bluuebunny/crossref_metadata_2025 huggingface dataset:
DEMONSTRATION AND CHARACTERIZATION OF A STEREOSPECIFIC OPIATE RECEPTOR IN THE NEUROBLASTOMA N18TG2 CELLS
Authors: Law P. Y. , Herz A. , Loh H. H.
Published: October 2006

Abstract— Morphine has been observed to have only a minor effect on the prostaglandin E1 (PGE1) stimulated adenylate cyclase or the basal cyclase activity in the neuroblastoma N18TG2 calls. However, this ineffectiveness of the opiates was not due to the absence of opiate receptor in this cell line. Contrary to previous observations, neuroblastoma N18TG2 cells possessed a high affinity, stereospecific opiate receptor. When [3H]dihydromorphine and [3H]naloxone binding were determined, a single component receptor with Kdiss= 25‐31 nm and with a capacity of 165 fmol/mg protein could be observed. This receptor has similar properties to those observed in the brain homogenates. The naloxone specific binding was dependent on the pH of the incubation medium and maximal binding occurred at pH 7.6. The agonist binding was inhibited by the alkali metal cations and divalent cations, while the antagonist binding was not affected by the cations significantly. There was no observable reversal of the Na+ inhibitory effect on agonist binding by the addition of Mn2+ to the incubation mixtures. Opiate binding to the neuroblastoma N18TG2 cells could be attenuated by pretreating the cells with N‐ethylmaleimide or proteolytic enzymes. Of the lipases tested, only phospholipase A2 has an inhibitory effect on the naloxone binding. Fractionation of the cell homogenates with differential centrifugation and purification of the membrane fractions by sucrose gradients suggested the localization of the receptor at the plasma membranes. Thus, the receptor in the neuroblastoma N18TG2 cells closely resembles those observed in the brain homogenates
[14C]AMINO ACID FORMATION FROM LABELLED GLUCOSE AND/OR ACETATE IN BRAIN CORTEX SLICES WITH EXPERIMENTALLY ELICITED PROLIFERATION OF ASTROGLIA. CORRELATION OF BIOCHEMICAL AND MORPHOLOGICAL CHANGES
Authors: Turský T. , Ruščaak M. , Laščánová M. , Rščáková D.
Published: October 2006

Abstract— Changes in morphology and in transformations of [U‐14C]glucose and [1‐14C]acetate into amino acids of the brain cortex were followed on the Sth, 10th and 21st days after production of mechanical lesions and compared with control tissue. In the experimental tissue, proliferation of astroglia and reduction of the number of neurons had taken place. On the 10th day, accumulation of mitochondria and of some gliofilaments in the cytoplasm of astroglia was observed. On the 21st day, the gliofilaments occupied a substantial portion of the astroglial cytoplasm and the mitochondria were reduced in number and compressed to the cell membrane. Incorporation of 14C from acetate into amino acids was substantially increased on the 10th day (up to 240% with respect to controls) and normalized again on the 21st day. Incorporation of [14C]glucose into amino acids decreased somewhat during the experimental period. It has been proposed that the proliferation of astrocytes and their ultrastructural changes may account for the increased transformation of [14C]acetate into amino acids, in particular into glutamine which is formed from the small glutamate pool.
A NOVEL ENZYME IN RAT BRAIN CONVERTING β‐ENDORPHIN INTO METHIONINE ENKEPHALIN: AFFINITY CHROMATOGRAPHY AND SPECIFICITY
Authors: Koida Masao , Aono Junichiro , Takenaga Keiaki , Yoshimoto Tadashi , Kimura Terutoshi , Sakakibara Shumpei
Published: October 2006

Abstract— Methionine enkephalin (met‐enk), an endogenous opioid, commonly occurs in sequences of lipotropins (LPH) and endorphins, implying a possible biosynthetic pathway for this pentapeptide. In search of the enzyme which generates met‐enk from human β‐endorphin [LPH(61‐91), β‐end], it was found that the soluble fraction of rat brain contained such activity. The enzyme was purified by ammonium sulfate fractionation (50‐80% saturation), ion‐exchange chromatography on DEAE‐Sephadex A‐50, and affinity chromatography for which LPH(64‐67) functioned as affinity ligand and eluant. The final preparation was essentially free of other peptidases, such as kininase, met‐enk degrading and post‐proline cleaving activities. Studies on specificity revealed that the enzyme selectively cleaved the Met‐Thr bond in both LPH(64‐67) and β‐end. It is possible that this enzyme participates in the biosynthesis of met‐enk in vivo.
GANGLIOSIDE COMPOSITION OF CHEMICALLY INDUCED RAT NEURAL TUMORS AND CHARACTERIZATION OF HEMATOSIDE FROM NEURINOMAS
Authors: Chou Kuo‐Howere , Ambers L. S. A. , Jungalwala F. B.
Published: October 2006

Abstract– Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20mg/kg body wt, in the tail vein of the mother. The ganglioside content and pattern in these tumors and the normal tissues from which the tumors originated are described. The ganglioside content in tumors was reduced, on wet tissue weight basis, compared to normal control. However, there was no significant difference of ganglioside content on dry weight or protein basis. Altered ganglioside composition was found in most of the neural tumors. In central nervous system tumors, there was some increase in GM3 and GT1b′ (nomenclature according to Svennerholm, 1963), a marked decrease in GM1 and some decrease in GD1a, but no apparent loss in GD1b. Extreme simplification of ganglioside pattern was seen in tumors originated from peripheral nervous system. Large accumulation of GM3 with concomitant loss of all the higher gangliosides was seen. GM3 from neurinomas as well as from normal gray matter was isolated and characterized. GM3 from neurinomas separated into two bands on thin layer chromatographic plates. Both these GM3 bands had identical sphingosine and carbohydrate composition but differed in their fatty acid composition. The fast moving band had 77% of the total fatty acids as C20:0 or longer chain while the slow moving band had only 22% of the long chain fatty acids. Normal gray matter GM3 had one major band containing 82% of and only 17% of the fatty acids as C20:0 or higher. It is suggested that in the tumor cells either the specificity of the enzyme cytidine monophosphate‐N‐acetyl neuraminic acid: ceramide dihexoside sialyltransferase for C18.0 fatty acid containing glycolipid was altered or that the compartmentation of precursor pools for the simpler glycolipids present in normal tissue did not exist in transformed cells.
AXOTOMY INDUCED CHANGES IN CHOLINERGIC ENZYMES IN RAT NERVE AND MUSCLES1
Authors: Ranish Norman A. , Kiauta Tomaz , Dettbarn Wolf‐D.
Published: October 2006

Abstract— The effect of axotomy on acetylcholinesterase (AChE. EC 3.1.1.7). butyrylcholinesterase (BuChE. EC 3.1.1.8) and choline acetyltransferase (CAT, EC 2.3.1.6) activities in sciatic nerve stumps above (proximal) and below (distal) the site of transection. as well as in soleus (SOL) and extensor digitorum longus (EDL) muscles, has been studied in rat. Within 2 weeks. AChE activity decreased on a per mm basis, in proximal nerve by 65%, in distal nerve by 80% and on a per whole muscle basis, in denervated muscle by 85%.

BuChE activity increased in proximal nerve and distal nerve to 150% of control and decreased in muscle to 51% of control. CAT activity in the proximal nerve stump was reduced to 70% of control and in the distal stump to less than 1% within 1 week. CAT activity in muscle decreased to 20 and 30% of control for soleus and EDL respectively during a 14 day period.

The reduction in AChE and CAT activity observed in the proximal nerve segment may reflect changes in the synthesis, as well as the transport and local degradation of these enzymes. Previous studies on axotomy induced changes have not examined the simultaneous changes in proximal and distal nerve as well as denervated muscle in a single animal species.
Nucleoside Uptake by Slices of Mouse Brain
Authors: Banay‐Schwartz M. , Guzman T. De , Lajtha A.
Published: October 2006

: The properties of the uptake of nucleosides and nucleotides by brain cells were examined in slices of mouse brain. Of the compounds tested, adenine and adenosine had the most rapid uptake and reached the highest levels. Uptake was mediated, as shown by saturability and strong inhibition, by low temperature, or by cyanide, and was only partially sodium‐ or calcium‐dependent. The inhibition pattern by analogues indicated the presence of several uptake systems (possibly four), as shown by differences between adenine and guanine uptake, between adenine and adenosine uptake, and between adenosine and cytidine uptake. The properties of uptake systems for nucleotides and nucleosides were somewhat different from those for amino acids.
Intraneuronal Site of Action for Imipramine in Rat Striatal Slices
Authors: Daniels A. J. , Gysling K. , Arqueros L.
Published: October 2006

: The uptake of 3H‐labeled imipramine ([3H]IMI) in rat corpus striatum slices was found to be strongly temperature‐dependent. The accumulation was shown to be saturable and two apparent Km's were found: 2.2 × 10−8 and 3.5 × 10−7m. Once incorporated, the labeled drug was released from superfused slices by K+ (55 mm) depolarization in the presence of calcium ions. Imipramine was also studied for its ability to induce the release of [3H]dopamine ([3H]DA) which had been previously accumulated by striatal slices. It was found that striatal slices superfused during 1 or 6 min with imipramine (10−6‐10−4m) release substantial amounts of radioactive dopamine, independently of the presence of Ca2+ in the medium. This release is completely abolished after reserpine pretreatment. It is proposed that imipramine enters the dopaminergic storage vesicles and displaces dopamine. An intraneuronal mechanism of action for imipramine is discussed.
Glycine in the Central Nervous System in Experimental Allergic Encephalomyelitis
Authors: Turecký L. , Líška B. , Pecháň I.
Published: October 2006

: The content of glycine, a possibly inhibitory neurotransmitter was studied in central nervous system of guinea pigs with experimental allergic encephalomyelitis (EAE). The glycine level was increased in spinal cord, but not in the brain of animals with EAE. The greatest increase in glycine concentration was in lumbosacral cord, and at the time of appearance of clinical signs of disease. The results are discussed in terms of possible connection between the changes of glycine concentration and clinical signs of EAE.
Modification of the Rate of Ouabain Binding to (Na++ K+)ATPase by Lithium Ions
Authors: Krishnan Nagaswamy , Albers R. Wayne
Published: October 2006

: We report on the interactions of Li+, a congener of K+ with the (Na++ K+)‐ATPase from E Electricus as measured by their effects on the rate of [3H]‐ouabain binding to this enzyme. Like K+, Li+ slows ouabain binding under both Type I (Na++ ATP) and Type II (Pi) conditions, but with lower affinity. In contrast to K+, the Li+ inhibition curve is hyperbolic, suggesting interaction at an uncoupled site. Also differing from the complete inhibition by high K+, a residual ouabain‐binding rate persists at high Li+. The interactions of Li+ and K+ are synergistic: the apparent K+ affinity increases 3 to 4‐fold in presence of Li+. These results are consistent with the conclusion that Li+ interacts with only one of the two K+ sites and may be of interest in interpreting lithium pharmacology.
Oxygen Affinity of Tyrosine and Tryptophan Hydroxylases in Synaptosomes
Authors: Katz Ira R.
Published: October 2006

: Tyrosine and tryptophan hydroxylase activities were studied in a synaptosome‐enriched (P2) preparation from the rat striatum. Care was taken to avoid the potential diffusional artifacts to which measures of oxygenase activities in respiring tissue are, in general, subject. Tyrosine hydroxylase exhibited a Km for oxygen of 2 to 3 mm Hg under a variety of conditions. Tryptophan hydroxylase exhibited a Km of 3 to 4 mm Hg at C.S.F. levels of tryptophan (10 μm). The Km increased to 8 to 10 mm Hg at 2 μm tryptophan. These values are all consistent with some degree of unsaturation with respect to oxygen in vivo.
Uptake of Vinca Alkaloids into Mammalian Nerve and its Subcellular Components
Authors: Iqbal Zafar , Ochs Sidney
Published: October 2006

Abstract: Three Vinca alkaloids, vinblastine (VLB), vincristine (VCR) and vindesine (VDS), were recently found to affect axoplasmic transport to different degrees, with VCR the most potent. The uptake of these three species by desheathed cat sciatic nerves in vitro was determined by using tritium‐labeled derivatives. In a sucrose medium, the uptake of VCR was found to be three to four times greater than that of VLB and VDS, which is in accord with the neurotoxicity of VCR. Uptake of VCR was dependent on Ca2+ concentration in the medium. Removal of Ca2+ from the incubation medium reduced the uptake of VCR, without having much effect on VLB or VDS uptake. The uptake of all three Vinca agents into nerve in a saline medium was about 50% of that in a sucrose medium and elimination of Ca2+ from the saline incubation medium did not result in any significant change in uptake. High Ca2+ concentrations (100 mM) in the incubation medium, which cause a block of axoplasmic transport, did not change the total uptake of the Vinca alkaloids to any significant degree. The amount of labeled alkaloid found in the soluble fraction was, however, decreased by 50%. There was an increase in the amount present in the particulate fraction, caused, most likely, by an aggregation of vinca‐binding components. The amount of VCR associated with tubulin‐containing components isolated by gel filtration of the soluble fraction increased twofold when the nerves were exposed to a high‐Ca2+ medium, as might be expected of a microtubule disassembly. Exposure of the nerve to low temperatures (0‐4°C) for 90 min did not show any effect on the total uptake of Vinca alkaloids.
Adrenergic Regulation of the Reduction in Acetyl Coenzyme A: Arylamine 7V‐Acetyltransferase Activity in the Rat Pineal
Authors: Alphs L. , Heller A. , Lovenberg W.
Published: October 2006

Abstract: Pharmacologically active agents were employed to study the mechanisms that control the reduction in levels of acetyl‐coA: arylamine N‐acetyltransferase activity (NAT) (EC 2.3.1.5) in the rat pineal. Pretreatment of rats with phenoxybenzamine or phentolamine prevented the rapid light‐mediated decrease in NAT activity, although pretreatment with yohimbine or atropine did not alter this effect of light. Administration of mecamylamine resulted in a rapid reduction in enzyme activity prior to light exposure. When clonidine was administered intraperitoneally to animals with elevated NAT levels, there was a rapid decrease in enzyme activity, mimicking the effects of light. However, intraperitoneal injections of norepinephrine, methoxamine and phenylephrine into similar groups of animals had no significant effect on enzyme acitivity. When clonidine and norepinephrine were administered intraventricularly, there was a rapid reduction in enzyme activity. On the other hand, intraventricular administration of phenylephrine did not result in reduced enzyme activity. Pretreatment of animals with phenoxybenzamine failed to block the reduction in NAT activity precipitated by low doses of clonidine. This clonidine‐mediated reduction in enzyme activity was, however, blocked by yohimbine. When animals were simultaneously exposed to light and administered clonidine, the rapid reduction in NAT activity was affected only when animals were pretreated with both yohimbine and phenoxybenzamine. In contrast to the decrease in pineal NAT activity observed in in vivo preparations, incubation of pineals with clonidine in an organ culture system produced a moderate, but consistent, rise in enzyme activity. These results suggest that stimulation of a receptor with α‐adrenergic characteristics mediates the reduction in NAT activity produced by light. Stimulation of yet a second adrenergic‐like receptor appears to mediate a reduction in pineal NAT activity precipitated by clonidine. Our evidence suggests that one or both of these receptors are located within the central nervous system.
5′‐Nucleotidase in Rat Brain Myelin
Authors: Cammer W. , Sirota S. R. , Zimmerman T. R. , Norton W. T.
Published: October 2006

Rat brain myelin showed substantial activity of 5′‐nucleotidase. The specific activity in myelin was enriched two‐ to threefold over that in rat brain homogenates, and the total activity in myelin accounted for approximately 24% of the activity in the homogenates. The 5′‐nucleotidase in the homogenates and in isolated myelin had optimum activity at pH 7.5‐9.0, was stimulated by Mg2+ and Mn2+, and was inhibited by Co2+, Zn2+, EDTA, and EGTA. 5′‐AMP, 5′‐UMP, and 5′‐CMP were the preferred substrates, and 5′‐GMP was hydrolyzed at approximately one‐half the rate of the other mononucleotides. The very low rates of cleavage of β‐glycerophosphate and 2′‐AMP ruled out any significant contribution of nonspecific phosphatase to the observed 5′‐nucleotidase activity in myelin. The 5′‐nucleotidase was inhibited by concanavalin A and was protected by α‐methyl‐d‐mannoside against inhibition by that lectin, suggesting that this enzyme in the CNS is a glycoprotein. It is concluded from these data, and from histochemical observations made in other laboratories, that the myelin sheath is one major locus of 5′‐nucleotidase in the rat brain.
Ultraviolet Irradiation Produces Loss of Saxitoxin Binding to Sodium Channels in Rat Synaptosomes
Authors: Weigele John B. , Barchi Robert L.
Published: October 2006

Ultraviolet irradiation (UV) has been shown to cause an electrophysiologically measured inactivation of the rapid, transient sodium conductance system in nerve. Tritiated saxitoxin ([3H]STX) was used as a structural probe to assess the possibility of a corresponding perturbation in the conformation of the STX binding site. UV irradiation caused an irreversible decrease in the total number of high‐affinity [3H]STX binding sites in rat synaptosomes, while the dissociation constant of the remaining sites did not change. The receptor loss followed first‐order kinetics, and the rate of loss was independent of temperature. The action spectrum for binding loss indicated a peak in spectral sensitivity near 280 nm. A22Na flux assay in irradiated synaptosomes directly demonstrated that [3H]STX binding sites and veratridinestimulated, STX‐blocked 22Na efflux had similar sensitivities to UV radiation. We conclude that the UV inactivation of functional channels includes a modification of the STX binding‐site structure.
Subcellular Distribution and Developmental Change of 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase in the Central Nervous System of the Myelin‐Deficient Shiverer Mutant Mice
Authors: Mikoshiba Katsuhiko , Nagaike Kazuhiro , Tsukada Yasuzo
Published: October 2006

Shiverer is an autosomal recessive mutant characterized by dysmyelination in the central nervous system. The myelin in the shiverer is devoid of the major dense line. The pattern of increase of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) in the various parts of the central nervous system during postnatal development was identical with that of controls. Subcellular fractionation of the brain from shiverer showed that the recovery of CNPase activity of the P2A was only 14% of the control, but those of P2B and P3 were several times higher than the controls. The specific activity of CNPase of the purified myelin and P2A from shiverer did not differ significantly from that of the controls. The combined P2 and P3(P2+ P3) fraction was subjected to linear sucrose density gradient (0.32‐1.3 m) centrifugation. By measuring CNPase activities of the fractions obtained after centrifugation, shiverer showed two peaks of CNPase activities in the sucrose gradient, although that of the control showed a single peak. The main peak obtained from shiverer was found in the region of higher sucrose concentration than that of the control, while a small peak was found in the region of lower sucrose concentration.
Turnover Numbers of γ‐Aminobutyrate Aminotransferase in Some Regions of Rat Brain
Authors: Ossola Lucienne , Maitre Michel , Blinderman Jean‐Marc , Mandel Paul
Published: October 2006

Using a radioimmunoassay to measure GABA (γ‐aminobutyric acid) transaminase in brain tissue homogenate, we have determined the turnover number of the enzyme in various regions of the rat brain. These numbers are not constant and vary from 3.5 to 30.6 s‐1 according to the regions studied, the highest numbers being found in regions rich in white matter. The significance of these results is discussed.
Lipid Requirement for Long‐lived Morphine‐dependent Activations of Adenylate Cyclase of Neuroblastoma × Glioma Hybrid Cells
Authors: Wilkening Douglas , Nirenberg Marshall
Published: October 2006

Morphine or norepinephrine at 10 μM inhibit adenylate cyclase of NG108‐15 neuroblastoma × glioma hybrid cells and gradually, over a period of 12 h or more, elicit a compensatory, persistent increase in adenylate cyclase activity. The cells are then dependent upon the receptor ligand for maintenance of normal cyclic AMP levels. Serum lipids or linoleic acid are required for the full expression of the long‐lived morphine‐dependent increase in adenylate cyclase activity, but serum lipids are not required for inhibition of the enzyme by morphine. Serum lipids are also not required for the full expression of the norepinephrine‐dependent increase in adenylate cyclase activity. In the NG108‐15 model system, therefore, the acquisition of dependence upon morphine can be dissociated from the inhibition of adenylate cyclase by morphine as well as from the acquisition of dependence upon another receptor ligand, norepinephrine.
Purification and Characterization of Rat Pheochromocytoma Dopamine β‐Hydroxylase
Authors: Fong Jim C. , Shenkman Louis , Goldstein Menek
Published: October 2006

Dopamine β‐hydroxylase (DβH) was purified from rat adrenal pheochromocytoma tumors by a procedure that requires only a few purification steps. Tumors obtained from a small number of rats yield sufficient quantities of pure DβH to raise antibodies to DβH in guinea pigs. The specificity of the antiserum was demonstrated by immunoelectrophoretic and Ouchterlony immunodiffusion analysis, as well as by the specific inhibition of DβH activity. Enzyme inhibition studies revealed complete cross‐reactivity between the rat pheochromocytoma antiserum and rat adrenal DβH, but interspecies cross‐reactivity was almost nonexistent. The molecular parameters and the kinetic properties of rat pheochromocytoma DβH are similar to those reported for bovine adrenal, human plasma, and human pheochromocytoma DβH.
Regional and Subcellular Distribution of ATP‐Citrate Lyase and Other Enzymes of Acetyl‐CoA Metabolism in Rat Brain
Authors: Szutowicz A. , Lysiak W. Z.
Published: October 2006

The activities of ATP‐citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP‐citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and Bp. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl‐CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP‐citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP‐citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.
Surface Proteins of Cultured Mouse Cerebellar Cells
Authors: Rohrer Hermann , Schachner Melitta
Published: October 2006

Surface proteins of cultured monolayer cells from embryonic and early postnatal C57BL/6J mouse cerebella were identified by a lactoperoxidase‐catalysed 131iodine labelling technique. Major iodinated polypeptides have molecular weights of approximately 200, 145, 120, 100, 85, 65, 50, and 30 X 103 (P200, P145, ⃛) as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis. Membrane glycoproteins, of apparent molecular weights 200, 145, 100, 85, and 50 X 103, are detected by biosynthetic labelling with [3H]fucose. The two major iodinated proteins are the glycoproteins P200 and P145. P145 is released from the cells into the medium together with other surface proteins. No changes in the patterns of labelled cerebellar cell surface proteins are detectable between embryonic day 17 and postnatal day 10. A pattern similar to the one seen with cerebellum is obtained with embryonic day 12 and 17 cerebral cortex. Cultured retinal cells from 2‐day‐old mice, skin fibroblasts, and l‐cells display a distinctly different pattern, which does not contain P145 as a major iodinated component. In granule cell‐enriched fractions of cerebellar cells the two glycoproteins P200 and P145 are proportionately increased, while three proteins, P100, P85, and P50, are more abundant in the glial cell‐enriched fraction. These three polypeptides are also enriched in cells obtained from staggerer mutant mice. An antiserum against 4‐day‐old cerebellar cells (anti‐NS‐4) precipitates the 145 and 200 X 103 molecular weight proteins, from lysates of both embryonic cerebral and postnatal cerebellar cells. From lysates of mouse retinal cells, anti‐NS‐4 antiserum precipitates two proteins with molecular weights of 140 and 210 X 103. Rohrer H. and Schachner M. Surface proteins of cultured mouse cerebellar cells. J. Neurochem.35, 792–803 (1980).
Isolation of Cholinergic Synaptic Vesicles from the Myenteric Plexus of Guinea‐Pig Small Intestine
Authors: Dowe G. H. C. , Kilbinger H. , Whittaker V. P.
Published: October 2006

The acetylcholine‐rich myenteric plexus‐longitudinal muscle preparation of the guinea‐pig small intestine has been subjected to subcellular fractionation using modifications of both classical methods and that originally devised for bulk isolation of cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo marmorata by means of density gradient centrifugation in a zonal rotor. The latter method gave a vesicle fraction with the highest acetylcholine content so far recorded for a mammalian particulate fraction, 30.9 × S.E.M. 1.8 (5) nmol of acetylcholine × mg of protein−1. Electron‐microscopical examination showed that it consisted of a homogeneous preparation of vesicles of mean spherical diameter 61 ×sd 4 (108) nm, with little or no contamination with other lipoprotein membrane structures, mixed how‐ever with considerable amounts of actomyosin fibrils, presumably derived from the longitudinal muscle. Slab‐gel electrophoresis in sodium dodecyl sulphate showed that, in addition to prominent peaks attributable to actin and myosin, there was a relatively simple pattern of (presumably) vesicle protein among which all the proteins thought to be characteristic of Torpedo synaptic vesicles were present. Dowe G. H. C. et al. Isolation of cholinergic synaptic vesicles from the myenteric plexus of guinea‐pig small intestine. J. Neurochem.35, 993–1003 (1980).
Acetylcholine Synthesizing Enzymes in Frog Skeletal Muscle
Authors: Molenaar P. C. , Polak R. L.
Published: October 2006

Acetylcholine synthesis in homogenates of frog sartorius muscle was measured by a radiometric method with a low blank. Choline acetyltransferase activity was very low (Vmax, 2 nmol g1 h−1, Kmfor choline, approx. 50 μ, m). The enzyme was found only in the endplate area and disappeared after denervation; it was inactivated by 4‐(1‐naphthylvinyl)pyridine. At high substrate concentrations its activity was overshadowed by the acetylcholine‐synthesizing activity of a different enzyme not saturated by 10 mm‐choline. The non‐specific enzyme was present at and away from the endplate area, and it was not affected by denervation.
Phosphorylation of Chromatin Proteins in Cerebral Anoxia and Ischemia
Authors: Yanagihara T.
Published: October 2006

Phosphorylation of nuclear protein was investigated in cerebral anoxia up to 30 min with rabbit brain and in cerebral ischemia up to 6 h with gerbil brain in vitro. Isolated nuclei were incubated in the presence of [γ‐32P]ATP and were then fractionated into the NaCl‐soluble, HCl‐soluble, and phenol‐soluble protein fraction. Each protein fraction was further separated by gel electrophoresis, and profiles of 32P incorporation were evaluated in these pathophysiological conditions. 32P incorporation of the acidic phenol‐soluble nonhistone chromatin protein became significantly suppressed in cerebral anoxia after 15 min, and there were decreases of 32P incorporation in protein with high molecular weight and increase in protein with low molecular weight on gel electrophoresis. With gerbil brain nuclei, 32P incorporation into the NaCl‐soluble and HCl‐soluble fraction was increased without significant de‐crease in the phenol‐soluble fraction after an ischemic period of 3 h. However, further separation of the phenol‐soluble fraction demonstrated decrease of 32P incorporation in protein with high molecular weight and increase in protein with low molecular weight. At present, the significance of these findings, particularly in relation to chromatin template activity or irreversibility of these pathophysiological conditions, is not clear.
Transient Concanavalin A‐Binding Glycoproteins of the Parallel Fibres of the Developing Rat Cerebellum: Evidence for the Destruction of Their Glycans
Authors: Reeber A. , Vincendon G. , Zanetta J‐P.
Published: October 2006

Abstract: The lifetime of the glycoprotein glycans of the rat cerebellum was followed in the 2nd and 3rd weeks of postnatal age, after injection of labelled glucosamine. It appears that a particular class of glycans binding to Concanavalin A synthesized at an early age has a short lifetime. These results indicate that the glycans of the Concanavalin A‐binding glycoproteins abundant on the newly formed parallel fibres are rapidly degraded between the 14th and the 18th postnatal day. Reeber A. et al. Transient Concanavalin A‐binding glycoproteins of the parallel fibres of the developing rat cerebellum: Evidence for the destruction of their glycans. J. Neurochem. 35, 1273–1277 (1980).
Cyclic AMP Enhancement of Depolarization‐Induced NAD(P)H Changes in Brain Cortical Slices
Authors: Keller E. , Cummins J. T.
Published: October 2006

Abstract: The increased levels of NAD(P)H effected by electrical depolarization are markedly augmented in the presence of cyclic AMP, isoproterenol, or RO 20–1724, agents known to elevate cyclic AMP in rat brain slices. The data presented indicate that the cyclic AMP effect on an important component of intermediate metabolism is not an enhancement of a basal response but a separate response that is activated by depolarization, is Ca2+–dependent, regulates cytochrome a‐a3 independently of its effects on NAD(P)H levels, and is dependent on a substrate other than glucose.
A Caution in the Use of Tritiated Substrates for Monoamine Oxidase Assays
Authors: May Michael E.
Published: October 2006

Abstract: A monoamine oxidase assay utilizing generally labeled [3H]‐serotonin as substrate became nonlinear after only ∼5% conversion of initial c.p.m. to product. Subsequent analysis showed that a significant proportion of the tritium label was readily exchangeable into water and that monoamine oxidase activity increased release of label as water. The use of generally labeled substrates for oxidase activities is not recommended.
Estimation of the Rate of Tryptophan Hydroxylation In Vivo: A Sensitive Microassay in Discrete Rat Brain Nuclei
Authors: Tappaz Marcel L. , Pujol Jean‐Franqois
Published: October 2006

Abstract: The rate of tryptophan hydroxylation in vivo was estimated in discrete rat brain nuclei by measuring L‐5‐hydroxytryptophan (5‐HTP) accumulated after pharmacological blockade of L‐5‐hydroxytryptophan decarboxylase by NSD 1015, using a sensitive radioenzymatic microassay. Endogenous serotonin, a major contaminant in this assay, was quantitatively removed by cationexchange chromatography prior to analysis. In non‐treated animals, endogenous 5‐HTP could be detected in small but measurable amounts. Following NSD 1015, accumulation occurred linearly for at least 30 min. At this time the recorded figures were two to six times higher when compared to values obtained in the same discrete structure from non‐treated animals. This allows an accurate estimation of the rate of tryptophan hydroxylation in vivo in small fragments of grossly dissected brain regions (e.g. cortex) as well as in discrete nuclei containing either serotoninergic (5‐HT) cell bodies (brain stem raphe nuclei) or 5‐HT‐terminals (e.g. catecholaminergic group A l, A2, A6.,. etc). Parachlorophenylalanine drastically reduced the rate of tryptophan hydroxylation in vivo in both terminal regions and raphe nuclei, with similar figures, 3 h or 3 days after injection. Chloral hydrate anaesthesia was attended by a transient decrease which appeared delayed in the raphe nuclei. Finally, pargyline pretreatment led to an 80% decrease in the forebrain, while no significant change appeared in the raphe nuclei. Thus, as illustrated by these few pharmacological manipulations, this method allows the study of the regulation of tryptophan hydroxylation in vivo with an improved anatomical resolution. Investigations can be carried out in the various raphe nuclei and their corresponding terminals in discrete brain areas simultaneously.
Studies on the Kinetic Mechanism and Salt Activation of Bovine Brain Choline Acetyltransferase
Authors: Hersh Louis B.
Published: October 2006

Abstract: :The kinetic mechanism of bovine brain choline acetyltransferase has been studied using acetylaminocholine as a dead‐end inhibitor and di‐methylaminoethanol as an alternate substrate. Acetylaminocholine inhibition is competitive with respect to acetylcholine and noncompetitive with respect to choline. Dimethylaminoethanol exhibits one‐sixth the Vmax obtained with choline. These results suggest that the reaction obeys a sequential random kinetic mechanism. Salt activation of the enzyme is nonspecific with respect to monovalent anions, and results in a parallel increase in the Km for choline and the Ki for acetylcholine. These results support the conclusion that salt activation of choline acetyltransferase is a nonspecific effect and that no specific chloride ion regulation of this enzyme occurs in vivo.
Temperature Dependence of Muscarinic Acetylcholine Receptor Activation, Desensitization, and Resensitization
Authors: El‐Fakahany Esam , Richelson Elliott
Published: October 2006

Abstract: The effects of temperature on muscarinic acetylcholine receptor activation, desensitization, and resensitization were studied with the use of intact mouse neuroblastoma cells (clone N1E‐115), which have muscarinic receptors that mediate cyclic GMP synthesis. Below 15‐20°C, activation or desensitization of muscarinic receptors by carbamylcholine and recovery from desensitization (caused by carbamylcholine at 37°C) did not occur. Above these temperatures, the apparent rates of receptor‐mediated cyclic GMP synthesis, desensitization, and recovery of sensitivity increased as the incubation temperature was increased. Arrhenius plots of the data yielded activation energies of 25, 14, and 23 kcal.mol−1 for activation, desensitization, and resensitization, respectively. These data suggest that a certain degree of membrane phospholipid fluidity is required for these processes to occur.
Evidence for the Presence of Two Types of Monoamine Oxidase in Rabbit Choroid Plexus and Their Role in Breakdown of Amines Influencing Cerebrospinal Fluid Formation
Authors: Lindvall M. , Owman C.
Published: October 2006

Abstract: The presence of monoamine oxidase (MAO) in the choroid plexuses from all brain ventricles of rabbit was established radiornetrically. Histochemical staining of MAO revealed the enzyme to be present primarily in the secretory epithelium. The mode of inhibition by the selective inhibitors clorgyline and deprenyl showed that type A and B MAO are present in approximately equal proportions in the plexus, Intraventricular administration of nialamide markedly reduced CSF production measured by [14C]inulin dilution during ventriculocisternal perfusion. The nialamide effect was significantly diminished after sympathetic denervation of the plexus, indicating that neuronal MAO is also present in the plexus; it probably represents a small fraction of the total MAO A activity in the tissue. In addition, nialamide tended to potentiate the reduction in CSF formation obtained by intraventricular noradrenaline.
Transport of G AB A, β‐Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum
Authors: East J. Malcolm , Dutton Gary R. , Currie D. Neil
Published: October 2006

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high‐affinity carrier‐mediated transport systems for [3H]GABA (Kt=1.9 μM, V = 1.8 pmol/106 cells/min) and [3H]glutamate (Kt= 10 μM, V = 7.9 pmol/106 cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [3H]β‐alanine (which is taken up specifically by the glial GABA transport system) and [3H]glutamate were concentrated by glial‐enriched fractions. However [3H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high‐affinity [3H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high‐affinity glutamate transport is a property of glial cells but not granule neurons.
Effect of Tetanus Toxin on Transmitter Release from the Substantia Nigra and Striatum In Vitro
Authors: Collingridge G. L. , Collins G. G. S. , Davies J. , James T. A. , Neal M. J. , Tongroach P.
Published: October 2006

Abstract: The effect of tetanus toxin on the uptake and release of radiolabelled transmitters from slices prepared from substantia nigra (SN) and striatum of rats has been investigated. Tetanus toxin‐500–750 mouse lethal doses (MLD)‐injected into the SN 6 h before preparing the slices significantly reduced the calcium‐dependent, potassium‐evoked release of [3H]GABA. Endogenous GABA levels in the SN and [3H]GABA uptake by nigral slices were unaffected by pretreatment with the toxin. Injections of tetanus toxin (1000–2000 MLD) into the striatum significantly reduced the calcium‐dependent, potassium‐evoked release of [14C]GABA and also [3H]dopamine, but had no effect on the K+‐evoked release of [3H]5‐hydroxytryptamine or [14C]acetylcholine. It is concluded that tetanus toxin inhibits GABA release directly and not by interference with synthesis or inactivation processes.
Incorporation of N‐Acetylmannosamine into Rat Brain Subcellular Gangliosides: Effect of Pentylenetetrazol‐Induced Convulsions on Brain Gangliosides1
Authors: Yohe Herbert C. , Ueno Kunihiro , Chang Nan‐Chen , Glaser Gilbert H. , Yu Robert K.
Published: October 2006

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N‐acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides GT1b and GQ1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, GT1b and GQ1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane‐bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water‐soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin‐layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin‐layer chromatogram.
Cross‐Linked 7S Nerve Growth Factor Is Biologically Inactive
Authors: Stach Robert W. , Shooter Eric M.
Published: October 2006

Abstract: The 7S nerve growth factor complex was cross‐linked with dimethylsuberimidate in a pH jump reaction and the cross‐linked product of highest molecular weight was separated from non‐cross‐linked subunits by gel filtration at acid pH. The amount of the cross‐linked 7S nerve growth factor generated was reduced by exposure of the complex to pH 9.5, where it dissociates. The cross‐linked 7S nerve growth factor migrated as a single species on electrophoresis under denaturing and reducing conditions and had a molecular weight, 132,000, identical to that of 7S nerve growth factor. All three subunits of the complex were released on deamidination. The biological activity of the cross‐linked 7S nerve growth factor complex was less than 0.3% that of native βNGF, suggesting that the biological activity of the latter is completely inhibited by interactions with other subunits in the complex.
Inhibition by Botulinum Toxin of Acetylcholine Release from Synaptosomes: Latency of Action and the Role of Gangliosides
Authors: Wonnacott S.
Published: October 2006

Abstract: Crude and crystalline botulinum toxin type A have been compared for their ability to inhibit [14C]ACh release from synaptosomes preloaded with [14C]choline. The toxin preparations exhibited similar dose‐response curves, with maximal inhibition at 105 mouse LD50/ml after 60 min preincubation. The time course for the inhibitory action of the toxin showed that inhibition develops almost linearly over this time period. However, free toxin could be removed from the synaptosome suspension after 15 min without altering the subsequent development of inhibition of [14C]ACh release, which suggests that the toxin is rapidly fixed by synaptosomes and that fixation alone cannot account for the latency of its action. Incorporation of gangliosides into synaptosomes by prior preincubation failed to increase the potency of the toxin, which implies that gangliosides do not serve as the membrane receptor for the toxin. Treatment of botulinum toxin with dithiothreitol greatly diminished its ability to inhibit [14C]ACh release and it is suggested that botulinum toxin may be analogous to other bacterial toxins in its structure and mode of action.
The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media
Authors: Snyder D. Stephen , Raine Cedric S. , Farooq Muhammad , Norton William T.
Published: October 2006

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from 10‐, 30‐ and 60‐day‐old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase‐bright rounded cells having diameters after fixation of 7‐12 μm. The contamination was primarily by red blood cells and phase‐dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pg/cell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals.
Neurochemical Studies of the Mesolimbic Dopaminergic Pathway: Somatodendritic Mechanisms and GABAergic Neurones in the Rat Ventral Tegmentum
Authors: Beart P. M. , McDonald Danielle
Published: October 2006

Abstract: The rat ventral tegmentum (containing dendrites and somata of mesolimbic neurones) contained 1.3 μg/g of dopamine, which was reduced to 40% of the control level by reserpine. Slices of ventral tegmentum were able to accumulate and release (elevated potassium or protoveratrine A) exogenous [3H]dopamine. In parallel studies the uptake mechanism in ventral tegmentum was shown to be virtually identical to the nerve terminal uptake of [3H]dopamine by slices of nucleus accumbens. The release of [3H]dopamine was indistinguishable from that observed in substantia nigra, where there is substantial evidence for dendritic mechanisms. Basal adenylate cyclase activity was present, but dopamine‐stimulated activity was not detected. A high GABA concentration (7.7 μmol/g) was present in ventral tegmentum, in conjunction with an uptake and a release mechanism for [3H]GABA. GABA and muscimol elicited a small, reproducible efflux of [3H]dopamine, but an interaction between dopamine and [3H]GABA efflux was not observed. The results are in accord with transmitter roles for dopamine and GABA in the somatoden‐dritic area of mesolimbic dopaminergic neurons.
Brain Glutamate Decarboxylase: Properties of Its Calcium‐Dependent Binding to Liposomes and Kinetics of the Bound and the Free Enzyme
Authors: Covarrubias Manuel , Tapia Ricardo
Published: October 2006

Abstract: In the present work we describe several properties of the Ca2+‐dependent binding of glutamate decarboxylase (GAD) to phosphatidylcholine‐phosphatidylserine liposomes. The binding occurs very rapidly, is dependent on temperature in the range 23–37°C, is inhibited up to 35% by K+ in a concentration‐dependent manner and is slightly increased when the dielectric constant of the medium is decreased by 3% ethanol. The association of GAD and liposomes is very firm, since EGTA displaces only 40% of the bound enzyme and Triton X‐100 about 55%. Since apparently only part of the total GAD is able to bind to the liposomes and in a previous study two forms of GAD activity have been identified kinetically, we compared the activations by pyridoxal 5′‐phosphate (PLP) of the soluble and the bound GAD, as well as their inhibition by PLP oxime‐O‐acetic acid. The bound GAD was activated 150–265% by 10−6 to 10−4m‐PLP, whereas the activation of GAD that remained soluble was only 65–110% in the same PLP concentration range. In the absence of PLP, the bound GAD was less inhibited by the PLP oxime‐O‐acetic acid than the soluble GAD, but the inhibition was similar when 0.1 mm‐PLP was added. In contrast, activity of both the soluble and the bound GAD was totally blocked by aminooxyacetic acid. Endogenous PLP did not bind to liposomes under the experimental conditions inducing GAD binding. We conclude that the binding of GAD to negatively charged liposomes is primarily ionic. Furthermore, the GAD molecules that bind to the liposomes seem to be deficient in free PLP and therefore, are probably more susceptible to regulation by the coenzyme. These conclusions may be relevant to the hypothesis of a coupling between synthesis and release of GABA in inhibitory nerve endings.