bluuebunny/crossref_metadata_2025 huggingface dataset:
PROPERTIES OF ACID β‐d‐GALACTOSIDASE ISOLATED FROM I‐CELL DISEASE BRAIN AND SPLEEN
Authors: Miller Arnold L. , Levitt Pat , Ingraham Holly , Converse James , Lewis Linda
Published: October 2006

Abstract— Acid 4‐methylumbelliferyl β‐d‐galactosidase activity from autopsied I‐cell disease brain and spleen tissues was 28% and 35% respectively of normal activity. Acid β‐d‐gatactosidase (β‐d‐galactoside galactohydrolase, EC 3.2.1.23) from two I‐cell disease brains demonstrated a 5‐fold increase over normal for the proportion of enzyme activity which did not adsorb to Concanavalin A‐Sepharose 4B, while acid β‐d‐galactosidase from two I‐cell disease spleens demonstrated a 21–35‐fold increase in the proportion of unadsorbed enzyme activity. Normal and I‐cell disease acid β‐d‐galactosidase present in crude brain and spleen supernatant fluids and in preparations partially purified on Concanavalin A‐Sepharose 4B had similar apparent Km values with respect to 4‐methylumbelliferyl β‐d‐galactopyranoside and GM1‐ganglioside. Isoelectric focusing profiles of normal and I‐cell disease acid β‐d‐galactosidase from crude brain and spleen‐supernatant fluids and partially purified preparations were similar. Neuraminidase treatment and subsequent isoelectric focusing of the partially purified normal and I‐cell disease enzyme preparations from brain and spleen revealed increases in the proportion of I‐cell β‐d‐galactosidases found at neutral pH values, suggesting that the electrophoretic variations observed for the I‐cell enzymes may not be attributed solely to changes in sialic acid composition.
BIOLOGICAL ACTIVITY OF THE β NERVE GROWTH FACTOR: THE EFFECTS OF VARIOUS ADDED PROTEINS
Authors: Stach Robert W. , Garian Nora , Olender Edward J.
Published: October 2006

—The biological activity of β nerve growth factor varies greatly when other proteins are added to the culture medium. We have found that the biological activity can vary by a factor of 7 in a bioassay using 9 day embryonic chick sensory ganglia by adding bovine serum albumin to the culture medium. Adding the α and γ subunits in the form of 7S nerve growth factor has no effect on the biological activity of the β nerve growth factor. On the other hand, cytochrome c has an intermediate effect. Most of the increase in the biological activity seen on the addition of other proteins seems to be due to the ability of the other proteins to keep β nerve growth factor in solution; i.e. to prevent its adsorption to the culture tubes. However, some of the increase may also be due to an interaction that causes an activation of the β nerve growth factor.
Preparation and Properties of Antibodies to GD3 and GM1 Gangliosides
Authors: Kundu Samar K. , Marcus Donald M. , Ven Rüdiger W.
Published: October 2006

Abstract: Gangliosides GD3 and GM1 were coupled to proteins by their car‐boxyl groups and antisera were raised against the complexes. Anti‐ganglioside antibodies were isolated by affinity chromatography on ganglioside‐amino‐propyl silica gel columns and the specificity of the antibodies was determined by a quantitative microcomplement fixation assay. Antibodies to GD3 were highly specific and did not crossreact with GM3, lactosyl ceramide, or other glycolipids. Purified antibodies to GM1, in contrast, crossreacted with asialo‐GM1, GD1b and to a lesser extent, GM2 and asialo‐GM2. A derivative of GM1, containing a C‐7 sialic acid residue produced by periodate oxidation, reacted with the anti‐GM1 antibodies almost as readily as with GM1. The specificities of anti‐GM1 antibodies elicited by the covalent ganglioside‐protein complexes were similar to those produced by immunization with noncovalent complexes of GM1 and methylated bovine serum albumin. The ganglioside‐protein complexes described here should be useful for preparing antibodies to polysialo‐gangliosides that contain neuraminidase‐sensitive linkages.
Thymidine Phosphorylase Activity in the Optic Tectum of Developing Chick Embryos and the Effects of 3′,5′‐Cyclic Nucleotides and Monosodium Glutamate on Enzyme Activity
Authors: Hyndman A. G.
Published: October 2006

Changes in activities of thymidine phosphorylase (EC 2.4.2.4.) in the optic tectum of developing chick embryos were determined and the effects of the addition of adenosine 3′,5′‐monophosphate or guanosine 3′,5′‐ monophosphate to the test medium were also determined. In addition, injection of 30 mg of monosodium glutamate onto the chorioallantoic membrane was assessed for effect on thymidine phosphorylase activity in the optic tectum. Thymidine phosphorylase activity was highest at the earliest times studied, peaking at the 10th day of incubation, but decreasing sharply until the 12th day, at which time a new level of activity was maintained until after the 18th day. Between the 18th day and 1 day post‐hatching there was a steady decrease in activity. The addition of adenosine 3′,5′‐monophosphate (cAMP) to the test medium resulted in decreased enzyme activity at 10 and 16 days of incubation, but no change at 1 day post‐hatching; guanosine 3′,5′‐monophosphate (cGMP) in the test medium resulted in decreased activity at 16 days, but in no change at 10 days or 1 day post‐hatching. The injection of monosodium glutamate into the egg before 10 days of incubation resulted in a decrease in enzyme activity only at 10, 18, and 20 days of incubation. Thymidine phosphorylase activity during optic tectum development has two phases (days 8‐10 and days 12‐18), each followed by a decrease in enzyme activity. The addition of cAMP to the test medium lowers activity during both phases, and cGMP only affects the second phase. This suggests two mechanisms for control of the enzyme, one present during both phases and one only during the second phase. That a single injection of MSG decreases enzyme activity at 10, 18, and 20 days of incubation, but not days 12‐16 or 1 day post‐hatching, suggests that MSG has not only immediate but also long‐term biochemical effects on prenatal brain development.
Differences Between Intracellular Platelet and Brain Proteins that Bind Serotonin
Authors: Tamir H. , Bebirian R. , Muller F. , Casper D.
Published: October 2006

Two proteins with binding capacity highly specific for serotonin are present in the 100,000 g supernatant obtained from rat platelets: a glycoprotein and albumin. They were purified by means of (NH4)2SO4 fractionation, Sephadex sieve chromatography, and affinity column chromatography. The two proteins differed in most of their physical and chemical properties: (a) migration on 7.5% acrylamide gel of the complex (protein‐Fe+2‐[3H]serotonin); (b) molecular weight (sodium dodecyl sulfate gels); (c) carbohydrate reaction; (d) binding capacity and binding constants; (e) effect of reserpine; (f) heat stability; and (g) isoelectric point. However, they showed two similar properties: sensitivity to trypsin and dependence on Fe +2 for serotonin binding. The properties of both the glycoprotein and albumin differ considerably from those of serotonin binding protein of brain (SBP), which was not detected in platelets. Since brain serotonin binding protein is thought to be involved in storage of the amine, these results suggest that the storage form of serotonin in rat platelets is different from that in rat brain. These results also imply that the storage of serotonin in platelets may not serve as a model for the storage of serotonin in nerve terminals of the brain.
Nervous System‐Specific Proteins in Developing Rat Cerebral Cells in Culture
Authors: Bock E. , Yavin Z. , Jørgensen O. S. , Yavin E.
Published: October 2006

Abstract: The nervous system‐specific proteins: synaptin, D1, D2, D3, glial fibrillary acidic protein (GFA) and 14‐3‐2, were quantified in dissociated cerebral cells from the foetal rat brain at various times of growth in culture. By approximately 1 week in culture, the neuronal membrane markers synaptin, D1, D2, and D3 could all be demonstrated. A maximum concentration of 10–20% for synaptin, D1, and D3 and 160% for D2, in comparison with the levels in adult forebrain, was attained during the 2nd week in vitro. The astroglial gliofilament marker GFA increased continuously, reaching by 38 days of cultivation an 18‐fold higher level than the concentration in adult forebrain. The neuronal cytoplasm marker 14‐3‐2 could be demonstrated in trace amounts, and only after more than 1 week in vitro. Neuronal cell bodies and processes stained by indirect immunofluorescence using an anti‐D2 serum were strongly fluorescent after 1 week in vitro. Immunofluorescence staining for GFA revealed a cytoplasmatic filamentous network in perinuclear areas and processes of, presumably, astroblasts.
A Prolactin Action on Acetylcholine Metabolism in Striatum, Hippocampus, and Thalamus
Authors: Wood Paul L. , Cheney D. L. , Costa E.
Published: October 2006

Abstract: Since prolactin can regulate the release of striatal dopamine, we have evaluated the functional implications of this effect by studying the action of injected prolactin on the turnover rate of acetylcholine (TRACh) in various brain areas. We selected striatum and hippocampus as two areas in which dopaminergic terminals are known to regulate TRACh and frontal and parietal cortex as areas where dopamine has little or no control on TRAch. Intraventricularly injected prolactin reduces the TRACh in striatum, hippocampus, and thalamus but not in the two cortical areas. Intraseptal injection of prolactin reduces TRACh in hippocampus, suggesting that this polypeptide acts on hippocampus by changing the activity of afferent neurons impinging upon the cell bodies of the cholinergic septal‐hippocampal neurons. The reductions in TRACh induced by intraventricular prolactin in hippocampus and striatum are nullified by 6‐hydroxydopamine‐induced lesions of dopaminergic neurons located in areas A9 and A10. These results suggest that the presence of dopaminergic neurons is required to obtain the prolactin‐elicited decrease of TRAch.
Glycogen Metabolism in Neonatal Rat Brain During Anoxia and Recovery
Authors: Vannucci S. J. , Vannucci R. C.
Published: October 2006

Abstract: Metabolic alterations in glycogen and in glycogen‐related metabo lites were studied in neonatal rat brain during controlled anoxia and recovery. One‐day postnatal rats were exposed to 100% N, at 37°C for up to 20 min; some rats were allowed to recover in air. Animals were frozen in liquid N, and the brains were prepared for fluorometric analysis of compounds involved in glycogen turnover. During anoxia, glycogen decreased by 29% and 42% at 10 and 20 min, respectively; the free (soluble) and bound (insoluble) components of glycogen decreased in nearly equal proportions. Brain glucose decreased by 72% at 10 min with little further change there after; G‐6‐P, G‐1‐P, and UDPG also declined. During recovery from anoxia, glucose and G‐6‐P increased above control levels for up to 60 min. G‐1‐P paralleled G‐6‐P levels, but UDPG remained low. Glycogen returned to control values by 4 h. The findings suggest that although glycogen is mobilized slowly in newborn rat brain, the metabolite contributes at least one‐third of the cerebral energy supply during anoxia. Presumably, readily available stores of glycogen combined with low cerebral metabolic requirements underscore the known tolerence of immature animals to hypoxic stress. Glycogen accumulation during recovery appears to be facilitated at the synthetase step, since equilibrium measurements of the phosphoglucomutase and pyrophosphorylase systems indicate that these reactions are not rate‐limiting for glycogen synthesis.
Effect of Organophosphorus Insecticides on the Oxidative Processes in Rat Brain Synaptosomes1
Authors: Sitkiewicz Dariusz , Skonieczna Maria , Krzywicka Krystyna , Dziedzic Elzbieta , Staniszewska Krystyna , Bicz Wlodzimierz
Published: October 2006

Abstract: This paper describes the effect of four organophosphorus insecticides: Dipterex, DDVP, Ronnel and its oxygen analogue on the respiration of rat brain synaptosomes. Dipterex and DDVP in the concentrations used, 5, 50, or 500 μM, did not change the rate of oxygen uptake and oxidative phosphorylation in rat brain synaptosomes. Ronnel in the highest concentration (500 μM) inhibited respiration in state 3 conditions and abolished respiratory control by ADP. This inhibition was correlated with a change of cytochrome c oxidase activity. The oxygen analogue of Ronnel (OAR) in micromolar concentrations (50 μM) increased the rate of respiration of synaptosomes utilizing glutamate plus malate as substrate. Higher concentrations of OAR produced inhibition of respiration, cytochrome c oxidase and NADH: cytochrome c reductase activities. These observations are typical for uncouplers of oxidative phosphorylation. Noteworthy is the fact that the uncoupling activity of OAR was observed at concentrations which did not inhibit acetylcholinesterase activity. These findings seem to suggest that disturbances in oxidative processes could play an important role in the toxicity of organophosphorus insecticides. The relation between chemical structure and the ability of insecticides to affect oxidative phosphorylation is discussed.
Specific Binding Sites for S‐100 Protein in Isolated Brain Nuclei
Authors: Donato Rosario , Michetti Fabrizio
Published: October 2006

Abstract: Isolated brain nuclei possess binding sites for S‐100 protein. The interaction of S‐100 with these sites is specific and time‐, temperature‐, and Ca+‐dependent. The profile of the 125I‐labelled S‐100 binding inhibition is biphasic, displaying a high‐affinity component and a low‐affinity component. The S‐100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S‐100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S‐100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high‐affinity component of S‐100 binding.
Demonstration and Characterization of Opiate Inhibition of the Striatal Adenylate Cyclase
Authors: Law P. Y. , Wu J. , Koehler J. E. , Loh H. H.
Published: October 2006

Abstract: The conditions in which Leu5‐enkephalin inhibition of striatal adenylate cyclase was observed were defined. It was determined that enkephalin inhibition was dependent on GTP. The apparent Km for GTP in opiate inhibition was determined to be 0.5 and 2 μM when 0.1 mM‐ and 0.5 mM‐ATP were used as substrate. ITP, but not CTP or UTP, could substitute for GTP in the reaction. Though the addition of monovalent cations—Na+,K+, Li+, Cs+, and choline+—stimulated striatal adenylate cyclase activity, enkephalin inhibition of striatal adenylate cyclase did not require Na+ when theophylline was used as the phosphodiesterase inhibitor. Under optimal conditions, i.e., 20 μM‐GTP and 100 mM‐Na+, Leu5‐enkephalin inhibited the striatal adenylate cyclase activity by 23–27%. When the enkephalin regulation of the cyclase activity was further characterized, it was observed that Leu5‐enkephalin inhibited the rate of the enzymatic reaction. Kinetic analysis revealed that the opioid peptide decreases Vmax values but not the Km values for the substrates Mg2+ and Mg‐ATP. Agents such as MnCl2, NaF, and guanyl‐5′‐ylimido‐diphosphate, which directly activated the adenylate cyclase, antagonized the opiate inhibition. Levorphanol and (–)naloxone were more potent than dextrorphan and (+)naloxone in inhibiting adenylate cyclase and in reversing the enkephalin inhibition, respectively. There were differences in the potencies of various opiate peptides in their inhibition of striatal adenylate cyclase activity, with Met5‐ > Leu5‐enkephalin > β‐endorphin. The opiate receptor through which the enkephalin inhibition was observed is most likely δ in nature, since in the presence of either Na+ or K+, the magnitude of the alkaloid inhibition was reduced, whereas the peptide inhibition was either potentiated or not affected.
Biochemical and Immunological Properties of the Mouse Brain Enolases Purified by a Simple Method
Authors: Keller Angelica , Scarna Hélène , Mermet Anne , Pujol Jean‐Francois
Published: October 2006

Abstract: A simple and rapid purification method is presented for the two mouse cerebral isozymes of enolase (EC 4.2.1.11), E1 and E3. The purity of the preparations was ascertained by electrophoresis under two different conditions. The biochemical and immunological properties of E1 and E3 were compared. The molecular weight of the cerebral enolases was analysed by column chromatography on Sephadex G 150 and by electrophoresis in the presence of SDS. Both E1 and E3 are homodimers with a subunit of molecular weight of 50,000. The procedure also yields a semi‐purified fraction of E2. Conditions of in vitro formation of E2 from pure or semi‐purified fractions of E1 and E3 show that it is likely to be a real hybrid, rather than an aggregate and that it is probably not an artefact formed during the purification. The Km values (Km= 3–4·10−5 M) for the substrate are not significantly different amongst the three forms. However, E1 and E2 but not E3 are inhibited by excess substrate. Antisera against E1 and E3 have been obtained from rabbit and goat, respectively. Antibodies against each protein do not show any cross‐reactivity with each other. There is, however, a broad species cross‐reactivity, showing conservation of each enolase form during evolution. Both anti‐E1 and anti‐E3 sera react with the E2 enolase fraction, in agreement with its hybrid structure. Anti‐E3 serum does not react with extracts of other tested organs. Brain enolase 1 resembles liver enolase in its biochemical and immunological properties. A slight cross‐reactivity of anti‐E1 serum with muscle extracts is observed. Heterogeneity of brain enolase 1 is observed by both biochemical and immunological methods; the nature of this heterogeneity is discussed.
Myo‐Inositol Turnover in the Intact Rat Brain: Increased Production after d‐Amphetamine
Authors: Barkai Amiram I
Published: October 2006

Abstract: Apparent turnover of myo‐inositol in the brain of urethane‐anesthetized rats was estimated in vivo from the rate of appearance of endogenous myo‐inositol in the cerebroventricular compartment. Ventricular‐cisternal perfusion technique combined with isotope dilution of [14C]myo‐inositol was used to determine the rate of appearance of brain‐produced myo‐inositol and its modification by d‐amphetamine. A mean value of 0.75 nmol/min was obtained for the rate of appearance in the cerebroventricular system. A dose dependent increase in this rate was seen after the administration of d‐ampheta‐mine. The endogenous removal of myo‐inositol from the perfusate was also studied and found to be mediated in part by a saturable transport system that was not influenced by d‐amphetamine. The rate of entry of myo‐inositol from blood to the erebroventricular system was very low and accounted for only 2% of the total rate of appearance, indicating that the majority of myo‐inositol in the rat cerebroventricular fluid originates in the brain.
Uptake of Amino Acids by Brain Microvessels Isolated from Rats after Portacaval Anastomosis
Authors: Cardelli‐Cangiano Patrizia , Cangiano Carlo , James J. Howard , Jeppsson Bengt , Brenner William , Fischer Josef E.
Published: October 2006

Abstract: The uptake of amino acids by microvessels isolated from brains of rats was studied. Previous studies have demonstrated alterations in blood‐brain amino acid transport after portacaval shunt in rats. In order to elucidate whether such changes in the blood‐brain barrier were located in the microvessels, brain microvessels were isolated from both rats with portacaval shunt and controls. Brain microvessels from rats 2 weeks after shunt operations took up significantly greater amounts of 14C‐labeled neutral amino acids, but not of glutamic acid. lysine, or α‐methylaminoisobutyric acid than microvessels from sham‐operated controls. Measurement of uptake kinetics showed a higher Vmax for phenylalanine and leucine uptake and a lower Vmax for lysine uptake in microvessels from shunted rats compared with control, whereas the respective Km's of uptake were similar in both preparations. The results suggest that changes in brain microvessel transport activity account for altered brain neutral amino acid concentrations after portacaval shunt and that such changes can be studied in vitro in isolated microvessels.
Purification of Rat 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase
Authors: Wells M. R. , Sprinkle T. J.
Published: October 2006

Abstract: 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl‐Sepharose CL‐4B, CM‐Sepharose CL‐6B, and 8‐(6‐aminohexyl) amino‐2′AMP‐Sepharose 4B. From 15 g of rat brain, approximately 400 μg of pure CNP was obtained, with a specific activity of 1.200 (2′,3′‐cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM. using 2′,3′‐cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5–9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48.000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.
Effects of Acute and Chronic Denervation on Release of Acetylcholinesterase and Its Molecular Forms in Rat Diaphragms
Authors: Carter Jonathan L. , Brimijoin Stephen
Published: October 2006

Abstract: Hemidiaphragms were removed from rats at various times after intrathoracic transection of the left phrenic nerve and were incubated in organ baths containing 1.5 ml of oxygenated, buffered physiologic saline solution, with added glucose and bovine serum albumin. After incubation, the acetylcholinesterase (AChE; EC 3.1.1.7) activities of the bath fluid and of the muscle were determined. Innervated left hemidiaphragms were found to release 107 units of AChE over a 3‐h period, corresponding to 1.9% of their total AChE activity. Denervation led to a rapid loss of AChE from the muscle coincident with a transient increase in the outpouring of enzyme activity into the bath fluid. Thus, 1 day after nerve transection the left hemidiaphragm contained only 68% of the control amount of AChE activity, but released 140% as much as control. After 3 or 4 days of denervation, the AChE activity of the diaphragm stabilized at 35% of the control value. Release also fell below control by this time, but not as far. One week after denervation the release, 69 units per 3 hr, corresponded to 3.3% of the reduced content of AChE activity in the muscle, indicating that denervation caused an increase in the proportion of AChE released. Sucrose density gradient ultracentrifugation showed that 10S AChE accounted for more than 80% of the released enzyme activity at all times. The results did not rule out the possibility, however, that the released enzyme originally stemmed from 4S or 16S AChE in the diaphragm.
The Effects of Kainic Acid Injections on Guanylate Cyclase Activity in the Rat Caudatoputamen, Nucleus Accumbens and Septum
Authors: Walaas Ivar
Published: October 2006

The activity of soluble and particulate guanylate cyclase (EC 4.6.1.2) has been compared with the distribution of neurotransmitter candidates in three rat forebrain nuclei, and the effects of local kainic acid injections into these nuclei have been tested. Soluble guanylate cyclase was highly concentrated in both the caudatoputamen and the nucleus accumbens, with lower activity found in the septum. This distribution coincided with markers for acetylcholine and monoamines, but not with markers for γ‐aminobutyrate (GABA) or glutamate neurons. In contrast, particulate guanylate cyclase was equally active in all regions. Local injections of kainic acid, which destroyed cholinergic and GABA neurons in the caudatoputamen and in the nucleus accumbens, caused a rapid (70–90%) decrease in the soluble guanylate cyclase and a slower 50‐60% fall in the particulate guanylate cyclase in these nuclei. In the septum, where kainate destroyed GABA cells but not cholinergic neurons, the guanylate cyclase activity was unchanged after the lesion. Thus, both the soluble and particulate guanylate cyclases appear to be concentrated in local neurons in the caudatoputamen and nucleus accumbens. In the septum, however, most of the guanylate cyclase activity is located outside kainate‐sensitive neurons.
Turnover In Vivo of Lactosyl‐ceramide and Other Glycosphingolipids in the Adult Rat Brain
Authors: Barkai Amiram I.
Published: October 2006

The quantitative contribution of glucose to the biosynthesis of lactosyl‐ceramide and other glycosphingolipids was studied in the adult rat brain in vivo using a semicompartmental model. Half‐lives of glucose carbon in both the total carbon pool and the carbohydrate residue of the lipid were calculated. In all glycolipids the half‐life of carbohydrate units was six to eight times shorter than the half‐life of carbon in the total carbon pool of the same lipid. This carbohydrate half‐life appears to be closely related to the turnover rate of the glycolipid. The shortest carbohydrate half‐life (2.2 days) was obtained for lactosyl‐ceramide followed by gangliosides. galactosyl‐ceramides, and sulphatides. The results indicate that lactosyl‐ceramide may serve as a branch point for the biosynthesis of cerebral gangliosides in vivo rather than occur as a breakdown product of more complex molecules.
Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane
Authors: Bader Marie‐France , Ciesielski‐Treska Jaroslava , Thierse Danièle , Hesketh John E. , Aunis Dominique
Published: October 2006

Abstract Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone‐like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine‐storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone‐like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.
Na+‐Dependent Uptake and Release of Taurine by Neuroblastoma × Glioma Hybrid Cells
Authors: Kürzinger Konrad , Hamprecht Bernd
Published: October 2006

Abstract In neuroblastoma × glioma hybrid cells, a cell line of neuronal character, a saturable uptake system for taurine is found which displays high affinity and high capacity (Km= 38 μm, V= 1.25 nmol mg−1 min−1)‐ Only the closely related structural analogues hypotaurine and β‐alanine are able to inhibit the transport of radioactively labeled taurine. Imipramine or haloperidol at 100 μmeffectively blocks taurine uptake. High‐affinity taurine uptake shows an absolute and highly specific requirement for Na+. The hybrid cells internalize taurine very slowly and, with 1 mm extracellular taurine, attain a plateau only after more than 20 h, at which time approximately 34 mm labeled taurine has accumulated in the cytosol. Generally there is hardly any spontaneous release of accumulated taurine. Efflux can, however, be induced by increasing the intracellular Na+ content and is then accelerated by lowering the extracellular Na+ concentration. The hypothesis is forwarded that taurine may exert its function by driving the extrusion of Na+ in emergency situations.
Kernicterus: Enzymatic Evidence for Difference Between the Development of Cholinergic and GABAergic Innervations in the Brain of the Gunn Rat
Authors: Ohno Takuo
Published: October 2006

Abstract The activities of choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD), markers of cholinergic and GABAergic terminals, were measured in growing and adult Gunn rats, which possess an autosomal recessive gene for jaundice or kernicterus. In the olfactory tubercle, hippocampus, neostriatum, thalamus, and hypothalamus of the animals with kernicterus, the development of the cholinergic pathways was delayed, but by the adult stage it was normal, while there was practically no action on the innervation by GABAergic neurons, at least as indicated by the chemically measured parameters.
Effects of Variations in Glutamic Acid Decarboxylase Activity on Acute Oxygen Poisoning
Authors: Segerbo Bengt
Published: October 2006

Abstract— A study was made to test the influence of rapid variations in glutamic acid decarboxylase (GAD) activity on the susceptibility of rats to hyperbaric oxygen (HBO). GAD was inhibited by the convulsant drug unsymmetrical dimethylhydrazine (UDMH) and reactivated by pyridoxine (PYR) after onset of convulsive activity. There was a relatively long induction period after UDMH injection until the onset of convulsions and the predictable interictal periods between successive periodic convulsions made it possible to study the impact of variations in GAD activity on survival rates, suspectibility to HBO and brain glycogen levels in a time sequence after UDMH administration. The experiments showed that UDMH interferes with aerobic metabolism in brain in such a way that profound alterations in resistance to acute oxygen poisoning resulted. An accumulation of substrate proximal to the enzyme block is assumed to develop during UDMH poisoning. The protective effect against HBO toxicity that was achieved after reactivation of GAD by PYR injection, as well as the rapid re‐establishment of glycogen levels, is believed to speak in favour of this hypothesis.
Alternative Pathways of Glucose Utilization in Brain; Changes in the Pattern of Glucose Utilization in Brain Resulting from Treatment of Rats with 6‐Aminonicotinamide
Authors: Hothersall John S. , Zubairu Salmata , McLean Patricia , Greenbaum A. Leslie
Published: October 2006

Abstract: The effect of 6‐aminonicotinamide (6AN) treatment on the activities of alternative pathways of glucose metabolism in 20‐day‐old rat brain was evaluated by measurements of yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, or 6 and uniformly labeled glucose, and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. At the highest dose of 6AN used (35 mg/kg body weight) there was a significant decrease in the 14CO2 yields via the pentose phosphate pathway, the glycolytic route, tricarboxylic acid (TCA) cycle, and via the glutamate‐γ‐aminobutyric acid pathway. Giving a graded series of doses (20–35 mg 6AN/kg body weight) revealed a hierarchy of responses in which the pentose phosphate pathway, lactate, glyceride‐glycerol, and fatty acid formation were most sensitive, followed, in sequence, by the pyruvate dehydrogenase reaction, the glutamate‐γ‐aminobutyrate route and, finally, the TCA cycle. The nature of the blocks in the various pathways was examined by the use of metabolite profiles.
Effects of Chloral Hydrate, γ‐Butyrolactone, and Equithesin on the Efflux of Dopamine and 5‐Hydroxytryptamine Metabolites into Cerebroventricular Perfusates of Rats
Authors: Nielsen J. A. , Moore K. E.
Published: October 2006

Abstract: Chloral hydrate, γ‐butyrolactone, and Equithesin are general anesthetics which have been employed in a variety of neurochemical and electrophysiological experiments. The effects of these anesthetics on the efflux of dopamine and 5‐hydroxytryptamine metabolites into lateral cerebroventricular perfusates were determined in rats bearing chronically implanted cannulas. Chloral hydrate and Equithesin increased, whereas γ‐butyrolactone decreased, the efflux of the dopamine metabolites 3,4‐dihydroxyphenylacetic acid and homovanillic acid. None of the anesthetics had a consistent effect on the efflux of the 5‐hydroxytryptamine metabolite 5‐hydroxyindoleacetic acid.
EDTA Inhibits In Vitro Increases in the GABA Content of Human CSF
Authors: Löscher Wolfgang , Ahnfelt‐Rønne Ian
Published: October 2006

Abstract: Aliquots of pooled samples of lumbar CSF from patients with neurological disorders were subjected to storage at room temperature for different periods of time up to 4 days. The determination of γ‐aminobutyric acid (GABA) levels in CSF by a radioreceptor assay showed a progressive increase in GABA content with time, which could be counteracted by addition of EDTA (5 mM). Similar results were obtained with cisternal CSF collected from dogs. The in vitro increases in GABA levels of untreated CSF might relate to the action of a metal‐enzyme which is inhibited by metal‐trapping substances such as EDTA.
Ca2+ and Calmodulin‐Dependent Phosphorylation of Endogenous Synaptic Vesicle Tubulin by a Vesicle‐Bound Calmodulin Kinase System
Authors: Burke Briant E. , DeLorenzo Robert J.
Published: October 2006

Abstract: Endogenous synaptic vesicle α‐and β‐tubulin were shown to be the major substrates for a Ca2+‐calmodulin‐regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two‐dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+‐calmodulin kinase. Evidence is presented indicating that the Ca2+‐calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. α‐Tubulin and β‐tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH‐L and DPH‐M. The Ca2+‐calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+‐activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization‐dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle α‐and β‐tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.
Stimulation of Na+‐Dependent, Veratridine‐Sensitive Synaptosomal Amino Acid Uptakes by Serum Albumin
Authors: Rhoads D. E. , Peterson N. A. , Raghupathy E. , Verity M. Anthony
Published: October 2006

Abstract: Bovine serum albumin (BSA) is shown to stimulate selectively the synaptosomal uptakes of those amino acids that are dependent on external Na+ and that are inhibited by veratridine. Thus, the stimulation can be seen in the case of aspartic acid, glutamic acid, glycine, proline, and γ‐aminobutyric acid, but not with serine and threonine. Further, studies on the interaction of veratridine, valinomycin, and BSA on the uptake of proline suggest that the primary action of the albumin is to increase the influx of proline. Such an action could result as a consequence of stabilization of the Na+ gradient by increased endogenous levels of ATP. Intrasynaptosomal ATP was increased in the presence of BSA but significantly decreased by veratridine.
Preservation of Oligodendroglial Cytoplasm in Cryopreservative‐Pretreated Frozen White Matter
Authors: Brammer M. J. , Ray P.
Published: October 2006

Abstract: Various cryoprotective agents [glycerol, dimethylsulphoxide, polyvinylpyrrolidone (PVP 40)], all dissolved in Krebs phosphate medium were tested for their effects on cytoplasmic preservation in oligodendroglia isolated from bovine white matter stored at ‐30°C. Of these agents, only PVP 40 (15% wt/vol) produced a significant improvement in recovery of oligodendroglial cytoplasm compared with untreated frozen brain. Cells isolated after PVP 40 pretreatment contained levels of membrane‐bound enzymes similar to those found in cells isolated from fresh white matter. There was, however, some loss of soluble protein. Studies of galactocerebroside synthesis in neuronal and oligodendroglial perikarya have shown that the glial cells contain ceramide galactosyltransferase at much higher specific activity than the neurones.
Demonstration of 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase in Cultured Human Schwann Cells
Authors: Reddy N. Boji , Askanas Valerie , Engel W. King
Published: October 2006

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2′3′‐Cyclic nucleotide 3′‐phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [3H]2′,3′‐cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme‐substrate kinetics, with an apparent Km of 1.6 mM for 2′,3′‐cyclic AMP, and the enzyme was stimulated by detergents such as Triton X‐100 and deoxycholate. It was inhibited by p‐chloromercuricbenzoate and 2′‐AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.
Purification and Characterization of a Ca2+‐ and Calmodulin‐Dependent Protein Kinase from Rat Brain
Authors: Fukunaga Kohji , Yamamoto Hideyuki , Matsui Kazuo , Higashi Kenji , Miyamoto Eishichi
Published: October 2006

Abstract: A Ca2+‐ and calmodulin‐dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800‐fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S‐300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited Km values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and Ka values of 12 nM and 1.9 μM for brain calmodulin and Ca2+, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine‐rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca2+‐ and calmodulin‐dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP‐dependent protein kinase. Our findings indicate that there is a multifunctional Ca2+‐ and calmodulin‐dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.
Turnover of the Molecular Forms of Acetylcholinesterase in the Rat Diaphragm
Authors: Brimijoin Stephen , Carter Jonathan
Published: October 2006

Abstract: The turnover of acetylcholinesterase (AChE) and its molecular forms was measured by following the loss of enzyme activity in the right hemidiaphragms of Sprague‐Dawley rats treated with cycloheximide, 20 mg/kg, every 4 h. This treatment inhibited 96% of the incorporation of [3H]leucine into muscle protein. After 8 h of treatment, the total AChE activity of the diaphragm decreased by 17% (P < 0.01). Assuming first‐order exponential kinetics, a half‐life of 30 h and an hourly turnover of 180 units were calculated. The measured accumulation of AChE activity at a ligature on the phrenic nerve indicated that axonal transport contributed trivially to this turnover. Sucrose density gradient experiments showed that the cycloheximide‐induced loss of AChE activity was restricted to the 4S enzyme, which had an apparent half‐life of 6.2 h.
Intermediate Filaments of Schwann Cells
Authors: Autilio‐Gambetti Lucila , Sipple Jack , Sudilovsky Oscar , Gambetti Pierluigi
Published: October 2006

Abstract: Intermediate filaments were prepared from distal stumps of rabbit sciatic nerve 5 weeks after nerve section, at which time Schwann cells account for 85–90% of the cell area. A polypeptide of molecular weight 58,000 was the main component of this fraction. An antiserum raised in guinea pig against this polypeptide stained all cells present in the distal stump, as well as Schwann cells and 3T3 cells in culture. The identity of the molecular weight 58,000 polypeptide obtained from distal stumps with vimentin was proved with one and two‐dimensional sodium dodecyl sulfate pol yacrylamide gel electrophoresis and with immunoautoradiography. It is concluded that the intermediate filament subunit of undifferentiated Schwann cells is vimentin. The possibility that Schwann cells in normal nerve may have another type of intermediate filament besides vimentin cannot be ruled out.
“On” and “Off” Responses of K+‐Induced Synaptosomal Proline Release: Involvement of the Sodium Pump
Authors: Nickolson Victor J.
Published: October 2006

Abstract: The K+‐induced release of the putative neurotransmitter proline in rat cerebral cortex synaptosomes was studied by measuring [5‐3H]proline released upon exposure to high‐K+ media. In contrast to previous experiments, K+ was not substituted for Na+, which was held constant. Under the resulting conditions of increased osmolarity, two peaks of proline release were obtained, one upon exposure to increased [K+], another upon restoring normal [K+]. The dependence of the latter peak on ionic composition of the medium, and its sensitivity to ouabain are discussed.
Ganglioside‐Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species
Authors: Byrne Mary C. , Ledeen Robert W. , Roisen Fred J. , Yorke Glee , Sclafani Joseph R.
Published: October 2006

Abstract: Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility that neurite‐stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three‐step purification procedure that eliminated at least 95% of the contaminating peptides. These purified preparations retained their capacity to induce extensive neurite growth in neuro‐2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of their inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.
Induction of Cholinergic Expression in Developing Spinal Cord Cultures
Authors: Brenneman Douglas E. , Warren Dale
Published: October 2006

Abstract: The induction of choline acetyltransferase (ChAT) by cAMP derivatives was studied in dissociated spinal cord cultures. Dibutyryl cAMP (dbcAMP) and 8‐bromo cAMP (1 mM) produced a 2–3‐fold stimulation of ChAT activity in developing cultures whereas 8‐bromo cGMP had no effect. A phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine, also increased (2‐fold) ChAT activity in immature cultures. Significant elevations in ChAT were detected after 2 h incubation with dbcAMP. Maximum enzyme induction was observed 24 h after dbcAMP supplementation to the culture medium. Developmental studies revealed that ChAT could be induced on days 2–16 in culture. The largest induction of ChAT activity was observed on day 7 in culture. After day 19, when control enzyme activity attained levels of mature cultures, cAMP‐mediated ChAT induction was no longer observed. Cycloheximide and actinomycin D blocked ChAT induction whereas basal enzyme activity remained unaffected. Culture protein content was not changed after 1‐day exposure to dbcAMP. 125I‐Tetanus toxin fixation after dbcAMP treatment revealed a 20% decrease from control in neuronal surface during days 7–9 in culture. These data indicated that cAMP derivatives produced a rapid increase in cholinergic expression during a specific period of development in spinal cord cultures. There appears to be specificity to this effect, as total neuronal surface does not respond in the same manner as ChAT activity. The ChAT induction mediated by dbcAMP appears to be regulated at the transcriptional level.
Dopamine‐β‐Hydroxylase Activity of the Cat Carotid Body Under Different Arterial O2 and CO2 Conditions
Authors: Starlinger H. , Acker H. , Heinrich R.
Published: October 2006

Abstract: To determine the importance of dopamine and noradrenaline as neurotransmitters during chemoreception in the cat carotid body we investigated the contents of both compounds as well as the activity of dopamine‐β‐hydroxylase (DBH) under different arterial Po2 and Pco2 conditions. The superior cervical ganglion was used as a control organ. In the carotid body and the ganglion an inverse relationship exists between the catecholamine content and the DBH activity. The carotid body has a high catecholamine content with a low DBH activity whereas the superior cervical ganglion has a low catecholamine content and high DBH activity. Hypercapnia did not produce any significant change in the catecholamine content or in the DBH content of the carotid body. However, in comparison with hyperoxia, hypoxia produced a significant change (p < 0.05) in the noradrenaline content without changing the DBH activity. The dopamine content under these conditions did not change significantly. The results may indicate that the high catecholamine content of the carotid body is the result of a high retention and/or low rate of degradation rather than of a high rate of synthesis.