bluuebunny/crossref_metadata_2025 huggingface dataset:
DIFFERENTIATION OF NEUROBLASTOMA, GLIOMA, AND HYBRID CELLS IN CULTURE AS MEASURED BY THE SYNTHESIS OF SPECIFIC PROTEIN SPECIES: EVIDENCE FOR NEUROBLAST‐GLIOBLAST RECIPROCAL GENETIC REGULATION
Authors: Rosenberg Roger N. , Vance Carol K. , Morrison Marcelle , Prashad Nagindra , Meyne Julianne , Baskin Fred
Published: October 2006

Abstract— Protein species from differentiating neuroblastoma, glioma, and hybrid neuroblastoma‐glioma cell lines in cell culture were separated and identified initially in the first dimension by the use of isoelectric focusing gels and were further separated in the second dimension by SDS‐acrylamide gels. There were two main classes of proteins identified: proteins which were dominantly expressed in neuroblastoma and also in hybrid cell cultures, and proteins which were expressed in glioma and also hybrid cell cultures. In general, proteins were identified which were significantly expressed in neuroblastoma cells and much reduced in glioma cultures, and also conversely so. The hybrid cell line expressed many of the neuroblastoma‐type proteins and relatively fewer of the glioma type proteins. A specific protein species (2) was identified in hybrid cells and was not present in either parental neuroblastoma or glioma cultures. Protein z was expressed however by the co‐culturing of neuroblastoma and glioma cells suggesting its induction is dependent on a soluble factor. Protein z in hybrid cells was demonstrated in both stained gels and by autoradiography. Chromosome analysis of hybrid cells confirmed the presence of both rat and mouse chromosomes. It is suggested that similar neuronal‐glial interaction may be functional in the intact brain, and that similar reciprocal modulation between neurons and glia may be a central mechanism of differentiation in the nervous system.
ELECTROPHORETIC ANALYSES OF PROTEINS TRANSPORTED TO THE RAT POSTERIOR PITUITARY
Authors: Gainer Harold , Brownstein Michael J.
Published: October 2006

Abstract— [35S]cysteine, [3H]methionine, or [3H]fucose were injected into the supraoptic nuclei (SON) of rats, and the labelled proteins that were transported to and accumulated in the posterior pituitary 24h post‐injection were analyzed electrophoretically. The transported, labelled proteins which were soluble in 0.1 m‐HCl were primarily of low molecular weight (about 12,000 on SDS gels). However, the selectivity of labelling of these proteins by the three different labelled precursors could be revealed by isoelectric focusing. The 0.1 m‐HCl insoluble labelled proteins, presumably reflecting membrane proteins transported from the SON to the pituitary, were more diverse and generally of higher molecular weight (> 43,000 on SDS gels).
CARBAMYLATION AND DECARBAMYLATION OF ACETYLCHOLINESTERASE: EFFECT OF CHOLINE, 3,3‐DIMETHYL‐l‐BUTANOL AND SOME ALLOSTERIC EFFECTORS
Authors: Dawson M.
Published: October 2006

Abstract— Choline is more effective than 3,3‐dimethyl‐l‐butanol (DMB) in protecting bovine erythrocyte AChE against carbamylation by physostigmine sulphate (an ester of methylcarbamic acid). Both of these alcohols bind to dimethylcarbamyl‐AChE and accelerate its decarbamylation. Choline has a higher affinity for the inhibited enzyme, and causes a more rapid reactivation than does OMB. At low ionic strength, various allosteric effectors also bind to the dimethylcarbamylenzyme and accelerate its reactivation, but not to the same extent as choline and DMB. The rate of reactivation in the presence of an allosteric effector and choline is less than the sum of the individual rates. However the rate of reactivation in the presence of an allosteric effector and DMB exceeds the sum of the individual rates. The results provide further support for a previous proposal that choline and DMB bind to different sites on the enzyme, despite their structural similarity.
THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID
Authors: Brunetti Marina , Giuditta A. , Porcellati G.
Published: October 2006

Abstract—

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion‐nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP‐choline in the intact ganglion‐nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base‐exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations.

Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.
IN VIVO RELEASE OF ENDOGENOUS GABA FROM RAT SUBSTANTIA NIGRA MEASURED BY A NOVEL METHOD
Authors: Van der Heyden J. A. M. , Venema K. , Korf J.
Published: October 2006

Abstract—

A sensitive GABA assay using HPLC coupled with fluorimetric detection with o‐phthalaldehyde is described. GABA, lysine and ethanolamine can be measured within approx 12 min. The detection limits for these compounds (signal/noise = 3) is 0.67 pmol, 1.8 pmol and 0.73 pmol respectively. Using this assay the in vivo release of GABA from rat substantia nigra was studied with a push‐pull perfusion technique.

A pronounced increase in the rate of endogenous GABA release was observed after addition of depolarizing amounts of K+ to the perfusion medium, whereas the concentrations of lysine and ethanolamine in the perfusate did not change. This enhanced release of GABA was not diminished after omission of Ca2+ and Mg2+ from the medium. Increasing the Mg2+ concentration and leaving out Ca2+ however, resulted in a marked depression in the K+‐induced GABA release.

Electrical stimulation of the striatum also produced an increase in release of GABA from the substantia nigra.

Inhibition of glutamic acid decarboxylase (with 3‐mercaptopropionic acid) caused an immediate decrease in GABA release. Inhibition of GABA transminase (with aminooxyacetic acid) leads to an increased release of GABA after approx 15 min.

These findings suggest that the technique is suitable for measuring neuronal release of endogenous GABA in vivo
ON THE COMPOSITION AND STRUCTURE OF INDIVIDUAL GANGLIOSIDES FROM THE BRAIN OF ELASMOBRANCHES
Authors: Avrova N. F. , Li Y.‐T. , Obukhova E. L.
Published: October 2006

Gangliosides with a short carbohydrate chain: II3(NeuAc)2‐LacCer, II3(NeuAc)2‐GgOse3Cer, II3(NeuAc)3‐LacCer, II3NeuAc‐LacCer, and II3NeuAC‐GgOse3 Cer, were found to be predominant in the brain of 8 species of cartilaginous fish, elasmobranches. N‐acetylneuraminic acid was the only sialic acid found in these gangliosides, the N‐glycolyl derivative being practically absent. 4‐Sphingenine was shown to be the predominant sphingoid in elasmobranch brain gangliosides.

The sequential enzymatic hydrolysis of II3(NeuAc)2 ‐LacCer from shark and ray brain by acylneur‐aminyl hydrolase (EC 3.2.1.18) and β‐D‐galactoside‐galactohydrolase (EC 3.2.1.23), as well as permethylation studies, provide further evidence for the following structure of this major elasmobranch brain ganglioside:
[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS
Authors: Nelson D. L. , Herbet A. , Pétillot Y. , Pichat L. , Glowinski J. , Hamon M.
Published: October 2006

A high‐affinity (Kd= 5.9 nM) specific binding site for [3H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [3H]serotonin ([3H]5‐HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra‐synaptosomal origin exhibited very similar properties with respect to [3H]harmaline binding characteristics and MAO A activity.

Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [3H]harmaline. Logit‐log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [3H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 102‐2 × 103 times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [3H]harmaline from its specific binding site. Ki and IC50 values for the inhibition of [3H]harmaline binding by MAO I and MAO substrates (tryptamine, 5‐HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [3H]5‐HT as the substrate. In conclusion, the specific binding site for [3H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [3H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.
TYROSINE HYDROXYLASE ACTIVITY IN NORADRENERGIC NEURONS OF THE LOCUS COERULEUS AFTER RESERPINE ADMINISTRATION: SEQUENTIAL INCREASE IN CELL BODIES AND NERVE TERMINALS
Authors: Zigmond R. E.
Published: October 2006

The effect of a single systemic injection of reserpine on tyrosine hydroxylase activity in the locus coeruleus, cerebellum, hypothalamus, and hippocampus was examined. Increases in enzyme activity were seen in all four brain areas; the time‐course of the changes, however, was different in each case. In the locus coeruleus the maximum change in enzyme activity was seen 3 days after drug administration; in the cerebellum, 7‐11 days; in the hypothalamus, 8‐11 days; and in the hippocampus, 21 days. Since tyrosine hydroxylase in the cerebellum and hippocampus is present in terminals of neurons whose cell bodies are located in the locus coeruleus, the delayed increase in enzyme activity in cerebellum and hippocampus probably depends upon the slow rate of transport of TH molecules in these neurons.
STUDIES ON THE BIOCHEMICAL BASIS OF DISTAL AXONOPATHIES–I. INHIBITION OF GLYCOLYSIS BY NEUROTOXIC HEXACARBON COMPOUNDS
Authors: Sabri M. I. , Moore C. L. , Spencer P. S.
Published: October 2006

Abstract— Neurotoxic hexacarbon compounds 2,5‐hexanedione (2,5‐HD) and methyl n‐butyl ketone (MnBK) inhibit crystalline and endogenous CNS and PNS glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Preincubation of the enzyme with the toxin was necessary for inhibition. The enzyme activity of GAPDH was preserved by the addition of dithiothreitol in the presence of either neurotoxin. By contrast, lactate dehydrogenase (LDH) activity was not inhibited by these neurotoxic chemicals. Neurologically inactive compounds 1,6‐hexanediol and acetone failed to inhibit GAPDH. The present data indicate that 2,5‐HD and M “BK block energy metabolism by inhibiting glycolysis at the site of GAPDH. These observations may account for the known failure of GAPDH‐dependent axonal transport and the axonal degeneration which occurs in hexacarbon neuropathy.
BIOGENIC AMINE‐MEDIATED ALTERATION OF PYRIDOXAL PHOSPHATE FORMATION IN RAT BRAIN
Authors: Ebadi M. , Govitrapong P.
Published: October 2006

Abstract— DOPA, dopamine, norepinephrine, tyramine, serotonin, histamine and GABA inhibited pyridoxal kinase; whereas, tyrosine, 5‐hydroxytryptophan, histidine, glutamic acid, hypotaurine and taurine were without inhibitory effects. Tetrahydroisoquinoline derivatives formed from Pictet‐Spengler condensation between DOPA, dopamine and norepinephrine with pyridoxal and pyridoxal phosphate did not inhibit pyridoxal kinase. These results are interpreted to indicate that interaction of biogenic amines and pyridoxal kinase may alter the formation of pyridoxal phosphate which in turn may influence the activity of numerous pyridoxal phosphate dependent enzymatic reactions in brain.
BRAIN EXTRACELLULAR POTASSIUM AND ENERGY METABOLISM DURING ISCHEMIA IN JUVENILE RATS AFTER EXPOSURE TO HYPOXIA FOR 24 h
Authors: Hansen A. J. , Nordströsm C.‐H.
Published: October 2006

Abstract— The ischemia‐induced change of brain extracellular potassium concentration [K+]e and brain energy metabolism was studied following decapitation of normal 7‐dav‐old rats and 7‐day‐old rats exposed to a simulated altitude of 6000 m (pO2= 70 mm Hg) for 24 h. Initially there was in both groups a slow rate of rise of [K +]e followed by a faster rise until a steep increase occurred. In the group exposed to hypoxia the initial rate of rise was lower and the duration until the steep increase twice as long as in the normal group. Energy consumption expressed as Δ#P and the rates of lactate accumulation were similar in the two groups in the first minute following decapitation. After 2 min the Δ#P and the rate of lactate accumulation was higher in the group exposed to hypoxia. These findings are explained by a doubling of the glucose concentration in the brain of the rats exposed to hypoxia, permitting anaerobic glycolysis to maintain ATP regeneration for a longer time during ischemia.
CHARACTERIZATION OF BENZODIAZEPINE BINDING SITES IN CULTURED CELLS OF NEURAL ORIGIN
Authors: Syapin Peter J. , Skolnick Phil
Published: October 2006

Abstract— The binding of [3H]diazepam to benzodiazepine receptors was investigated in cultured cell lines of neural origin. Two cell lines, the rat C6 glioma and mouse NB41A3 neuroblastoma possess large numbers of benzodiazepine binding sites, while the other neural cell lines examined had significantly fewer benzodiazepine binding sites. [3H]diazepam binding to membranes prepared from C6 or NB41A3 cells was saturable and of a relatively high affinity (KD± 12 and 20 nm, respectively) when compared with rat cerebral cortex (KD± 4.6 nm). A single class of binding sites in both cell lines was demonstrated by Scatchard analysis. The maximum binding capacities (Bmax) in the C6 and NB41A3 cell lines were found to be 10 and 3.5 fold higher than in rat cerebral cortex, respectively. In contrast to the rat cerebral cortex, binding of [3H]diazepam in cultured cells was not displaced by the clinically active benzodiazepines clonazepam and oxazepam while the clinically inactive benzodiazepine Ro 5‐4864 potently inhibited the binding of [3H]diazepam in both neural cell lines. In toto, this data suggests a change in the benzodiazepine binding sites in cultured cells of neural origin to that found in peripheral (kidney) tissue. The observation that cell lines derived from both neuronal and glial elements contain large numbers of benzodiazepine binding sites also suggests benzodiazepine receptors in the central nervous system may not be confined to a single cell type.
MAXIMUM ACTIVITIES, PROPERTIES AND DISTRIBUTION OF 5’NUCLEOTIDASE, ADENOSINE KINASE AND ADENOSINE DEAMINASE IN RAT AND HUMAN BRAIN
Authors: Phillips E. , Newsholme E. A.
Published: October 2006

Abstract— The maximum activities of 5’nucleotidase, adenosine kinase and adenosine deaminase have been measured in several areas of rat and human brain. There is no major difference between the activities of nucleotidase and kinase between rat and human brain, but the activity of deaminase is considerably higher in human brain. The activities of all these enzymes are similar in three areas of rat brain and nine areas of human brain, except for hind brain of the human, which has a low activity of adenosine deaminase. This variation may indicate the existence of different steady‐state concentrations of adenosine in certain areas of the brain.

Subcellular fractionation of different areas of rat brain showed that, whereas adenosine kinase and deaminase activities were located mainly in the soluble fractions, 5’nucleotidase was present in all subcellular fractions (i.e. membrane, synaptosomal, mitochondrial and soluble). In particular, there was no major localisation within the synaptosomal fraction. Thus it is unlikely that the regulation of the activities of these enzymes is dependent upon changes within a specific compartment (e.g. synaptosomes) in the brain.
DEVELOPMENTAL CHANGES IN LEVELS AND FORMS OF CHOLINESTERASES IN MUSCLES OF NORMAL AND DYSTROPHIC CHICKENS
Authors: Lyles Joan M. , Silman Israel , Barnard Eric A.
Published: October 2006

Abstract— Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were studied in vivo and during the first several months of development of pectoral and posterior latissimi dorsi (PLD) muscles in normal and dystrophic chickens. Muscle extracts were prepared in a high ionic strength‐nonionic detergent medium in the presence of protease inhibitors, in order to obtain complete solubilization and to prevent degradation of intrinsic molecular forms of both enzymes.

In both normal and dystrophic pectoral muscles levels of AChE and °ChE increase rapidly in vivo, °ChE accounting for 5–10% of total cholinesterase activity. In the normal pectoral muscle the concentration of both enzymes drops rapidly after hatching with increasing muscle mass; total AChE per muscle remains relatively constant for 30 days post‐hatch. In the dystrophic pectoral muscle both AChE and °ChE accumulate after hatching, resulting in greatly elevated levels (approx 10–25‐fold) of both enzymes throughout the period studied.

Multiple molecular forms of AChE and °ChE are observed in the pectoral muscle by sucrose gradient centrifugation. Four principal forms are distinguished: two light (L1, L2), one medium (M), and one heavy (H2). The °ChE forms are 0.5–1.0 S units lighter than the corresponding AChE forms. L2 is the predominant light form of AChE, whereas L1 is the major light °ChE form detected. The lighter forms of AChE predominate in normal and dystrophic embryonic pectoral muscle at day 14, being replaced by the H2 form by day 19. H2 is the major °ChE form detected at day 19. After hatching, H2 AChE is the predominant form found in both of the normal muscles studied. In the dystrophic pectoral muscle, progressive accumulation of the L2 form of AChE is detected as early as day 4 post‐hatch; this form eventually becomes predominant, although the heavier forms are also elevated. In PLD muscle the same phenomenon occurs, but with a slower time course. In dystrophic pectoral muscle a similar rise in the L1 form of °ChE is first observed by day 4, with heavier forms also elevated in the mature muscle. Thus the alteration in the control of these two enzymes in dystrophic fast‐twitch muscles results in an accumulation of the light forms of AChE and °ChE.
ADENOSINE REGULATES VIA TWO DIFFERENT TYPES OF RECEPTORS, THE ACCUMULATION OF CYCLIC AMP IN CULTURED BRAIN CELLS
Authors: Calker Dietrich van , Müller Margarete , Hamprecht Bernd
Published: October 2006

Abstract— In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β‐adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.
EFFECTS OF EXCITATORY NEUROTRANSMITTER AMINO ACIDS ON SWELLING OF RAT BRAIN CORTICAL SLICES1
Authors: Chan Pak H. , Fishman Robert A. , Lee Jane L. , Candelise Livia
Published: October 2006

Abstract— With the single rat brain cortical slice serving as an in vitro bio‐assay system, the effects of neurotransmitter amino acids (1 mm) on brain swelling, water, sodium and potassium content, inulin space, and lactate production were studied. The putative dicarboxylic amino acid neurotransmitters, l‐glutamic acid and l‐aspartic acids, greatly increased intracellular brain swelling with increased intracellular Na+, water content and lactate production, and decreased inulin space and intracellular K+. Equimolar GABA, taurine, glycine, the putative inhibitory neurotransmitter amino acids, and equimolar α‐amino‐isobutyric acid had no effect. Brain swelling and intracellular Na+/K+ ratios were greatly increased by l‐glutamate and l‐aspartate at a concentration of 10 mm. However, l‐aspartate at these concentrations greatly depleted the K+ content and lactate production as compared to l‐glutamate.

Further studies indicated that only the structural analogs and isomers of the dicarboxylic amino acids possessing two acidic groups and an α‐amino group had a similar effect on the induction of brain swelling. Among the analogs of glutamic acid, dl‐homocysteic acid and kainic acid had a greater effect on brain swelling, as observed from the total adenosine 5′‐triphosphate (ATP) levels and the time‐course and dose‐response. A biphasic response in lactate production was induced by dl‐homocysteic acid and kainic acid, suggesting that these analogs had a neurotoxic effect on cellular metabolism at higher concentrations.
A MODEL OF HIGH AFFINITY CHOLINE TRANSPORT IN RAT CORTICAL SYNAPTOSOMES
Authors: Wheeler D. D.
Published: October 2006

Abstract— Initial velocity of choline uptake by cortical synaptosomes from the Long‐Evans rat has been measured as a function of both choline and sodium concentration. These data were then fitted to the rate equation for each of several possible models which characterize the participation of sodium in the transport process, and the models giving best fit were identified. Although one cannot unequivocally distinguish between a model including a high affinity carrier component plus diffusion and one including both high affinity and low affinity carriers, the conclusions concerning the high affinity component are the same in both cases. The major conclusions from the model are as follows: (1) The carrier may first combine with either choline or sodium; if the first reaction is with sodium, then there is an obligatory reaction with a second sodium before choline can interact with the carrier. (2) Translocation may occur as either CS or CNa2S (C= carrier; S= choline; CS= carrier‐substrate complex). (3) The apparent maximal velocity (Va) is dependent on the sodium concentration. (4) K1, the choline concentration giving Va/2. is also dependent on the sodium concentration. K1 increases with [Na] from 0 to 38.41 mm; above 38.41 mm‐[Na]. K1 declines with [Na]. (5) There is a sigmoidal relationship between velocity of uptake and [Na]; however, uptake is not zero at [Na] = 0. (6) Jm. uptake at a given [choline] and infinite [Na]. is hyperbolically related to the choline concentration, but changes slowly over the range of 0.5–5.0 ± 10‐6m. (7) KNa, the sodium concentration giving a velocity equal to Jm/2, is related to the choline concentration by a quadratic equation, and was found to be greater than physiological [Na] at choline concentrations of 0.5, 0.6, or 1.0 ± 10‐6m. but less than physiological [Na] at choline concentrations of 2.0 or 5.0 ± 10‐6m.

The best fit model for the high affinity uptake of choline is very similar to what has been found in previous studies for the high affinity uptake of glutamic acid and GABA, thus raising the question of whether or not all high affinity synaptosomal mechanisms may be variations of a common model.
LOCALIZATION OF GLUTAMIC ACID DECARBOXYLASE WITHIN LAMINAE OF THE RAT OLFACTORY TUBERCLE
Authors: K.RIEGER NEIL R. , HELLER JOHN S.
Published: October 2006

—There are three histological layers within the rat olfactory tubercle: plexiform, pyramidal and polymorphic. We have assayed glutamic acid decarboxylase (GAD) and GABA in homogenates of frozen sections cut parallel to these layers. Consecutive sections (16 μm) were homogenized in groups and assayed for GAD or GABA. Every seventh section was stained with Toluidine Blue to monitor the depth and orientation of the plane of section. Steep variations in GAD activity (up to 6‐fold) were observed as a function of depth in the tubercle. These were paralleled by corresponding but less marked variations in GABA levels. The lowest values were found in the plexiform layer. This suggests that GAD may play a very limited role there. The highest activities were found in sections from the deepest lamina of the polymorphic layer. This is the only lamina of the tubercle that does not receive a dopaminergic input.
Regulation of Rat Pineal Hydroxyindole‐O‐Methyltransferase: Evidence of S‐Adenosylmethionine‐Mediated Glucocorticoid Control
Authors: Sandrock Alfred W. , Leblanc Gabrielle G. , Wong Dona L. , Ciaranello Roland D.
Published: October 2006

: Rat pineal hydroxyindole‐O‐methyltransferase is controlled similarly to adrenal medullary phenylethanolamine N‐methyltransferase. S‐adenosylmethionine (SAM), the in vivo cofactor utilized by the enzyme to convert N‐acetylserotonin to melatonin, protects this methyltransferase against tryptic proteolysis in vitro. Furthermore, in vivo studies suggest that the nucleoside itself is controlled by glucocorticoids. Hypophysectomy decreases hydroxyindole‐O‐methyltransferase levels as compared with control animals, while dexamethasone and SAM administration restore enzyme levels toward control values. In vitro proteolytic studies further demonstrate that, although N‐acetylserotonin does not stabilize the enzyme against trypsinization, this substrate acts synergistically with SAM to confer greater stabilization than observed with SAM alone.
The Synthesis and Degradation of Poly(A)‐containing mRNAs in Mouse Neuroblastoma Cells Treated with Dibutyryl cAMP or with Ro20‐1724 *
Authors: Morrison Marcelle R. , Hall Catherine L. , Pardue Sibile , Brodeur Rick , Baskin Fred , Rosenberg Roger N.
Published: October 2006

Abstract: The relative rates of synthesis and turnover of poly(A)‐containing mRNAs were determined in the S‐20 (cholinergic) and NIE‐115 (adrenergic) neuroblastoma clones after 3 days treatment with dibutyryl cAMP or the phos‐phodiesterase inhibitor N‐4‐(3‐butoxy‐4‐methoxybenzyl)‐2‐imidazolidinone (Ro20‐1724) in an attempt to correlate transcriptional and posttranscriptional changes with observed cAMP‐induced changes in the differentiated state. Treatment of S‐20 cells with dibutyryl cAMP for 3 days caused a fourfold increase in intracellular cAMP and a 30% decrease in growth rate compared with the levels in control cells. However, there was no difference in the synthesis of the poly(A)‐containing RNAs, relative to the ribosomal RNAs, after either 2‐ or 18‐h labeling with 3H‐adenosine. There was also no increase in the steady‐state mRNA levels. Treatment with Ro20‐1724 caused only a transient twofold increase in intracellular cAMP level when S‐20 cells were plated at a low density and no increase at higher densities although growth was inhibited by 60%. There was, again, no increase in the relative synthesis of poly(A)‐mRNAs with 2‐h label in cells treated with Ro20‐1724, but there was a twofold increase after labeling for 18 h. Optical density measurements, however, showed that this increased incorporation of 3H‐adenosine did not result in an increase in the absolute amount of unlabeled poly(A)‐containing mRNAs. NIE‐115 cells showed an 8‐fold increase in intracellular cAMP after 3 days of treatment with Ro20‐1724 and an 80% inhibition of growth rate. However, the relative rates of synthesis of the poly(A)‐containing mRNAs were identical to those obtained using S‐20 cells. These results show that neither increased intracellular cAMP levels nor growth inhibition necessarily result in a relative increase in the synthesis of the poly(A)‐containing mRNAs in these neuroblastoma clones. There was no change in the synthesis or processing of the poly(A) regions of the mRNAs in the S‐20 control and treated cells. In contrast to results with other cells, the poly(A) profiles were heterogeneous, even after 1 hour of labeling. The average size of the poly(A) region decreased similarly in control and drug‐treated cells after 1, 2 and 18 h labeling.
Regulation of the Cycle in Chick Pineal Serotonin N‐Acetyltransferase Activity In Vitro by Light
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: October 2006

The diurnal cycle in vivo in the level of serotonin N‐acetyltransferase (NAT) activity found in the pineal gland of chicks kept under diurnal lighting was reproduced in vitro with glands incubated in organ culture under the same diurnal lighting conditions. We have examined the effects of varying the lighting conditions of culture upon development of NAT activity in vitro with pineal glands from birds which were killed at different times during the photoperiod in vivo. The ability of chick pineal glands to develop increased NAT activity in culture during the dark period of a diurnal cycle of illumination was determined primarily by the proportion of the photoperiod in vivo elapsed at the time of sacrifice of the birds. The time of development of maximal NAT activity in cultured chick pineal glands could be advanced or delayed by corresponding changes in time of start of the dark period. However, the ‘settings’of the pineal‘biological clock’remained unchanged. Glands from birds sacrificed in the final 30 min of the photoperiod in vivo developed increased NAT activity without lag when cultured in the light. Similarly, pineal glands cultured under continuous illumination developed an increased photostable NAT activity beginning at the time of the „subjective” end of the photoperiod in vitro. Ability to develop additional NAT activity in the dark was lost rapidly at the „subjective” time in vitro of start of the next photoperiod. The maximal NAT activity developed in the dark consisted of both photolabile and photostable components.
Alterations in γ‐Aminobutyric Acid Receptor Sensitivity Following Acute and Chronic Ethanol Treatments
Authors: Ticku Maharaj K. , Burch Troie
Published: October 2006

The effects of acute and chronic ethanol ingestion on the binding of the inhibitory neurotransmitter 3H‐γ‐aminobutyric acid (GABA) was investigated in inbred strains of mice. Acute effects were analyzed in DBA/2J (alcohol avoiding) and C57BL/6J (alcohol preferring) mice, while chronic effects were analyzed in C57 mice. Acute ethanol (4 g/kg) produced a significant increase in the binding capacity of the low‐affinity γ‐aminobutyric acid binding site in both C57 (12%) and DBA (23%) mice, without significantly altering other binding parameters. The enhanced binding capacity may be responsible for the behavioral depression associated with ethanol use. Chronically treated C57 mice exhibited a decrease in the density of low‐affinity GABA receptor sites. These results suggest the possible involvement of γ‐aminobutyric acid synaptic function in the neuropharmacological effects of ethanol. The possibility that chronic ethanol administration may result in adaptation of γ‐aminobutyric acid receptors to the continuous presence of ethanol‐induced central nervous system depression is discussed.
Regional Protein Synthesis in Rat Brain Following Acute Hemispheric Ischemia
Authors: Dienel G. A. , Pulsinelli W. A. , Duffy T. E.
Published: October 2006

Regional protein synthesis was measured in rat brain at intervals up to 48 h following occlusion of the four major arteries to the brain for either 10 or 30 min. Four‐vessel occlusion produces ischemia in the cerebral hemispheres and oligemia in the midbrain‐diencephalon and brainstem. During the hour following 10 min of ischemia, protein synthesis, measured by incorporation of [14C]valine into protein, was inhibited in the cerebral cortex by 67%. Normal rates of protein synthesis were attained within 4 h of recirculation. In rats subjected to 30 min of ischemia, protein synthesis was inhibited by 83% during the first hour of recirculation in the cortex, caudate‐putamen, and hippocampus. Recovery of protein synthesis in these regions was slow (25–48 h). The midbrain‐diencephalon showed less inhibition, 67%, and faster recovery (by 12 h). Protein synthesis was unaffected in the brainstem. [14C]Autoradiography revealed that the pyramidal neurons of the hippocampus and areas of the caudate and cortex failed to recover normal rates of protein synthesis even after 48 h.

The accumulation of TCA‐soluble [14C]valine was enhanced (55–65%) in the cortex, caudate, and hippocampus after 30 min of ischemia; the increase persisted for 12 h. A smaller rise in [14C]valine content (30%) and more rapid normalization of valine accumulation (by 7 h) were observed in the midbraindiencephalon; no changes were found in the brainstem. In the cortex, recovery was more rapid when the duration of ischemia was reduced. Thus, the degree of inhibition of protein synthesis, the accumulation of valine in the tissue, and the length of time required to reestablish normal values for these processes were dependent on both the severity and the duration of the ischemic insult. Restoration of normal rates of protein synthesis after ischemia was slow compared with the normalization of cerebral energy metabolites.
Lipid Peroxidation in Rat Brain Cortical Slices as Measured by the Thiobarbituric Acid Test
Authors: Kovachich G. B. , Mishra O. P.
Published: October 2006

Abstract: Malonaldehyde formation by cortical brain slices from rat brain was determined as a function of incubation time and of oxygen pressure. This substance, a byproduct of lipid peroxidation, was detected by the thiobarbituric acid test. Significant amounts of malonaldehyde were formed by brain slices during incubation in the 0.2 (air) to 10 atm oxygen range, and a portion of it was released into the medium. The rate of malonaldehyde formation was the highest during the first 10 min. Elevation of oxygen pressure above 1 atm caused further increments in malonaldehyde production with kinetic properties similar to that seen at 1 atm pressure, but the increments per additional oxygen pressure were diminishing. The formation of a given amount of malonaldehyde can be expressed as a function of atm oxygen × min. This function has the shape of a saturation curve approaching a maximum at around 300 atm × min. The results indicate extensive lipid peroxidation in brain slices under standard incubation conditions.
Morphological and Biochemical Studies on the Cerebral Cortex from Reeler Mutant Mice: Development of Cortical Layers and Metabolic Mapping by the Deoxyglucose Method
Authors: Mikoshiba Katsuhiko , Kohsaka Shinichi , Takamatsu Ken , Aoki Emiko , Tsukada Yasuzo
Published: October 2006

Abstract: Cerebral cortex from reeler mutant mice was examined morphologically and biochemically. The sequential process of postnatal cell migration in the cerebral cortex of reeler (rl/rl) was examined morphologically. The dense cellular cortical plate lies below the molecular layer near the cerebral surface just after birth in normal mice while in reeler most of the cells are concentrated in the center of the cortex. In the cortex of adult reeler, the broad laminar structure of the neurons could be seen to form inverted positions in the cortical layers. The total wet weight, and the concentration of DNA and RNA in the pallium cerebri from reeler did not differ significantly from those in the control. As to the protein profiles of the pallium cerebri detected by SDS‐ polyacrylamide gel electrophoresis, no significant differences were observed. Activities of CNPase (2′,3′‐cyclic nucleotide 3′‐phosphohydrolase), which is a myelin enzyme of CNS, and choline acetyltransferase were at the same level in both the reeler and the control. Therefore, reeler mutation does not appear to affect the genetically determined cell numbers, number of cholinergic fibers, and myelination. By autoradiographic observation of the cerebral cortex after intraperitoneal injection of [14C]2‐deoxyglucose, it was revealed that 2‐deoxyglucose was incorporated intensively into the fourth layer (granular layer) of the cerebrum from the control. In reeler it was also incorporated into the granular layer but in a more widespread distribution. We conclude that terminals to the granular layer make metabolically active synapse, perhaps even in a manner inverted from normal.
Posttranslational Processing of Brain Actin
Authors: Palmer Elizabeth , de la Vega Humberto , Graña Dolores , Saborio José L.
Published: October 2006

Abstract: Two short‐lived isoforms of actin, named δ‐ and ε‐actin, have been detected in brain extracts from rats labeled in vivo with [35S]methionine. These two molecular species have PI values slightly more basic than β‐ and γ‐actin, the stable isoforms of the protein found in brain tissue. Under the appropriate incubation conditions δ‐ and ε‐actin, synthesized in vivo, can be converted in vitro into β‐ and γ‐actin. This posttranslational processing of δ‐ and ε‐actin requires acetyl coenzyme A, suggesting that an acetylation step, presumably of the NH2‐terminal end, is involved in the transformation of these proteins into β‐ and γ‐actin.
Improved Method for Prufication of 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase from Bovine Cerebral White Matter
Authors: Suda Haruhiko , Tsukada Yasuzo
Published: October 2006

Abstract: An improved procedure of the solubilization and purification of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) from bovine cerebral white matter is reported. To remove easily extractable protein, the tissue was homogenised in 10 vol. of 0.5 M‐ammonium acetate containing 10 mM‐Tris. HCI, pH 6.9, at 4°C and centrifuged at 105,000 g for 60 min. The precipitate was extracted with 10 vol. of 0.5% Triton X‐100 containing 10 mM‐Tris. HCI, pH 6.9, and centrifuged, By this extraction, over 70% soluble protein could be removed in the supernatant and most CNPase activity was kept in the precipitate. The precipitate was extracted with 10 vol. of 1% Triton X‐100 and 1 M‐ammonium acetate mixture containing 10 mM‐Tris.HCI, pH 8.2, and centrifuged at 105,000 g for 60 min. The extract contained 54% of CNPase and the specific activity was fivefold that of the original homogenate. Subsequently, the extractions were carried out with 2% Triton X‐100‐2 M‐ammonium acetate and 4% Triton X‐100‐4 M‐ammonium acetate at pH 8.2. The recovery of CNPase was found to be nearly 90% from the original homogenate, without loss of enzyme activity during extraction, while much CNPase activity was lost when guanidinium chloride was used as the extraction medium. Using the Triton X‐100‐ammonium acetate extract, several column chromatography techniques were applied to purify the enzyme. In the first step, Phenyl‐Sepharose CL‐4B column chromatography was performed by eluting with a double‐linear gradient of ammonium acetate and Triton X‐100. In the second step, the fraction containing CNPase after Phenyl‐Sepharose CL‐4B column chromatography was applied to a Sepharose 6B column and the enzyme was eluted with 1% Triton X‐100‐ I M‐ammonium acetate, pH 8.2. The peak containing CNPase was applied to CM‐Sepharose CL‐6B column chromatography in the final step. The enzyme was eluted with a linear gradient of KCI. In this step, CNPase eluted as a sharp peak and the specific activity was approximately 2300 pmol 2′‐AMP formed/min/mg protein. The recovery of CNPase from the original homogenate was about 50%. By the isoelectrofocusing technique, the pI of CNPase was found to be 8.6. When Reisfeld polyacrylamide gel electrophoresis and SDS‐polyacrylamide gel electrophoresis were carried out on the purified CNPase, only one protein band, corresponding to CNPase activity, was detected. Its molecular weight was estimated to be approximately 51,000 as the active enzyme form. K, value of the purified enzyme for 2′,3′‐CAMP calculated from a Lineweaver‐Burk plot was 3.13 mM.
The Effect of Acute Hypoxia on Synaptosomes from Rat Brain
Authors: Rafajowska Urszula , Erecifiska Maria , Wilson David F.
Published: October 2006

Abstract: Synaptosomes have been isolated from the brains of nonanesthetized and nembutal‐anesthetized rats subjected to 30 min hypoxia induced by breathing 7% oxygen in nitrogen. The respiratory rate was depressed in synaptosomes from starved hypoxic animals but was not significantly different from the respective control values in preparations from fed hypoxic animals, anesthetized animals, and hypoxic nonanesthetized animals allowed to recover from the hypoxic episode by 60 min of normoxic conditions. Observations are also reported concerning the levels of various metabolites in the synaptosomes isolated from the brains of the same groups of animals. It is suggested that hypoxia results in damage to the synaptosomal and/or mitochondrial membrane, which modifies substrate oxidation in the mitochondria and decreases availability of reducing equivalents for the respiratory chain. Results obtained on afflicted and recovered animals indicate that synaptosomal preparations provide a useful model for the study of hypoxic damage.
Effect of Aging on the Metabolism of Pyruvate and 3‐Hydroxybutyrate in Nonsynaptic and Synaptic Mitochondria from Rat Brain
Authors: Deshmukh D. R. , Owen O. E. , Patel M. S.
Published: October 2006

Abstract: Age‐dependent changes in the oxidative metabolism in nonsynaptic and synaptic mitochondria from brains of 3, 12, and 24‐month‐old rats were investigated. When pyruvate and malate were used in conjunction as substrates, a significant reduction in State 3 respiration was observed in both mitochondrial populations from 12‐and 24‐month‐old rats compared with 3‐month‐old animals. A similar age‐dependent reduction in the oxidation of [1‐11C]pyruvate was also observed in nonsynaptic and synaptic mitochondria from senescent rats. Pyruvate dehydrogenase complex activity (both active and total) was, however, not decreased in the two mitochondrial populations from brains of 3, 12, and 24‐month‐old rats. When DL‐3‐hydroxybutyrate plus malate were used as substrates, a decrease in State 3 respiration was observed only in synaptic mitochondria from 24‐month‐old rats compared with 3‐ month‐old animals. Similarly, an age‐dependent reduction in the oxidation of 3‐hydroxy[3‐11C]butyrate was also observed only in synaptic mitochondria from 12‐and 24‐month‐old rats. However, a significant reduction in the activities of ketone body‐metabolizing enzymes, namely, 3‐hydroxybutyrate dehydrogenase, 3‐ketoacid CoA transferase, and acetoacetyl‐CoA thiolase was observed in both mitochondrlal populations from 12‐ and 24‐month‐old rats compared with 3 month‐old animals. These findings show that specific alterations in oxidative metabolism occur in nonsynaptic and synaptic mitochondria from aging rats. The data also suggest that in addition to alterations in enzyme activities, permeability of anions (e.g. pyruvate) across the inner mitochondrial membrane may be altered in nonsynaptic and synaptic mitochondria from senescent animals.
Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems
Authors: Sinicropi D. V. , Dombrowski A. , Montgomery C. W. , Evans R. K. , Kauffman F. C.
Published: October 2006

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [3H]leucine into trichloroacetic acid‐precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation‐reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within 3h in vitro. Ganglion explants prepared by cutting the tissue into 300‐pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α‐ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.
Kinetic Measurements of the Turnover Rates of Phenylethylamine and Tryptamine In Vivo in the Rat Brain
Authors: Burden D. A. , Philips S. R.
Published: October 2006

Abstract: The turnover rates of phenylethylamine and tryptamine have been estimated as their rates of accumulation after inhibition of monoamine oxidase by pargyline and were found to be 1.53 nmol/g/h and 0.24 nmol/g/h, respectively. Rate constants for the substrate activities of these amines towards monoamine oxidase were calculated as 100 h−1 and 150 h−1, respectively. Tryptamine was found to exhibit a biphasic response to increasing pargyline dosage, demonstrating its dual activity to type B and type A monoamine oxidase, for which two rate constants were obtained, kB=100 h−1 and kA=50 h−1.