bluuebunny/crossref_metadata_2025 huggingface dataset:
Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats
Authors: Cousin M. A. , Lando D. , Moguilewsky M.
Published: October 2006

Abstract: Ornithine decarboxylase (ODC), the rate‐limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [3H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.
Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character
Authors: Grassi Jacques , Vigny Marc , Massoulié Jean
Published: October 2006

Abstract: In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (∼ 15%) of collagen‐tailed forms (mostly A12 and A8), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m‐NaCl, 5 mm‐EDTA, 1% Triton X‐100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G4 and the monomeric G1 forms, with a smaller proportion of the dimeric G2 form. (3) The tetrameric enzyme exists as a minor soluble component (GS4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G1 form presents a marked hydrophobic character, as indicated by its interaction with Triton X‐100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent‐free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris‐HCl buffer produce a larger overall yield of G1 form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G1 form apparently derives in part from a pool of GH4 form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G4) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex‐tracts, in a manner that depends on the ionic conditions. In Tris‐HCl buffer, G1 is converted into a well‐defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm‐Ca2‐ to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X‐100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.
Dopamine β‐Hydroxylase and Other Glycoproteins from the Soluble Content and the Membranes of Adrenal Chromaffin Granules: Isolation and Carbohydrate Analysis
Authors: Fischer‐Colbrie R. , Schachinger M. , Zangerle R. , Winkler H.
Published: October 2006

Abstract: Chromogranin A and two other proteins (A1 and A2) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N‐acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N‐acetylgalactosamine, and sialic acid were the main sugars. In addition membrane‐bound dopamine β‐hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane‐bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β‐hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.
Mass Fragmentographic Analysis of Monoamine Metabolites in the Spinal Cord of Rat After the Administration of Morphine
Authors: Commissiong John W.
Published: October 2006

Abstract: A mass fragmentographic method was used in which homovanillic acid (HVA), methoxyhydroxyphenylglycol (MHPG), and 5‐hydroxyindoleacetic acid (5HIAA) were measured from a single sample. The results describe the effect of morphine on the metabolism of the major monoamines, dopamine (DA), noradrenaline (NA), and 5‐hydroxytryptamine (5‐HT) in the spinal cord. Morphine has very little effect on the metabolism of DA and NA in the spinal cord. However, morphine causes a significant increase in the metabolism of spinal 5‐HT. The increase in 5‐HIAA induced by morphine is not restricted to the dorsal horn. The three main functional regions of the cord—dorsal horn (sensory), zona intermedia (auto‐nomic), and ventral horn (somatic motor)—are affected to the same degree. The results indicate that morphine causes a generalized activation of serotonin neurons in the spinal cord. There appears to be little or no selectivity for those serotonergic neurons that innervate the dorsal horn. The results are discussed with reference to current data which indicate a fairly strong link between descending serotonergic nerves and the mechanism of action of morphine‐induced analgesia.
Is Lysolecithin an In Vivo Constituent of Chromaffin Granules?
Authors: Frischenschlager Inge , Schmidt Wolfgang , Winkler Hans
Published: October 2006

Abstract: It was recently claimed that lysolecithin (lysophosphatidylcholine) in chromaffin granules is a postmortem artefact. We have, therefore, determined catecholamine/lysolecithin ratios in adrenal tissues and isolated chromaffin granules. In rat adrenals and bovine medulla the ratios in both tissues and granules were similar. This indicates that even in rapidly frozen rat adrenal glands, sufficient lysolecithin is present in the total tissue to account for its presence in isolated organelles. Owing to the high cortex/medulla ratios such studies cannot be performed with guinea pig or rabbit adrenals. However, isolated chromaffin granules from guinea pig, in contrast to a previous study, do contain lysolecithin. We conclude that lysolecithin is an in vivo constituent of chromaffin granules of all species so far investigated.
Cyclic AMP Induction of the Myelin Enzyme 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase in Rat Oligodendrocytes
Authors: McMorris F. Arthur
Published: October 2006

Abstract: Cyclic AMP (cAMP) is known to induce the activity of the myelin enzyme 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP; EC 3.1.4.37) in C6 rat glioma cells. This report shows that CNP is also inducible in oligodendrocytes explanted from 1‐day‐old rat cerebrum and grown in tissue culture. Induction was observed after a 1‐day treatment with 1 mM N6, O2′ ‐dibutyryl cyclic AMP (dbcAMP) and was maximal after 5 days, reaching 200–240% of control. Induction was observed both in mixed cerebral cell cultures containing oligodendrocytes and astrocytes, and in purified cultures of oligodendrocytes prepared by a differential shakeoff procedure. Addition of dbcAMP to the cultures 3–9 days after the cells were explanted from rat brain induced CNP activity, but no induction was observed when dbcAMP treatment was begun 13 or more days after explantation. These results demonstrate that one component of myelin, CNP, is inducible in oligodendrocytes by a cAMP‐mediated mechanism, and suggest a role for cAMP in the regulation of the myelin‐associated functions of oligodendrocytes.
Endogenous γ‐Hydroxybutyrate in Rat Brain Areas: Postmortem Changes and Effects of Drugs Interfering with γ‐Aminobutyric Acid Metabolism
Authors: Eli M. , Cattabeni F.
Published: October 2006

Abstract: The possibility that γ‐hydroxybutyrate (GHB), a metabolite of γ‐aminobutyric acid (GABA), may play a role in the CNS has recently come to attention. We describe here a sensitive and specific mass fragmento‐graphic technique that allows the measurement of picomole amounts of GHB in single rat brain areas. Moreover, we show that GHB can accumulate postmortem, an effect that is blocked by the use of microwave irradiation to kill the animals. To understand further the relationship between GABA and GHB formation, we treated rats with drugs known to inferfere with GABA metabolism at different levels and concomitantly measured GABA and GHB in cerebral cortex and cerebellum. Isoniazide, which blocks the formation of GABA, also decreases GHB. Blockers of the catabolism of GABA, such as aminooxyacetic acid and γ‐acetylenic GABA, increase GABA levels and decrease those of GHB. Sodium dipropylacetate increases both GABA and GHB, supporting the hypothesis that this effective antiepileptic drug also blocks in vivo the enzyme that converts succinic semialdehyde to succinic acid.
Stimulation of Tubulin‐Dependent ATPase Activity in Microtubule Proteins from Porcine Brain by Taxol
Authors: Fujii Toshihiro , Kondo Yoshiyuki , Kumasaka Michiyo , Suzuki Tatsuo , Ohki Kosuke
Published: October 2006

Abstract: Taxol, an antimitotic agent that induces microtubule assembly, stimulated tubulin‐dependent Mg2+ ‐ ATPase activity of microtubule‐associated proteins (MAPs). A concentration‐dependent increase in the rate of ATP hydrolysis was observed. Taxol acted through its binding to the tubulin molecule on MAP ATPase, and maximal stimulation, which was found at approximately equal concentrations of taxol and tubulin, reached about 140% of the original level in the absence of taxol. Taxol enhanced ATP hydrolysis by a mixture of MAPs and tubulin, and this continued at a steady linear rate even when the polymerization had approached a plateau. In the presence of taxol, a large portion of ATPase activity and protein was recovered in the pellet after centrifugation at 70,000 g for 60 min at 25°C. Both colchicine and podophyllotoxin inhibited taxol‐stimulated ATPase activity via the same mechanism by which they inhibited taxol‐induced microtubule polymerization. The stimulation by taxol was not found in the presence of Ca2+ alone but required Mg2+ . We conclude that tubulin effectively stimulates Mg2+‐ATPase activity of MAPs under conditions that induce tubulin polymerization.
Temporal Characteristics of Potassium‐Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes
Authors: Suszkiw J. B. , O'Leary M. E.
Published: October 2006

Abstract: The time course of Ca2+‐dependent [3H]acetylcholine ([3H]ACh) release and inactivation of 45Ca2+ entry were examined in rat brain synaptosomes depolarized by 45 mM [K+]o. Under conditions where the intrasynaptosomal stores of releasable [3H]ACh were neither exhausted nor replenished in the course of stimulation, the K+‐evoked release consisted of a major (40% of the releasable [3H]ACh pool), rapidly terminating phase (t1/2= 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K+‐stimulated Ca2+ entry suggests the presence of fast‐inactivating, slow‐inactivating, and noninactivating, or very slowly inactivating, components. The fast‐inactivating component of the K+‐stimulated Ca2+ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K+ stimulus. The noninactivating Ca2+ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K+]o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage‐dependent Ca channels.
Anticonvulsant Activity of Glycylglycine and δ‐Aminovaleric Acid: Evidence for Glutamine Exchange in Amino Acid Transport
Authors: Samuels Stanley , Fish Irving , Schwartz Stephen A.
Published: October 2006

We have proposed that glutamine serves in a facilitated diffusion process, mediated by the enzyme γ‐glutamyl transferase (γ‐glutamyl transpeptidase; γGT) and that it leaves the brain in exchange for entering amino acids. Glutamine is also a precursor of γ‐aminobutyric acid (GABA). Thus, providing an alternate substrate for γGT should spare brain glutamine, raise GABA, and cause an anticonvulsant effect. We have found that glycylglycine, the best‐known substrate for γGT, and δ‐aminovaleric acid (DAVA), a structural analog, have anticonvulsant activity in DBA/2J mice. Both compounds can decrease the incidence and severity of seizures induced by l‐methionine‐rs‐sulfoximine or electroconvul‐sive shock. DAVA was also tested and found to be active against seizures caused by pentylenetetrazol or picrotoxin. [14C]DAVA entered the brain at the rate of 18.7 nmol/g/min. The activity of DAVA as a substrate of γGT was intermediate to that of glycylglycine and glutamine. Preliminary studies have shown that brain glutamine and perhaps GABA are elevated 3 h after administration of DAVA (7.5 mmol/kg). These findings support the theory that glutamine exchange plays a role in amino acid transport across the blood‐brain barrier and suggests a new concept in anticonvulsant therapy. Key Words: Amino acids, transport—γ‐Aminobutyric acid—δ‐Amino valeric acid—Anticonvulsant—Glutamine, exchange—Seizures.
ß‐Adrenergic Receptor Activation Increases Acetylcholine Receptor Number in Cultured Skeletal Muscle Myotubes
Authors: Blosser James C.
Published: October 2006

Treatment of embryonic chick muscle myotubes with the ß‐adrenergic agonist isoproterenol increased the number of surface membrane nicotinic cholinergic receptors. Receptor degradation was unaffected by isoproterenol, suggesting that receptor synthesis was increased. The effect of isoproterenol appears to be mediated by the ß‐adrenergic receptor adenylate cy‐clase system for the following reasons: (a) The response to isoproterenol was dose‐dependent and stereospecific. (b) The response to catecholamines followed the order isoproterenol > epinephrine > norepinephrine. (c) Al‐prenolol, a ß‐adrenergic antagonist, but not phen‐tolamine, an a‐antagonist, abolished the effect, (d) The maximal effects of isoproterenol and cholera toxin, an activator of adenylate cyclase, were not additive. These results suggest that under certain physiological states catecholamines may play an important role in the regulation of cholinergic receptors.
Comparative Thermodynamics of Benzodiazepine Receptor Ligand Interactions in Rat Neuronal Membranes
Authors: Doble Adam
Published: October 2006

The effects of temperature on the interaction of various ligands with the benzodiazepine receptor were studied in rat brain membrane preparations. The affinities of all ligands studied were reduced on raising the temperature from 4 to 37°C. The variation of affinity constant with temperature deviated from the classical relationship for both the anticonvulsant ligand [3H]flunitrazepam and the proconvulsant ligand [3H]ethyl β‐carboline‐3‐carboxylate. This imples a variation of observed enthalpy change of binding with temperature. Possible reasons for this are discussed. γ‐Aminobutyric acid and sodium chloride both enhance the binding of [3H]flunitraze‐pam—the former by an increase in the entropic component of the binding energy, and the latter by an increase in the enthalpic component. In a series of ligands of different biological activities, no simple correlation was observed between biological activity and temperature dependence of binding.
The S100‐b Protein: Tyrosine Residues Do Not Exhibit an Abnormal Fluorescence Spectrum
Authors: Baudier Jacques , Gérard Dominique
Published: October 2006

The β subunit of the bovine brain S100‐b protein (ββ) lacks tryptophyl residue but contains one tyrosine. Our experiments show that this protein is characterized by a typical tyrosine fluorescence spectrum, with a maximum at 303 nm. Identical fluroescence properties were found for the rat brain S100‐b protein. Comparison with the fluorescence spectrum of the bovine brain S100‐a’ protein (α'β), which contains a tryptophan residue in the α’ subunit, enables us to demonstrate that the recent report describing an abnormal fluorescence spectrum for the bovine brain S100‐b protein may result from a contamination of the S100‐b by the S100‐α’ protein.
Axonal Polypeptides Cross‐Reactive with Antibodies to Neurofilament Proteins
Authors: Brown Beverly A. , Majocha Ronald E. , Staton Donna M. , Marotta Charles A.
Published: October 2006

Abstract: Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross‐reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000 and 70,000 NFPs (200K, 140K and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase‐conjugated indirect antibody procedure, however, differences were detected in the cross‐reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and thus, was suitable for the detection of cross‐reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti‐200K, anti‐140K, or anti‐70K was applied to overloaded two‐dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross‐reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Tilers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a variety of conditions.
Tryptophan Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes
Authors: Herrero E. , Aragón M. C. , Giménez C. , Valdivieso F.
Published: October 2006

Abstract:Tryptophan uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on the Na+gradient [Na+] outside > [Na+] inside and is maximal when both Na+and Cl−are present. The uptake represents transport into an os‐motically active space and not a binding artifact, as indicated by the effect of increasing the medium osmo‐larity. The uptake of tryptophan is stimulated by a membrane potential (interior negative) as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m‐chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tryptophan is accumulated by two systems with different affinities. Ouabain, an inhibitor of Na+, K+‐activated ATPase, does not affect tryptophan transport. The uptake of tryptophan is inhibited by high concentrations of phenylalanine, tyrosine, leucine and 3, 4‐dihydroxyphenylalanine.
Thyroid Hormones and Derivatives Inhibit Flunitrazepam Binding
Authors: Nagy A. , Lajtha A.
Published: October 2006

Abstract: Thyroid hormones and their derivatives were found to inhibit [3H]flunitrazepam binding stereospe‐cifically and in a monophasic manner. Among the compounds tested, D‐thyroxine was the most potent inhibitor (IC50= 0.5 μM). The naturally occurring L‐thyroxine was about 40‐fold less potent (IC50= 20 μM). The structure‐activity relationships seem to imply that the thyronine base has the principal role in the inhibition of benzo‐diazepine receptor binding. The type of inhibition was examined with the most potent inhibitor, D‐thyroxine, by Scatchard analysis. The apparent dissociation constant (KD) of the [3H]flunitrazepam binding increased and the receptor density (Bmax) decreased as a function of D‐thyroxine concentration; this is characteristic of mixed‐type inhibition.
Kinetics of Tryptophan Influx into Brain Slices Depleted of Sodium and Loaded withl‐Histidine
Authors: Korpi E. R.
Published: October 2006

Abstract:The kinetics of tryptophan influx were studied with rat brain slices preloaded withl‐histidine and/or depleted of sodium ions. The best fits of the data (velocity of influx versus tryptophan concentration) were computed by use of a model consisting of a saturable (Michaelis‐Menten type) and an unsaturable (diffusional) component with an iterative nonlinear regression analysis. Sodium depletion of the slices reduced the maximal velocity of saturable influx. In histidine‐preloaded slices, depleted or not depleted of sodium ions, the most marked alteration again occurred in the maximal velocity, which more than doubled. Slices preloaded with histidine contained greatly elevated levels of glutamine and histidine, which may have stimulated the influx by exchange with extracellular tryptophan even in the absence of sodium ions. The maximal velocity was higher with increasing concentration of large neutral amino acids in slices at the start of the influx measurements. The influx of tryptophan in brain cells is apparently modified by changes in the intracellular amino acid pool, which, when increased, also counteracts the effect of sodium depletion on the tryptophan influx.
Induction of Cytosolic Progestin Binding Sites by Catecholestrogens in Rat Pituitary Gland and Uterus: Different Potencies of 2‐ and 4‐Hydroxyestradiol
Authors: Kirchhoff Josef , Reinhardt Walter , Grünke Wolfgang , Ghraf Rüdiger , Ball Peter , Knuppen Rudolf
Published: October 2006

Abstract: The ability of Catecholestrogens to induce cytosolic progestin binding sites in the hypothalamus, pituitary gland and uterus of ovariectomised‐adrenal‐ectomised rats was demonstrated by the increase in high‐affinity [3H]promegestone binding sites (KD 1.39,0.50 and 0.54 n M, respectively) following a single subcutaneous injection (26.4 μg/animal) of the 3, 4‐dibenzoate ester of 4‐hydroxyestradiol. The affinity and the time course of induction of these binding sites were very similar to those after a single injection of an equivalent dose (20 (μg/animal) of estradiol 3‐benzoate, exhibiting maximal receptor levels after 44 h. Widely differing efficacies in the induction of progestin binding sites were observed between the dibenzoate esters of 2‐ and 4‐hydroxyestradiol. 2‐Hydroxyestradiol 2, 3‐dibenzoate was ineffective in the pituitary gland up to a dose of 132 μg/ animal, whereas 4‐hydroxyestradiol dibenzoate was equi‐potent to estradiol benzoate, showing a maximal induction of progestin binding sites at single doses in the range of 13.2–26.4 μg/animal (equivalent to 10–20 μg of estradiol benzoate). As compared to the pituitary gland, the uterus was much more sensitive to the systemic administration of estrogen benzoates. At single doses in the range of 1.32–6.6 μg/animal (equivalent to 1–5 μg of estradiol benzoate), 4‐hydroxyestradiol dibenzoate induced maximal levels of progestin receptors and even 2‐hydroxyestradiol dibenzoate, when given at a high dose (132.4 μg/animal, equivalent to 100 μg of estradiol benzoate), produced a slight increase in progestin binding sites.
Levels and Synthesis of Glutamate and Aspartate in the Olfactory Cortex Following Bulbectomy
Authors: Scholfield C. N. , Moroni F. , Corradetti R. , Pepeu G.
Published: October 2006

Abstract: Guinea pigs were unilaterally bulbectomised and the contents of aspartate, glutamate and GABA measured in slices of olfactory cortex taken from the lesioned and intact hemispheres. Two days after the operation there was a fall in the aspartate and glutamate levels, which persisted for over 120 days, whereas γ‐aminobutyric acid (GABA) showed a transient fall followed by a small rise. The fall in glutamate and aspartate was much greater in small, thin slices containing a high density of nerve terminals. The synthesis of 13C aminoacids from [13C]glucose during electrical stimulation was greater in the slices taken from the normal side than in those from the operated side. The GABA synthesis, however, was four times greater on the lesioned side. This time‐course for the fall in acidic amino acids correlates with the fall in electrical responses, and this lends weight to the idea that aspartate and/or glutamate mediate synaptic transmission in the area.
Norepinephrine Metabolism in Humans Studied by Deuterium Labelling: Turnover of 4–Hydroxy‐3–methoxyphenylglycol
Authors: Mårdh Göran , Sjöquist Birgitta , Änggård Erik
Published: October 2006

Abstract: D, L(±)‐4‐Hydroxy‐3‐methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study turnover of plasma free HMPG following an intravenous injection. Ten healthy men were given a pulse dose of either 4.3 μmol or 2.2 μmol of labelled HMPG ([2H3]HMPG piperazine salt). Plasma and urine levels of both endogenous and labelled HMPG were subsequently followed by gas chromatography‐mass spectrometry with selected ion detection. Kinetic calculations based upon a single‐compartment model were consistent with a monoexponential elimination of plasma free HMPG. The half‐life of HMPG was 0.46 and 0.78 h (mean values in the two dose groups). The HMPG production rate was 2.01 and 2.35 μmol/hour, and the urinary excretion rate of HMPG (free and conjugated) was 0.48 and 0.47 μmol/h. The endogenous plasma level of free HMPG was 25 and 33 nmol/L. The results show that HMPG turns over rapidly and that HMPG is further metabolized extensively. About one‐fourth of the HMPG produced is excreted in urine as free and conjugated HMPG.
Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G‐adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re‐dy Mutant
Authors: Watkins W. Jean , Watts D. C.
Published: October 2006

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G‐adr and 129Re‐dy, with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G‐adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re‐dy muscle confirm the findings of other workers.
Membrane Anomalies in Huntington's Disease Fibroblasts
Authors: Schroeder Friedhelm , Goetz Ingeburg E. , Roberts Eugene
Published: October 2006

Abstract: Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+‐ATPase indicated a 10‐fold purification of the isolated plasma membrane. The specific activity of Na+,K+‐ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+‐ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6‐diphenyl‐l,3,5‐hexatriene and of trans‐parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans‐Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans‐parinaric acid and 1,6‐diphenyl‐1,3,5‐hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20° and 30°C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD.
Calmodulin Binding Proteins of the Cholinergic Electromotor Synapse: Synaptosomes, Synaptic Vesicles, Receptor‐Enriched Membranes, and Cytoskeleton
Authors: Walker J. H. , Stadler H. , Witzemann V.
Published: October 2006

Abstract: Calmodulin binding proteins (CBPs) have been identified using a gel overlay technique for fractions isolated from Torpedo electromotor nerve endings. Different fractions possessed characteristic patterns of CBPs. Synaptosomes showed five major CBPs—Mr 220,000, 160,000, 125,000, 55,000, and 51,000. Polypeptides of Mr 55,000 and 51,000 were found in the cytoplasm and the others are membrane‐associated. The Triton X‐100‐insoluble cytoskeleton of synaptosomes was isolated in the presence or absence of calcium. The major CBPs had Mr of 19,000, 18,000, and 16,000. In the presence of calcium, no other CBPs were seen. In the absence of calcium, an Mr 160,000 polypeptide was present in the Triton cytoskeleton. Synaptic vesicles showed CBPs of Mr 160,000, 25,000, and 20,000. Membrane fragments enriched in acetylcholine receptors contained two major CBPs, Mr 160,000 and 125,000, together with a less prominent protein at Mr 26,000. A protein of Mr similar to that of fodrin was present in synaptosomes and acetylcholine receptor membrane fragments, but only in small amounts relative to the other polypeptides observed. The heavy and light chains of clathrin‐coated vesicles from pig brain did not bind calmodulin, although strong labelling of an Mr 47,000 polypeptide was found. Results showed that calelectrin does not bind calmodulin. The possible identity of the calmodulin binding proteins is discussed.
Phospholipid Biosynthesis in Myelin: Presence of CTP: Phosphoethanolamine Cytidylyltransferase in Purified Myelin of Rat Brain
Authors: Kunishita Tatsuhide , Ledeen Robert W.
Published: October 2006

Abstract: Highly purified myelin from rat brain was previously shown to contain the ethanolaminephosphotransferase which completes the synthesis of phosphatidyl ethanolamine. We have now obtained evidence for the presence in myelin of CTP:phosphoethanolamine cytidylyltransferase, the enzyme catalyzing formation of CDP‐ethanolamine. Myelin was isolated by two different procedures, one based on the Norton‐Poduslo method and the other involving repetitive gradients with osmotic shocking deferred to the end. The fact that activity remained constant through all but the earliest steps suggested that the enzyme is intrinsic to myelin. Comparison of subcellular fractions revealed that approximately half the total activity was in the supernatant, the remainder being distributed among the particulate fractions. Relative specific activity of myelin was 27–31% that of microsomes, thus eliminating the possibility of appreciable contamination by the latter. The possibility of adsorption of the soluble enzyme by myelin was rendered unlikely by retention of activity after washing the myelin with buffered sodium chloride or sodium taurocholate. Furthermore, relative specific activity of the cytidylyltransferase was 10‐fold higher than that of lactate dehydrogenase (a cytosolic marker) in myelin. The apparent Km for CTP was approximately the same for myelin and microsomes, but that for phosphoethanolamine was significantly higher for myelin.
Age‐Dependent Variations in Peroxide‐Utilizing Enzymes from Rat Brain Mitochondria and Cytoplasm
Authors: Vitorica Javier , Machado Alberto , Satrústegui Jorgina
Published: October 2006

Abstract: The location of peroxide‐utilizing enzymes has been studied in rat brain. Glutathione peroxidase and glutathione reductase distributions indicate that both enzymes are located in the cytoplasm and in the matrix space of “synaptosomal” and “free” mitochondria. On the other hand, catalase distribution parallels that of NADH‐cytochrome c reductase (rotenone‐insensitive), and appears to be associated with the outer membrane of brain mitochondria. Whereas no gross age‐dependent changes in various marker enzymes were found, a gradual but significant increase in glutathione peroxidase from the soluble fraction of free mitochondria was detected. The consequences of such increase are discussed with regard to the reducing potential of the cell.
Combined Stereological and Biochemical Analysis of Storage and Release of Catecholamines in the Adrenal Medulla of the Rat
Authors: Nordmann Jean J.
Published: October 2006

Abstract: Adrenal medullae from rats injected with insulin 1 h prior to decapitation were analyzed by stereology and their catecholamine content was determined. In control animals the ratio between adrenaline‐ and noradrenaline‐ containing cells was 4.06 whereas the ratio between adrenaline and noradrenaline contents was 4.11. The glands from insulin‐treated animals showed a 45% reduction in adrenaline content and a 42% decrease in the volumetric density of the adrenaline‐containing granules. However, neither the noradrenaline content nor the volumetric density of noradrenaline‐containing granules was modified in glands from insulin‐treated animals. From these data, the amount and concentration of adrenaline or noradrenaline in a single granule have been calculated. The results are consistent with the view that chromaffin granules are the source of the released catecholamines. The present paper demonstrates that stereology combined with biochemistry is a useful tool for resolution of problems at the cellular and subcellular levels.
Competitive Inhibition of Human Brain Hexokinase by Metrizamide and Related Compounds
Authors: Bertoni John M. , Weintraub Susan T.
Published: October 2006

Abstract: We have compared the competitive inhibitory effects of 2‐deoxyglucose, glucosamine, N‐acetylglucosamine, N‐benzoylglucosamine, and the commonly used radiographic and density gradient agent metrizamide (2‐[3 ‐ acetamido ‐ 2,4,6 ‐ triiodo ‐5‐(N‐ methylacetamido) benzamido]‐2‐deoxyglucose) on the mitochondrial and soluble forms of human brain hexokinase. Metrizamide produces a classical competitive inhibition with glucose for human brain hexokinase, with Kis of 2.8 and 2.5 mM, respectively, for the mitochondrial and soluble forms. Glucosamine exhibited Kis of 0.58 and 0.29 mM, while 2‐deoxyglucose exhibited Kis of 0.074 and 0.15 mM and N‐acetylglucosamine 0.098 and 0.092 mM for these two forms, respectively. N‐Benzoylglucosamine was by far the most effective inhibitor tested, with Ki values of 0.0086 and 0.022 mM, respectively. In order of increasing potency as a competitive inhibitor for mitochondrial hexokinase are metrizamide, glucosamine, N‐acetylglucosamine, 2‐deoxyglucose, and N‐benzoylglucosamine. For the soluble form of the enzyme in increasing potency are metrizamide, glucosamine, 2‐deoxyglucose, N‐acetylglucosamine, and N‐benzoylglucosamine. Since N‐benzoylglucosamine was over 100 times more potent than metrizamide, some of the effects of metrizamide could be due to contamination by N‐benzoylglucosamine. However, gas chromatography‐mass spectrometry analysis of metrizamide did not indicate the presence of N‐benzoylglucosamine. In addition, column chromatographic separation of commercially available metrizamide and reconstitution of freeze‐dried eluate fractions localized the inhibitory effect to the metrizamide peak.
Inhibition of GABAB Receptor Binding by Guanyl Nucleotides
Authors: Hill D. R. , Bowery N. G. , Hudson A. L.
Published: October 2006

Abstract: GTP and GDP decreased the saturable binding of [3H]baclofen or [3H]γ‐aminobutyric acid ([3H]GABA) to GABAB but not GABAA receptors whereas GMP displayed negligible activity. This effect was specific to guanyl nucleotides and was not mimicked by high concentrations of ATP. The inhibition of ligand binding was the result of a diminished receptor affinity with no change in receptor number. The use of a complete physiological saline solution rather than Tris buffer plus Ca2+ or Mg2+ increased the potency of GTP at the GABAB receptor. The results are discussed in relation to the effects of GABA and GTP on adenylate cyclase activity in the brain.
Distribution of Spinal Cord Nerve Endings Containing Various Neurotransmitters on a Continuous Density Gradient
Authors: Bucsics A. , Mayer N. , Pabst M. A. , Lembeck F.
Published: October 2006

Abstract: Homogenates of rat dorsal or ventral spinal cord were subjected to centrifugation on a continuous density gradient. The gradient was generated according to a new method with the aid of a microprocessor‐controlled HPLC pump. The distribution of substance P‐like immunoreactivity (SPI) and somatostatin‐like immuno‐reactivity (SRIFI) across the gradient showed two peaks. The SPI peak seen at lower density was found only in dorsal spinal cord tissue. No peak of SPI was seen at this position in homogenates prepared from the spinal cords of capsaicin‐pretreated rats. The second peak of SPI, found at a higher density, was accompanied by peaks in the levels of endogenous 5‐hydroxytryptamine (5‐HT), [14C]glycine, and [3H]norepinephrine uptake. This peak was seen at the same density in the dorsal and the ventral spinal cord. Tissue derived from capsaicin‐pretreated rats exhibited one peak of SPI, accompanied by a maximum of [14C]glycine uptake. The uptake of [3H]γ‐aminobutyric acid ([3H]GABA) was found to have a maximum at a somewhat lower density than that of [14C]glycine. It is concluded that the peak of SPI found at lower density in the dorsal spinal cord is associated with nerve endings belonging to capsaicin‐sensitive primary afferents, while other endings, including those also containing 5‐HT, are probably associated with the peak of SPI found at higher density.
Inhibition of the UDP‐N‐Acetylgalactosamine: GM3, N‐Acetylgalactosaminyl Transferase by Gangliosides
Authors: Nores G. A. , Caputto R.
Published: October 2006

Abstract: Gangliosides in the range of 0.1–0.4 mM inhibited the UDP‐N‐acetylgalactosamine:GM3, N‐acetyl‐galactosaminyl transferase (EC 2.4.1.79) of chicken retina. Other lipids such as phosphatidylethanolamine, sphingomyelin, sulfatides, and phosphatidic acid in concentrations similar to those of gangliosides did not affect the enzyme activity significantly. GM3 has an inhibition capability slightly less than that of gangliosides with two or three sialyl groups in their molecules, while asialo‐GM1 is clearly less inhibitory. The inhibitory effect of a constant amount of GT1 ganglioside was higher at low concentrations of membrane preparation, but the inhibition was similar at different concentrations of the substrates GM3 or UDP‐N‐acetylgalactosamine and at all incubation times studied. The added gangliosides were found attached to the membranes. In this attached state they may act either as substrate or inhibitor. The inhibitory effect of gangliosides was not apparent when a mixture of Triton CF 54‐Tween 80 was added to the incubation medium at concentrations >0.33%.
High Specific γ‐Aminobutyric Acid Binding to Membranes of the Human Ovary
Authors: Erdö Sándor L. , László Ádám
Published: October 2006

Abstract: The concentration of γ‐aminobutyric acid (GABA) in the human ovary and the capacity of a membrane preparation from the same organ to bind [3H]GABA specifically were examined. The GABA concentration in the ovary was found to be 214 ± 66 nmol/g frozen tissue (mean ± SEM of six independent determinations). Moreover, a single population of high‐affinity GABA binding sites has been identified in the ovarian membranes. The apparent dissociation constant (Kd) and maximum binding capacity (Bmax) were 38.3 nM and 676 fmol/mg protein, respectively. The specific binding of [3H]GABA was displaced by muscimol, unlabelled GABA, or (+)bicuculline, but was unaffected by (±)baclofen and picrotoxin. The present results show that GABA and an extremely high density of GABAA receptor binding sites are present in the human ovary, indicating a physiological significance of this amino acid in the female reproductive system.
Calcium, ATP, and Magnesium Activate Soluble Tyrosine Hydroxylase from Rat Striatum
Authors: Iuvone P. Michael
Published: October 2006

Abstract: Soluble tyrosine hydroxylase (TH) prepared from rat striatum by sonication, centrifugation, and gel filtration on Sephadex G‐25 was activated by preincubation with Ca2+, ATP, and Mg2+. Activation occurred with micromolar concentrations of Ca2+ and required the presence of both ATP and Mg2+. The activation was reversible and was characterized by a large decrease of apparent Km for the pteridine cofactor and a small increase of Vmax‐Ca2+‐induced activation was small when TH activity was assayed at pH values near the optimum, but the magnitude of the activation increased with increasing assay pH. The activation apparently did not involve Ca2+‐activated protease because it was not affected by the protease inhibitor leupeptin. Nor did it involve cyclic AMP‐dependent protein kinase, as evidenced by the failure of the heat‐stable inhibitor of this kinase to decrease Ca2+‐induced TH activation. Furthermore, the activation of TH evoked by Ca2+ and that produced by cyclic AMP was additive. These experiments indicate that striatal TH can be activated in vitro by an endogenous Ca2+‐dependent mechanism. The similarity of the Ca2+‐induced activation of TH to that elicited by increased neuronal activity and terminal depolarization is discussed.
Chloride Ions Preferentially Mask High‐Affinity GABA Binding Sites
Authors: Madtes Paul
Published: October 2006

Abstract: Chloride ions (Cl−) act to regulate the appearance of γ‐aminobutyric acid (GABA) binding sites in synaptic membranes from bovine retinas. Scatchard analysis indicates that the presence of Cl−results in a preferential decrease (85%) in the number of high‐affinity binding sites, with no statistically significant change in the affinity (KD) of either the high‐or low‐affinity sites. The finding that the appearance of this binding site is altered by the presence of Cl−may indicate that Cl−acts, either directly or indirectly, to regulate the appearance, and hence function, of the high‐affinity GABA receptor.
Protein Synthesis Rates in Rats with Portacaval Shunts
Authors: Dunlop D. S. , Kaufman H. , Zanchin G. , Lajtha A.
Published: October 2006

Abstract: Protein synthesis rates were measured (33 days postoperatively) in rats with portacaval shunts and in unoperated controls. In brain, no change in the rate of protein synthesis was evident in shunted rats. These data thus do not support the hypothesis that an inhibition of brain protein synthesis is a factor in the etiology of hepatic encephalopathy. The synthesis rate in forebrain at 82 days of age was 0.52%/h. Though brain wet weight was the same in both groups, rats with shunts grew relatively slowly, and their testicles probably decreased in weight. However, no inhibition of muscle, liver, or testicular protein synthesis could be detected. The mechanism of slower or negative growth in these tissues might thus involve an increase in the degradation rate, although a transient inhibition of synthesis at an earlier period is also possible.
Comparative Characterization of Glutamate Decarboxylase in Crude Homogenates of Oviduct, Ovary, and Hypothalamus
Authors: Erdő Sándor L. , Kiss Béla , Szporny László
Published: October 2006

Some biochemical characteristics of l‐glutamate decarboxylase (GAD) were compared using crude homogenates of the rat oviduct, ovary, and hypothalamus. As estimated by the measurement of CO2 production, the specific activities of oviductal and ovarian GAD were about 10 and 3% of the hypothalamic value, respectively. Stoichiometric formation of γ‐aminobutyric acid (GABA) and CO2 from l‐glutamate could be observed in oviduct and hypothalamus, while in ovarian homogenates the production of C02 was more than nine times that of GABA. Hypothalamic and tubal GAD showed similar time course, temperature dependence, and pH dependence. The dependence on protein concentration and on exogenous cofactor supply was also similar in these two tissues. The kinetic constant for l‐glutamate as a substrate was nearly the same in oviduct (1.30 mM) and hypothalamus (1.64 mM). The responsiveness of tubal and hypothalamic GAD to various inhibitors was also similar. The present findings indicate that the oviductal and the hypothalamic GAD may be identical, and they suggest a possible GABAergic innervation of the Fallopian tube.