variety of other cerebral tissues. Within 3 h after unilateral olfactory nerve section, ODC activity in the epithelium declines to 50% of control followed by a slow return to basal activity by 6 days. In the same animals, ODC activity increases severalfold in bulb (1 day) with a gradual decline to normal (9 days). Except for an early transient increase, the effects of unilateral bulbectomy on epithelial ODC activity are similar to those seen after nerve section. The changes in ODC activity following intranasal irrigation with 10 mm‐colchicine also closely mimic those seen after nerve section. The effects of intranasal irrigation on ODC activity with 0.5% Triton X‐100 or 0.17 m‐ZnSO4 are more complex. Thus, when the mature neuronal population is degenerating after surgery or chemical treatments, ODC activity decreases in the epithelium. The subsequent increase of ODC activity prior to reconstitution of the mature neuronal population probably reflects the regeneration mechanism of the olfactory epithelium. The increase of ODC activity in the olfactory bulb after nerve section is best interpreted as a cellular injury response. These alterations in ODC activity in olfactory tissues after chemical and surgical treatments constitute the earliest biochemical events observed in these tissues in response to cellular damage.
Changes in RNA Synthesis and Messenger RNA Content in the Cerebellum of Rats with Graft Versus Host Disease
Authors: Griffin W. Sue T. , Head Judith R. , Pardue Sibile , Morrisont Marcelle R.
Published: October 2006

Cerebellar RNA accumulation, synthesis, and functional capacity was studied in 14‐day‐old F, hybrid rats subjected to neonatally induced graft versus host disease (GVHD). There was a decrease in RNA synthetic rate as measured by the uptake of labeled precursors into RNA. The decrease in total cerebellar RNA synthesis was reflected both in a reduced amount of Nissl substance, visible in cresyl violet‐stained 10‐μ‐thick sections of cerebella, and in the total amount of cytoplasmic RNA isolated from individual cerebella from diseased animals compared with control littermates. Analysis of the RNA translational capacity in wheat germ protein synthesizing systems showed that RNA from experimental animals was also biologically less active. Qualitative differences between protein populations in control and diseased animals were analyzed by two‐dimensional gel electrophoresis. There were few alterations in the steady state levels of cerebellar protein. However, two‐dimensional gel electrophoresis of the peptides synthesized in vitro by RNA from control and diseased animals showed that there were several changes in the relative abundance of some mRNAs between the two RNA populations. These data show that the cerebellar RNA from rats with GVHD differs both qualitatively and quantitatively from that of controls.
Rate of Movement and Composition of Rapidly Transported Proteins in Regenerating Olfactory Nerve
Authors: Cancalon P. , Elam J. S.
Published: October 2006

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 × 9 mm/day at 23°C, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a lighter axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high‐molecular‐weight polypeptides.
Membrane Potentials in Cell‐free Preparations from Guinea Pig Cerebral Cortex: Effect of Depolarizing Agents and Cyclic Nucleotides
Authors: Creveling C. R. , McNeal E. T. , McCulloh D. H. , Daly J. W.
Published: October 2006

The distribution of [3H]triphenylmethylphosphonium ion between the medium and vesicular entities was examined in a cell‐free, particulate preparation from guinea pig cerebral cortex. This distribution followed the Nernst relationship with regard to the external potassium ion concentration and, in physiological media, indicated the maintenance of a mean trans‐membrane potential ranging from –58 to –78 mV. The neurotoxins batrachotoxin, veratridine, and grayanotoxin I, partially depolarized the preparation. Tetrodotoxin blocked the depolarization by batrachotoxin, veratridine, and grayanotoxin I. The depolarization by these neurotoxins was potentiated by the presence of anemone toxin II and presumably reflected the response of vesicular components of neuronal origin. An additional potassium‐sensitive depolarization probably represented the response of vesicular components of glial origin with an apparent transmembrane potential of –8 to –35 mV. No correlation could be demonstrated between changes in transmembrane potential and stimulation of cyclic AMP generation by a variety of agents in this preparation.
N‐Ethyl Analogues of Choline as Precursors to Cholinergic False Transmitters
Authors: Boksa P. , Collier B.
Published: October 2006

Cat superior cervical ganglia perfused with the choline analogue, diethylcholine, acetylated the analogue and acetyldiethylcholine was sub‐sequently released from the ganglia in response to preganglionic nerve stimulation by a Ca2‐‐dependent mechanism. Rat cerebral cortical slices incubated with monoethylcholine or diethylcholine acetylated the choline analogues, and both acetylmonoethylcholine and acetyldiethylcholine were released from the slices in response to stimulation by a high K+. concentration. In brain slices pre‐incubated with monoethylcholine, diethylcholine, or triethylcholine, acetylated derivatives of the choline analogues were found in both free and bound nerve‐ending stores, similar to the subcellular localization of [3H]acetyl‐choline (ACh) in brain slices pre‐incubated with [3H]choline; the relative distribution of each of the acetylated choline analogues between the two nerve‐ending stores differed only slightly from that of [3H]ACh. Nerve‐ending free and bound stores of acetyldiethylcholine were equally depleted when brain slices were stimulated under conditions that cause depletion of releasable acetyldiethylcholine stores; similar results were obtained with acetyltriethyl‐choline. It is concluded that the three acetylated ethyl analogues of choline fulfill the necessary criteria for identification as cholinergic false transmitters, and that, under the conditions of the present experiments with brain, the false transmitters are able to distribute similarly to ACh between nerve‐ending stores.
Studies on a Molecular Basis for the Heparin‐Induced Regulation of Enzymatic Activity of Mouse Striatal Tyrosine Hydroxylase In Vitro. Inhibition of Heparin Activation and of the Enzyme by Poly‐L‐lysyltyrosine and Poly‐L‐lysylphenylalanine and Their Constituent Peptides
Authors: Stone Audrey L.
Published: October 2006

Tyrosine hydroxylase was purified up to 10‐fold from hypotonic extracts of mouse striatum by heparin affinity chromatography. The purified enzyme (a) had a low Km for tyrosine (around 15 μm) and was not inhibited by tyrosine at concentrations up to 0.2 mm when tetrahydrobiopterin was cofactor and (b) was activated by heparin. The interaction of heparin with tyrosine hydroxylase was studied in ways relating to the known interaction with anti‐thrombin. Heparan and keratan sulfates failed to activate tyrosine hydroxylase in place of heparin; several fractions of the bulk heparin (constituting 5 and 15%) had enriched tyrosine hydroxylase‐activating potency; and two lysine copolypeptides (polylysyltyrosine and polylysylphenylalanine) inhibited the activation of tyrosine hydroxylase by heparin. The lysine copolymers also directly inhibited the enzyme. Heparin (but not heparan and keratan sulfates) protected tyrosine hydroxylase from this inhibition. The constituent lysyltyrosyl (but not lysylphenylalanyl) peptide inhibited tyrosine hydroxylase, and heparin also reversed this inhibition, which was sigmoidal (IC50 of 490 μM) and partially competitive with tyrosine. Tyrosine hydroxylase was purified up to sevenfold by lysyltyrosyl‐affinity chromatography. This enzyme preparation exhibited an eightfold greater sensitivity to lysyltyrosylamide than tyrosine hydroxylase purified by heparin affinity. The data indicate that tyrosine hydroxylase is regulated in vitro by a negatively charged site. Occupancy of this site by cationic effectors results in allosteric inhibition which mediates changes in the apparent Km for tyrosine.
Effect of Capsaicin Pretreatment on Substance P Binding to Synaptic Vesicles
Authors: Mayer N. , Gamse R. , Lembeck F.
Published: October 2006

Newborn or adult rats were pretreated with 50 mg kg–1 capsaicin. At the age of 2 to 4 months, binding of 1251‐labelled Tyr8‐substance P to synaptic vesicles prepared from different regions of the nervous system was examined. In both groups, capsaicin pretreatment led to a significant decrease in the number of binding sites in dorsal roots and spinal cord without having an effect on affinity. This decrease parallels the depletion of the substance P content (Gamse et al., 1980) and can be explained by degeneration of primary sensory neurons in newborn treated rats and by depletion of vesicles in adult treated rats.
Development and Regional Distribution of Methionine Synthetase in Rabbit Brain
Authors: Suleiman Suleiman A. , Spector Reynold
Published: October 2006

The development and regional distribution of methionine synthetase (EC 2.1.1.13) in rabbit brain was determined. In adult rabbits, the specific activity (units per milligram protein) of methionine synthetase in cortex, cerebellum, brain stem, and corpus striatum was comparable to the specific activity in whole brain (0.5 units/mg). In the first few weeks of life, the specific activity of methionine synthetase in whole rabbit brain declined from a value of 1.1 units/mg at 1 day of age to 0.5 units/mg at 6–10 weeks. Two‐year‐old rabbits had 0.6 units/mg in whole brain. These results show that: (a) methionine synthetase is distributed widely in mammalian brain and (b) methionine synthetase activity in brain declines relatively little with development.
Quantitative Relationships of Five Putative Neurotransmitter Receptor Sites in Rat Hippocampal Formation
Authors: Salvaterra Paul , Matthews Dee Ann , Foders Renée
Published: October 2006

The hippocampus is well suited for studies of the interrelationships of various neurotransmitter systems in the CNS by reason of its simple laminated organization, defined connections, and variety of identified neurotransmitters. We have studied the biochemical and pharmacological properties of five radiolabeled ligand binding sites in a membrane fraction prepared from rat hippocampal formation. These binding sites are thought to identify recognition sites for neurotransmitter receptors. The rank order of ligand binding sites is [3H]muscimol > [3H]quinuclidinyl benzilate > [3Hdihydroergocryptine > [3H]dihydroalprenolol > 125I‐labeled α‐bungarotoxin. All ligands have a single, saturable, high‐affinity binding site. Pharmacological characterization of the ligand binding sites indicates properties consistent with the identification of these sites as neurotransmitter receptors.
Influence of Synchronized Sleep on the Biosynthesis of RNA in Neuronal and Mixed Fractions Isolated from Rabbit Cerebral Cortex
Authors: Giuditta Antonio , Rutigliano Bruno , Vitale‐Neugebauer Anna
Published: October 2006

Abstract: Adult male rabbits implanted with dural electrodes were injected intraventricularly with [3H]orotate and killed 1 h later. During the period of incorporation they were left undisturbed while their EEG activity was continuously monitored. In the fraction of neuronal perikarya prepared from cerebral cortex by a method developed by Satake and Abe in 1966, the relative content of radioactive RNA of the nuclear particulate showed a twofold increase in the transition from 0 to 100% synchronization. On the other hand, a slight but significant decline was observed in the corresponding cytoplasmic compartment. A marked increase in the relative content of radioactive RNA was similarly observed in the nuclear particulate prepared from the mixed cellular fraction. The corresponding cytoplasmic compartment showed a nonsignificant increase. These results indicate that during sleep neuronal nuclei accumulate newly synthesized RNA (presumably hnRNA) at a faster rate. Under the same conditions the process of RNA transfer to the cytoplasm (presumably rRNA) may be reduced. These effects may be only partly shared by other cerebral cells.
The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells
Authors: Sellinger O. Z. , Der O.
Published: October 2006

Abstract: The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3‐day‐old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [3H]guanosine for 1.5–7.5 h; 3‐day‐old rats were injected with [3H]guanosine and were killed 5–45 min later. [3H]tRNA was extracted, partially purified, and hydrolyzed to yield [3H]‐guanine and [3H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [3H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [3H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [3H]1‐methylguanine represented up to 35% of the total [3H]methyl guanine radioactivity in astrocyte [3H]tRNA, while it became only negligibly labeled in brain [3H]tRNA. Conversely, brain [3H]tRNA contained more [3H]N2‐methylguanine than did astrocyte [3H]tRNA. Approximately equal proportions of [3H]7‐methylguanine were found in the [3H]tRNAs of both neural systems. The [3H]methylguanine composition of brain [3H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE‐cellulose and reverse phase chromatography (RPC‐5), and differences were found between the [3H]methylguanine composition of RPC‐5 fractions containing, respectively, tRNAlys and tRNAphe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell‐specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell‐specific tRNA modifications may be a prerequisite for the successful synthesis of cell‐specific neural proteins.
The Distribution of Mg2+ ATPase and Its Loss of Specific Activity upon Gradient Centrifugation of Isolated Chromaffin Granules
Authors: Konings Frank , De Potter Werner
Published: October 2006

Abstract: Preparations of purified chromaffin granules were subjected to isopycnic sucrose gradient centrifugation. Determination of Mg2+ ATPase activity and catecholamines showed that the distribution of ATPase almost parallelled the distribution of catecholamines. The distribution of ATPase was slightly shifted to lower densities and was suggested to be caused by the heterogeneity of the chromaffin granules. The results therefore provide evidence that ATPase is associated with chromaffin granules. Determination of the recovery of ATPase activity upon gradient centrifugation revealed losses of enzyme activity which were found to be proportional to the dilutions of the granule preparation subjected to gradient centrifugation.
Decreased Benzodiazepine Receptor Density in Rat Cerebellum Following Neurotoxic Doses of Phenytoin
Authors: Mimaki Takashi , Deshmukh Pushpa P. , Yamamura Henry I.
Published: October 2006

Abstract: The effect of acute and chronic administration of phenytoin on [3H]‐flunitrazepam binding was examined in the rat cerebellum. There was no significant effect of phenytoin on [3H]flunitrazepam binding in the rat cerebellum 1 and 6 h after a single i.p. injection of 200 mg/kg of phenytoin. However, after 14 days and 28 days of chronic phenytoin administration, significant de‐creases in [3H]flunitrazepam receptor density were observed, with no changes in apparent affinity constants in the rat cerebellum. This effect of phenytoin was dose‐dependent, as lower doses of phenytoin (100 mg/kg/day) for 14 or 28 days produced no alterations in [3H]flunitrazepam binding in the rat cerebellum. Light‐microscopic examination of the rat cerebellum treated with 200 mg/kg/day of phenytoin for 14 days showed degeneration of the Purkinje cells, with edematous Bergmann astrocytes. These data provide evidence for the neuronal localization of benzodiazepine receptors on cerebellar Purkinje cells.
Regulation of GABA Metabolism in Discrete Rabbit Brain Regions under Methoxypyridoxine—Regional Differences in Cofactor Saturation and the Preictal Activation of Glutamate Decarboxylase Activity1
Authors: Nitsch Cordula
Published: October 2006

Abstract: The activities of the enzymes of the GABA system, glutamate decarboxylase (GAD) and GABA‐transaminase, were measured in discrete regions of the rabbit brain before the onset and during the course of sustained epileptiform seizures induced by the vitamin B6, analogue methoxypyridoxine (MP). GAD activities were measured in a reaction mixture alternatively containing the cofactor pyridoxal‐5′‐phosphate (PLP) in excess or containing no PLP (holoenzyme of GAD). A comparison between these two estimations showed that the apoenzyme of GAD is only partially saturated with cofactor and that the degree of saturation varied from brain area to brain area, being highest in cerebellar cortex and lowest in substantia nigra. Holoenzyme activity fell steeply after administration of 100 mg/kg MP. The regional degree of enzyme inhibition by MP was a function of the saturation of the apoenzyme with cofactor; i.e., a low rate of saturation resulted in a high degree of inhibition, and vice versa. That GAD from the regio inferior of the hippocampus did not fit into the scheme (strong inhibition is present although the degree of saturation is high) is discussed in view of the role of the hippocampus in seizure genesis and generalization. Inhibition of GAD activity by MP was completely reversible in vitro by excess PLP. Before the onset of seizures but not during their course, apoenzyme activity surpassed control levels. This preictal activation is significant in regio inferior of hippocampus, in superior colliculus, and in cerebellar cortex. GABA‐transaminase activities were not significantly altered. The present study demonstrates that only investigation during the preictal period and in regional brain areas can reveal changes specific for the drug and perhaps representing the cause for seizure development, without being masked by additional alterations resulting from the severe functional and metabolic derangement during the ictal events. Thereby, it was disclosed that a decrease in vivo in the level of the enzyme product, GABA, is able to activate GAD.
Glycosidases and Cerebellar Ontogenesis in the Rat
Authors: Zanetta J‐P. , Federico A. , Vincendon G.
Published: October 2006

Abstract: Five glysosidases (α‐ and β‐D‐mannosidase, β‐D‐galactosidase, β‐N‐acetyl‐glucosaminidase, and α‐L‐fucosidase) were studied during the postnatal development of the rat cerebellum. Each glycosidase has a particular developmental pattern. Transient decreases in the specific activities of β‐mannosidase, β‐D‐galactosidase, and β‐D‐N‐acetyl‐glucosaminidase could be correlated with the phase of massive cell multiplication. A possible more specific role for α‐D‐mannosidase is discussed.
Stimulation of Carbonic Anhydrase Activity and Phosphorylation in Primary Astroglial Cultures by Norepinephrine
Authors: Church G. A. , Kimelberg H. K. , Sapirstein V. S.
Published: October 2006

Abstract: The carbonic anhydrase activity of 3‐week‐old primary astroglial cultures started from the dissociated cerebral hemispheres of neonatal rats was increased up to twofold after treatment of the cultures with 0.1 mM‐norepinephrine or histamine. Stimulation due to addition of norepinephrine was inhibited by propranolol. The carbonic anhydrase activity of primary cultures derived from the cerebellum plus brain stem regions was about fourfold greater than the activity of primary cultures started from cerebral hemispheres, but in contrast was not stimulated by norepinephrine. Treatment of the cerebral cultures with norepinephrine in the presence of 32P resulted in a two‐ to threefold increased incorporation of 32P into carbonic anhydrase purified from the same cultures, and this increased incorporation was inhibited by propranolol. It is suggested that one of the consequences of the stimulation of 3′,5′‐cyclic AMP levels in brain by norepinephrine is activation of astroglial carbonic anhydrase activity due to 3′5′‐cyclic AMP‐stimulated phosphorylation of the enzyme.
Regional Alterations in Rat Brain Neurotransmitter Systems Following Chronic Lithium Treatment
Authors: Maggi A. , Enna S. J.
Published: October 2006

Abstract: Chronic, but not acute, consumption of lithium leads to a significant decrease in serotonin and GABA receptor binding in selected regions of the rat brain, with no changes noted in P‐adrenergic or cholinergic muscarinic receptor binding. In addition, the concentration of β‐methoxytyramine, a dopamine metabolite, in the corpus striatum was increased in the animals treated chronically with lithium, suggesting a possible enhancement in dopamine release, or inhibition of uptake, in this brain area. In contrast, chronic consumption of rubidium had no effect on any of the parameters studied. The results suggest that lithium administration causes selective changes in brain neurotransmitter receptor systems and that the net result of these changes may be a decrease in GABAergic and serotoninergic activity. The fact that these alterktions are noted only after chronic administration suggests that they may be related to the therapeutic action of lithium in the prophylactic treatment of recurrent manic‐ depressive psychosis.
Changes in the Levels and Forms of Cholinesterases in the Blood Plasma of Normal and Dystrophic Chickens
Authors: Lyles Joan M. , Barnard Eric A. , Silman Israel
Published: October 2006

Abstract: Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were analysed in the blood plasma of developing chickens, both normal and those with inherited muscular dystrophy. The amounts and the molecular forms of each were examined. °ChE concentration rises in the plasma of normal and dystrophic chicks at the end of embryonic development and is maintained after hatching at a constant, relatively high level, accounting for 90‐95% of total cholinesterase activity in normal plasma. This level is maintained in normal and dystrophic chickens. In embryonic plasma of both normal and dystrophic chicks, on the other hand, the levels of AChE are higher than those of °ChE. Immediately after hatching the AChE level decreases rapidly in normal plasma, reaching a very low level by 2‐3 weeks ex ovo. The AChE level in plasma from dystrophic birds, although less than normal from day 19 in ovo to 2 weeks ex ovo, subsequently increases to peak around 4 months at levels 15‐20‐fold of those in normal birds. There is virtually no enzyme of either type in the erythrocytes of normal or dystrophic chickens. The changes of AChE in plasma were correlated with the alterations of AChE in dystrophic fast‐twitch muscles, suggesting that the latter pool is a precursor of the plasma AChE. Both the AChE and the °ChE in plasma exist in multiple molecular forms, which are similar to certain of those found previously in the muscles of these birds. The major form (60‐80%) of both enzymes in the plasma is the M form (sedimentation coefficient ≥11 S) in all cases, but it is accompanied by certain other forms. In no case is there any of the heaviest form (H2, 19‐20 S) of AChE or of °ChE found in normal and dystrophic muscle, which is attached at the synapses in normal muscle. The pattern of forms of plasma °ChE is constant at all ages, and in normal and dystrophic chickens. The pattern of forms of AChE in the plasma, in contrast, varies with age and with dystrophy in a characteristic manner. The sedimentation coefficients and the amounts of the enzymes in fast‐twitch muscle of dystrophic animals are compared with those of the plasma forms, and an interpretation is given of the characteristic patterns of AChE and of χE in their blood.
A GABAergic Influence on the Light‐Induced Increase in Dopamine Turnover in the Dark‐Adapted Rat Retina In Vivo
Authors: Morgan William W. , Kamp Cylia W.
Published: October 2006

Abstract: Light stimulates tyrosine hydroxylase activity and dopamine (DA) turnover in the dark‐adapted rat retina in vivo. The DA neurons are located in the amacrine cell layer and form numerous connections with other cells in this layer. Conceivably, alterations in neurotransmission in these other cells could influence the light‐responding parameters of the DA neurons. Evidence presented in this paper shows that in vivo pharmacologic manipulation of the GABA system modifies the light‐induced change in DA turnover. The decline in DA content following inhibition of tyrosine hydroxylase by α‐methyl‐p‐tyrosine (αMPT, 250 mg/kg, i.p.) was used to estimate DA turnover. The decline in DA content in retinas of the μMPT‐treated rats was significantly enhanced by light exposure for 30 or 60 min. Two doses of the potent GABA agonist muscimol (13.2 or 26.4 μmol/kg, i.v., cumulative) significantly inhibited the light‐induced increase in DA turnover (p <.001). This action was selective for GABA because the GABA antagonist picrotoxinin (1.88 mg/kg, i.v., cumulative) reversed the muscimol‐mediated blockade of the light‐induced stimulation. In fact, DA turnover in the presence of light, muscimol, and picrotoxinin was not different from DA turnover in light alone. These data suggest that there is either a direct or indirect GABAergic input to the DA system of the rat retina. Current studies are aimed at clarifying the physiological role, if any, that this input plays in the normal light response of the retinal DA system.
Proteins of Rapid Axonal Transport: Polypeptides Interacting with the Lectin from Lens Culinavis
Authors: Karlsson J.‐O.
Published: October 2006

Abstract: Rapidly transported fucose‐labelled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The labelled glycoproteins carried hydrophobic sites as revealed by a reversible binding to a hydrophobic matrix. A major portion of the labelled glycoproteins carried α‐D‐mannopyranoside (or glucopyranoside) residues, as shown by a specific interaction with the lectin from Lens culinaris. Three major groups of labelled polypeptides capable of binding to the lectin are described.
Morphological and Biochemical Changes in the Cerebellum Induced by Kainic Acid In Vivo
Authors: Foster A. C. , Roberts P. J.
Published: October 2006

Abstract: Injection Of nanomole quantities Of kainic acid into The Rat cerebellum resulted in a rapid and selective destruction Of neurons. At all times Studied, the purkinje cells were depleted following injection, and this loss was paralleled by a decrease in GABA levels, high‐affinity neuronal GABA uptake, and in glutamate decarboxylase activity. For the first 3 days following injection The granule cells were not reduced in numbers and the high‐affinity uptake of glutamate was similarly not decreased. After 4‐6 days, however, the granule cells were substantially depleted, although No clear changes in glutamate up‐take were observed. The normal laminar structure of the cerebellum was extensively disrupted and glial proliferation was evident. Measurement of endogenous amino acid levels showed a decrease in glutamate and aspartate levels after kainic acid injection. glycine levels were consistently increased while glutamine and alanine were unchanged. GABA receptor binding was decreased after injection, but glutamate binding was enhanced.
Actions of β‐Bungarotoxin on Amino Acid Transmitter Release
Authors: Smith C. C. T. , Bradford H. F. , Thompson E. J. , MacDermot J.
Published: October 2006

Abstract: The actions of purified β‐bungarotoxin (5 or 10 μg/ml) on the metabolic and transmitter‐releasing properties of rat cortical synaptosomes was studied. The toxin stimulated control respiratory rates, but prevented the respiratory response to veratrine. Tissue potassium levels were greatly reduced (54%) and endogenous glutamate, aspartate and GABA showed increased levels of release (10‐ to 25‐fold), but other amino acids were unaffected or showed much smaller changes. Tissue levels of these amino acids were reduced in proportion. The toxin inhibited the uptake of [U‐14C]GABA (35%) and [U‐14C]glutamate (53%) over 5‐min incubation periods. This uptake‐inhibition was Ca2+‐dependent and was reduced by tetrodotoxin. Miniature‐end‐plate‐potential (m.e.p. p.) frequencies at the locust neuromuscular junction (extensor tibialis) were greatly (four‐ to sevenfold) accelerated by local application of the toxin (5 μg/ml). This effect was reversible and occupied about 20 min. Amplitudes of m.e.p. p.'s were also increased and muscle membrane depolarization occurred. The results are interpreted as being due to a depolarizing action of the toxin.
Potassium Movements in Relation to Synaptosomal Morphology
Authors: Csillag András , Hajós Ferenc
Published: October 2006

Abstract: The uptake of K+ by rat cortical synaptosomes was followed using a K+ ‐selective membrane electrode. A parallel morphological study was carried out based on the previous description of two different configurations of incubated synaptosomes (S and M configurations). Synaptosomes were able to take up K+ against a concentration gradient; the average amount of cation taken up at equilibrium was 42.3 ± 3.1 (7) nmol/mg protein. Following K+ uptake the particles displayed a configuration corresponding to that of intact nerve endings in situ (‘M‐configuration’). In conditions where the uptake of K+ was inhibited or a net loss was elicited the development of M‐configuration was prevented. Agents which affected both the plasma membrane Na+ ‐K+ transport system and mitochondrial energy conservation brought about the ‘S‐configuration’ characteristic of unincubated synaptosomes, whereas in conditions where the plasma membrane transport system was preferentially inhibited, transitional forms were found. It was concluded that the S to M transition found in incubated synaptosomes is primarily associated with the inward movement of potassium.
Similarities Between A.P.U.D. Cells and Monoaminergic Neurons: Evidence for a Dopamine Uptake Mechanism in C Cells of Chicken Ultimobranchial Gland
Authors: Di Lauro A. , Popoli M. , Campanella G. , Avati U.
Published: October 2006

Abstract: The uptake of [3H]dopamine was studied in the C cells of chicken ultimobranchial gland (UBG). DA uptake appears to be an active and saturable mechanism (Km= 8.6 μM). The C cells' affinity for DA is intermediate between dopaminergic synaptosomes and such extraneural tissues as platelets and heart. UBG incubation with DA leads to the synthesis of a noticeable quantity of DOPAC, indirectly showing the presence of MAO activity. Thus UBG C cells, besides having both an embryological derivation from the neural crest and some morphological aspects similar to synaptosomes, also possess an active uptake mechanism for a central neurotransmitter.
Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats
Authors: Wiggins R. C. , Morell P.
Published: October 2006

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P0, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [32P]orthophosphate or [3H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [32P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2‐day‐old rats could incorporate radioactive fucose and [32P]‐phosphate into several proteins at the P0 region of polyacrylamide gels. Specific radioactivity of [3H]fucose in P0 protein was highest in preparations from 5‐day‐old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [32P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.
Reverse Phase High‐Performance Liquid Chromatography of Cerebrosides, Sulfatides and Ceramides: Microanalysis of Homolog Composition Without Hydrolysis and Application to Cerebroside Analysis in Peripheral Nerves of Adrenoleukodystrophy Patients
Authors: Yahara Shoji , Moser Hugo W. , Kolodny Edwin H. , Kishimoto Yasuo
Published: October 2006

Abstract: Reverse phase high‐performance liquid chromatographic analyses of nonhydroxy‐and hydroxycerebrosides and sulfatides, as well as ceramides, have been developed. Cerebrosides, ceramides and sulfatides were perbenzoylated then desulfated before chromatographic analysis. These sphingolipid derivatives can be separated according to their fatty‐acid composition by using an ODS 5‐μ, column (reverse phase column containing octadecylsiloxane derivative of silica, of 5‐μm particle size) and solvent systems consisting of various proportions of acetonitrile and methanol. The retention time of an individual homolog appears to be unaffected by the 4‐trans double bond of the sphingosine moiety. These methods allow us to determine fatty‐acid compositions of ceramides, cerebrosides and sulfatides without hydrolysis. Homolog composition of cerebrosides in peripheral nerve from adrenoleukodystrophy patients was investigated by using reverse phase high‐performance liquid chromatography and was found not to be significantly different from control nerves.
Topographical Distribution of Base Exchange Activities in Rat Brain Subcellular Fractions
Authors: Buchanan A. G. , Kanfer J. N.
Published: October 2006

Abstract: Enrichment in the base‐exchange activities was found in the micro‐somal fraction of rat brain, with less activity being associated with nuclei, mitochondria and synaptosomes. The distribution of the choline base exchange in microsomal subfractions differed from that for serine and ethanolamine and these three activities seemed asymmetrically distributed in the microsomes. Choline exchange activity was trypsin‐sensitive and presumably was located on the cytoplasmic side of the microsomes, while serine and ethanolamine exchange activities were trypsin‐insensitive and were assumed to be located on the luminal side of the microsomes. Treatment of rat brain microsomes with phospholipases A, C and D produced significant losses of membrane‐bound base exchange activities. Some activity was restored in phospholipase C‐treated microsomes by exogenous phospholipid, but significant restoration was not observed in phospholipase A‐treated microsomes by such additions. Exogenous phospholipid stimulated choline and ethanolamine exchange activities, but not serine exchange activity of phospholipase D‐treated microsomes. The exchange activities of rat brain microsomes differed in their responses to treatment with phospholipases, choline exchange activity in general being more sensitive than either serine or ethanolamine activities.
The Measurement of Spinal Cord Tissue pH Using a Diffusible, Lipid‐Soluble, pH‐Sensitive Fluorescent Indicator
Authors: Delheimer Steven C. , Anderson Robert E. , Sundt Thoralf M.
Published: October 2006

Abstract: Spinal cord tissue pH was measured in cats at normocapnia, hypocapnia, hypercapnia and death from anoxia using a pH‐sensitive fluorescent indicator (umbelliferone) with both molecular and ionic fluorophors. A ratio analysis of the indicator's calibrated 450 nm fluorescent tissue clearance curves from 340 and 370 nm excitation permitted direct in vivo tissue pH determinations. Fifteen animals were divided into three equal groups according to different arterial carbon dioxide tensions (Pa co2):five animals at Pa co2 20, five animals Pa co2 40 and five animals Pa co2 60 torr. Spinal cord tissue pH varied linearly with arterial pH, but within narrower limits. These values (arterial versus cord pH) were: 7.46 versus 7.15; 7.21, 7.09; and 7.04, 7.00. At death from hypoxemia the arterial pH fell to 6.99 and spinal cord to 6.67. The clearance curves of umbelliferone in spinal cord varied according to Pa co2 and appeared to reflect spinal cord blood flow.
Comparative Effects of Ethanol and Malnutrition on the Development of Catecholamine Neurons: Changes in Neurotransmitter Levels
Authors: Detering N. , Collins R. M. , Hawkins R. L. , Ozand P. T. , Karahasan A.
Published: October 2006

Abstract: The comparative effects of exposure to ethanol and malnutrition on the concentrations of tyrosine and catecholamines in whole brain and selected regions of brain have been studied in the developing rat. These animals were the offspring of optimally nourished rats (control pups), of rats fed a diet with 35% of the calories supplied by ethanol (ETOH pups), or of animals fed a diet calorically equivalent to the latter but lacking ethanol (iso‐caloric, 1C pups). These diets were administered to dams either during the last week of gestation (prenatal) or during lactation (postnatal). Tyrosine levels were elevated prior to birth in the prenatal ETOH or IC pups or at 1 and 2 weeks of age in postnatal ETOH or 1C pups as compared with values found in the control offspring. Dopamine concentration in whole brain was significantly lower in prenatal ETOH pups than in prenatal IC pups at 3 weeks of age. Levels in the brains of postnatal ETOH pups were lower than control values, but not relative to animals exposed to 1C diet. Investigation of corpus striatum showed a significant decrease in dopamine concentration compared with control or IC pup values as a result of postnatal exposure to ethanol. Norepinephrine levels in the whole brain of prenatal ETOH pups were consistently 30–40% lower than either control or matched 1C pups during development. At 3 weeks of age, the norepinephrine levels in the hypothalamus of animals exposed to ethanol pre or postnatally were 30–60% lower than values in the corresponding region in either control or 1C pups. In the rat model described, ethanol caused a decrease in catecholamine levels, perhaps solely by affecting the norepinephrine neurons.
An Analysis of γ‐Aminobutyrate Receptors and Uptake by Isolated Purkinje Cells
Authors: Henn Fritz A. , Venema Rick , Anderson David , Sellstrom Ake
Published: October 2006

Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [3H]GABA receptor binding and [3H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high‐affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β‐alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation.
Goldfish Brain (Na+, K+)‐ATPase: Purification of the Catalytic Polypeptide and Production of Specific Antibodies
Authors: Schwartz Michal , Siegel George J. , Chen Nelson , Agranoff Bernard W.
Published: October 2006

Abstract: The denatured catalytic polypeptide of (Na+, K+)‐ATPase of goldfish brain was purified and identified as the 32P‐labeled phosphoprotein. The protein served as immunogen for the preparation of rabbit antisera for immunohistochemical application to goldfish tissue sections, using the peroxidase‐antiperoxidase indirect method. Labeling in brain cross‐sections appears primarily in fibers of the optic nerve layer of the tectum. In optic nerve cross sections, labeling is restricted to fiber bundles.