bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐Term Effects of RU24722 on Tyrosine Hydroxylase of the Rat Brain
Authors: Labatut Rene , Richard Francoise , Milne Brian , Quintin Luc , Lecestre Dominique , Pujol Jean‐François
Published: October 2006

Abstract: The effects of RU24722 (14,15‐dihydro‐20,21‐di‐noreburnamine‐14‐ol) on tyrosine hydroxylase in central catecholaminergic neurons were studied in rats treated with different quantities of the molecule, and a time course was done for the minimal dose that gave the maximal effect. RU24722 induced increases in tyrosine hydroxylase activities and specific protein content in noradrenergic cells of the locus ceruleus and decreased all these parameters in dopaminergic neurons of the substantia nigra and ventral tegmental area. The results pointed out that the specific activity of newly synthesized tyrosine hydroxylase in the loci cerulei was potentially greater but was not expressed “in vivo” except 7 days after injection. The phenotypic specificity and the time course pattern of the action could be considered as a consequence of an induction mechanism. The comparison of long‐term change in tyrosine hydroxylase values after pipe‐roxane, RU24722, clonidine, and combined RU24722‐clon‐idine treatment demonstrated that an activation during a few hours did not induce tyrosine hydroxylase in central noradrenergic neurons. Clonidine antagonized the activating effect of RU24722 following its injection but did not affect its long‐term induction properties.
Similar Time Course Changes in Striatal Levels of Glutamic Acid Decarboxylase and Proenkephalin mRNA Following Dopaminergic Deafferentation in the Rat
Authors: Vernier Philippe , Julien Jean‐Francois , Rataboul Pierre , Fourrier Odile , Feuerstein Claude , Mallet Jacques
Published: October 2006

Abstract: The time course changes in levels of mRNA encoding glutamic acid decarboxylase (GAD) and proenkephalin (PPE) was analyzed in the rat striatum following unilateral lesion of substantia nigra with 6‐hydroxydopamine. The levels of both GAD and PPE mRNAs increased after the dopaminergic deafferentation, reaching concomitantly a maximal twofold increase on day 25. Thereafter, the mRNA levels declined; at 4 months, the amount of PPE mRNA remained slightly elevated whereas GAD mRNA had returned to the control value, suggesting the action of a compensatory mechanism. We also observed a rise of glial fi‐brillary acidic protein mRNA level which reflects a reactive astrocytosis. In contrast, α‐tubulin mRNA level remained unchanged, indicating that no significant synaptogenesis occurs in this experimental situation. No obvious modification in mRNA levels was detected in the striatum contralateral to the lesion. These results highlight the role of the modulation of gene expression in adaptive processes to dopamine deficiency in striatal efferent pathways. Its relevance to the pathophysiology of Parkinson's disease is discussed.
Detection of the L2/HNK‐1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina
Authors: Dennis Roger D. , Antonicek Horst , Wiegandt Herbert , Schachner Melitta
Published: October 2006

Abstract: The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid‐containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin‐layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK‐1 (recognizing an epitope on human natural killer cells); and a human IgM M‐protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M‐protein. After de‐sulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK‐1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK.‐1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.
In Adrenal Medulla Synaptophysin (Protein p38) Is Present in Chromaffin Granules and in a Special Vesicle Population
Authors: Obendorf Dieter , Schwarzenbrunner Ulrike , Fischer‐Colbrie Reiner , Laslop Andrea , Winkler Hans
Published: October 2006

Abstract: We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one‐dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two‐dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the “large granules” and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (<10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.
Evaluation of the Ca2+ Concentration in Purified Nerve Terminals: Relationship Between Ca2+ Homeostasis and Synaptosomal Preparation
Authors: Verhage Matthijs , Besselsen Elly , Lopes Da Silva Fernando H. , Ghijsen Wim E. J. M.
Published: October 2006

Abstract: The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin‐2 and fura‐2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 ± 2 nM for Percoll (C‐band) synaptosomes, loaded at 30°C with the acetoxymethyl ester of fura‐2 (fura‐2‐AM)], a high stability during prolonged incubations at 37°C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30° or 37°C). Loading quin‐2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin‐2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura‐2 is preferable in synaptosomes, although above a critical fura2‐AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura‐2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37°C. This study shows the applicability of measuring steady‐state [Ca]i and dynamic [Ca]i changes quantitatively in fura‐2‐loaded synaptosomes. The possible involvement of different synaptosomal pools to explain the divergence in [Ca]i between different preparations and the interpretation in physiological terms of [Ca]i measured in synaptosomes are discussed.
Platelet‐Activating Factor Antagonist BN52021 Decreases Accumulation of Free Polyunsaturated Fatty Acid in Mouse Brain During Ischemia and Electroconvulsive Shock
Authors: Birkle Dale L. , Kurian Pawels , Braquet Pierre , Bazan Nicolas G.
Published: October 2006

Abstract: We have investigated the effects of the specific platelet‐activating factor (PAF; 1 ‐alkyl‐2–acetyl‐glycero‐phosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol‐4,5–bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave‐fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)‐treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS‐treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22: 6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury‐induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C‐mediated degradation of PIP2.
Vascular Perfusion and Blood‐Brain Glucose Transport in Acute and Chronic Hyperglycemia
Authors: Harik Sami I. , LaManna Joseph C.
Published: October 2006

Abstract: We studied the effects of acute and streptozotocin‐induced chronic hyperglycemia on regional brain blood flow and perfusion characteristics, and on the regional transport of glucose across the blood‐brain barrier in awake rats. We found (1) a generalized decrease in regional brain blood flow in both acute and chronic hyperglycemia; (2) that chronic, but not acute, hyperglycemia is associated with a marked and diffuse decrease in brain l‐glucose space; and (3) that chronic hyperglycemia does not alter blood‐to‐brain glucose transport. Taken together, these results suggest that in streptozotocin‐induced chronic hyperglycemia, there is a reduction in the proportion of perfused brain capillaries and/or an alteration in brain endothelial membrane properties resulting in decreased noncarrier diffusion of glucose.
Rapid Postmortem Increase in Extracellular Dopamine in the Rat Brain as Assessed by Brain Microdialysis
Authors: Vulto Arnold G. , Sharp Trevor , Ungerstedt Urban , Versteeg Dirk H. G.
Published: October 2006

Abstract: Extracellular dopamine (DA) and its metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanil‐lic acid (HVA) in rat nucleus accumbens were determined before and shortly following death using microdialysis. A maximal 400‐fold increase in the output of DA was observed within the first 5 min of death. DA output remained elevated over the following hour at a level of approximately 70‐fold above pre‐death values. In contrast to that of DA, DOPAC and HVA output gradually declined. Before death the extracellular DOPAC/DA ratio was about 250; after death this ratio dropped to 0.44 at 5 min. These observations may have important implications for experiments measuring the output of (endogenous) DA and its metabolites from brain tissue in vitro: autoregulation of, e.g., transmitter release and synthesis in vitro may be seriously disrupted by the observed depletion of transmitter storage granules.
Dopamine Receptor Stimulation Does Not Affect Phosphoinositide Hydrolysis in Slices of Rat Striatum
Authors: Kelly Eamonn , Batty Ian , Nahorski Stefan R.
Published: October 2006

Abstract: The effect of dopamine receptor stimulation on the accumulation of labelled inositol phosphates in rat striatal slices under basal and stimulated conditions was examined following preincubation with [3H]inositol. Incubation of striatal slices with the selective D‐1 agonist SKF 38393 or the selective D‐2 agonist LY 171555 for 5 or 30 min did not affect the basal accumulation of labelled inositol mono‐, bis‐, tris‐, and tetrakisphosphate. Resolution by HPLC of inositol trisphosphate into inositol‐1,3,4‐tris‐phosphate and inositol‐1,4,5‐trisphosphate isomers revealed that under basal conditions dopamine did not influence the accumulation of inositol‐1,4,5‐trisphosphate. Depolarisation evoked by KC1, or addition of the muscarinic receptor agonist carbachol, produced a marked increase in the accumulation of labelled inositol phosphates in both the presence and absence of lithium. Addition of dopamine did not reduce the ability of KC1 or carbachol to increase inositol phospholipid hydrolysis. In the presence of lithium, dopamine (100 μM) enhanced KCl‐stimuIated inositol phospholipid hydrolysis, but this effect appears to be mediated by α1 adrenoceptors because it was blocked by prazosin. SKF 38393 (10 μM) or LY 171555 (10 μM) also did not affect carbachol‐stimulated inositol phospholipid hydrolysis. These data, in contrast to recent reports, suggest that striatal dopamine receptors do not appear to be linked to inositol phospholipid hydrolysis.
A Point Mutation in the Coding Sequence of the β‐Hexosaminidase α Gene Results in Defective Processing of the Enzyme Protein in an Unusual GM2‐Gangliosidosis Variant
Authors: Nakano Takeshi , Muscillo Michele , Ohno Kousaku , Hoffman Andrew J. , Suzuki Kunihiko
Published: October 2006

Abstract: cDNA clones were isolated from cultured fibro‐blasts of a patient previously reported as having GM2‐gangliosidosis due to defective processing of the precursor β‐hexosaminidase α chain. Sequence analysis of a clone containing the entire protein coding sequence showed a single nucleotide substitution, from G to A, at nucleotide residue no. 1444, which resulted in a change in amino acid residue no. 482, from the normal glutamic acid to lysine. This transversion was confirmed in two other cDNAs from the same unamplified library. The results collectively indicate that the change from the strongly negative to strongly positive charge at amino acid residue no. 482 is responsible for the defective processing of the enzyme in this patient.
Increase in Extracellular Dopamine in the Striatum During Cerebral Ischemia: A Study Utilizing Cerebral Microdialysis
Authors: Slivka Actam , Brannan Timothy S. , Weinberger Jesse , Knott Peter J. , Cohen Gerald
Published: October 2006

Abstract: Unilateral ligation of the left common carotid artery in anesthetized Mongolian gerbils resulted in a steep rise in extracellular dopamine in the ipsilateral striatum in 9 out of 19 animals. Extracellular dopamine was measured by cerebral dialysis in vivo and reached a peak of 0.19 mM at 40 min. At the same time, the level of homovanillic acid fell, whereas the levels of ascorbate and 3,4‐dihydroxyphenylacetic acid remained relatively constant. In a separate group of animals studied with a combined dialysis/electrochemistry probe, a rise in the in vivo chronoamperometric signal in three out of six animals correlated with a rise in extracellular dopamine. The number of animals responding in these experiments (roughly 50%) corresponds to the frequency of incompetent Circle of Willis, as well as literature reports of the frequency of signs of stroke in unanesthetized gerbils. These results show a remarkable accumulation of dopamine in extracellular fluid in response to cerebral ischemia. Released dopamine appears to be responsible for the elevated in vivo electrochemical signal previously reported.
Potentiation by Kainate of Excitatory Amino Acid Release in Striatum: Complementary In Vivo and In Vitro Experiments
Authors: Young A. M. J. , Crowder J. M. , Bradford H. F.
Published: October 2006

The effect of kainate on extracellular levels of amino acids in corpus striatum was investigated in vitro and in vivo, to elucidate the mechanism underlying its neurotoxicity. Kainate increased extracellular glutamate and aspartate in both striatal slices in vitro and intact striatum in vivo, as previously reported. Both in vitro and in vivo, DL‐threo‐3‐hydroxyaspartate increased extracellular glutamate and aspartate levels (to between 150 and 200% of basal), and also enhanced their kainate‐evoked release. The action of kainate in vivo was reduced by prior frontal decortication, whereas in vitro the kainate‐evoked responses were only slightly reduced by tetrodotoxin. and remained above control values. These results confirm that kainate increases extracellular glutamate and aspartate. and provide evidence that this is due to synaptic release evoked by an action on receptors on glutamatergic neurone terminals. These findings may be relevant to the understanding of epilepsy.
Effects of ATP on Phosphatidylinositol‐Phospholipase C and Inositol 1‐Phosphate Accumulation in Rat Brain Synaptosomes
Authors: Huang H.‐M. , Sun G. Y.
Published: October 2006

Incubation of rat brain synaptosomes prelabeled with [2‐3H]inositol resulted in a time‐dependent release of labeled inositol 1 ‐phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1‐phosphate formation three‐ to fivefold. Using [l‐14C]arachidonoyl‐phosphatidylinositol which was introduced into saponin‐permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (˜20 μM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2‐3H]inositol‐phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1‐phosphate, but also inhibited the conversion of inositol 1‐phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol‐phospholipase C. Inhibition of the conversion of inositol 1‐phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor‐mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.
Localization of Glycogen Synthase in Brain
Authors: Inoue Nobuhiro , Matsukado Yasuhiko , Goto Satoshi , Miyamoto Eishichi
Published: October 2006

Antisera against glycogen synthase from canine brain were prepared and used for investigation of the localization of the enzyme in the brain. Antisera cross‐reacted only with the 88‐kilodalton protein that is the subunit of brain glycogen synthase. Immunoreactivity of glycogen synthase was universally distributed in all regions of the brain, although hippocampus, cerebral cortex, caudatoputamen, and cerebellar cortex had relatively high immunoreactivity. Light microscopic examination revealed that the immunoreactivity was found in all cell types, such as neurons in several regions, astrocytes, ependymal cells surrounding the ventricle, oligodendrocytes, and epithelial cells of the choroid plexus in the ventricle. Immunoreactive intensity was more prominent in neurons than glial cells. Immunostaining may be a useful tool for investigation of the state of glycogen metabolism under normal and pathological conditions.
Fatty Acid Transport Through the Blood‐Brain Barrier
Authors: Spector Reynold
Published: October 2006

Across the cerebral capillaries, the anatomical locus of the blood‐brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate‐to‐brain fatty acid transport, we found that both [14C]octanoic and [14C]myristic acids were transported through the blood‐brain barrier in vivo, in large part, by a specific, probenecid‐sensitive transport system. However, the transport of [14C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability‐surface area constant was higher for myristic acid (4.8 × 10‐‐2× s‐1) than for octanoic and linoleic acids (1.5 and 1.2 × 10‐‐2× s‐1, respectively). Approximately 70, 30, and 25% of the [14C]myristic, [14C]octanoic, or [14C]linoleic acids, respectively, were extracted from the perfusate.
Biochemical and Autoradiographic Study of Cerebral Protein Synthesis with [18F]‐ and [14C]Fluorophenylalanine
Authors: Bodsch W. , Coenen H. H. , Stocklin G. , Takahashi †K. , Hossmann K.‐A.
Published: October 2006

Fluorine‐18‐labeled ortho or para isomers of l‐fluorophenylalanine were used in double‐label experiments together with l‐[3H]phenylalanine for amino acid incorporation into cerebral proteins of Mongolian gerbil brain. It was demonstrated by qualitative regional comparison of the 18F and 3H autoradiographic images that l‐p‐[18F]fluo‐rophenylalanine is incorporated into proteins and exhibits a regional cerebral protein synthesis pattern. To a minor extent, l‐p‐fluorophenyl[3–14C]alanine and l‐o‐[18F]fluo‐rophenylalanine are hydroxylated in vivo to form labeled tyrosine or tyrosine analogues that are incorporated into cerebral proteins as well. The advantage and validity of the application of l‐p‐[18F]fluorophenylalanine with positron emission tomography for noninvasive studies of cerebral protein synthesis in humans are evaluated on the basis of an experimental animal approach.
Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey
Authors: Hirano Arlene A. , Greengard Paul , Huganir Richard L.
Published: October 2006

Abstract: The rat CNS contains high levels of tyrosine‐specific protein kinases that specifically phosphorylate the tyrosine‐containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis‐Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern vas observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well‐characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity‐purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine‐containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine‐specific protein kinase activity, and in phosphotyrosine‐containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.
Effects of Daily Cocaine and Morphine Treatment on Somatodendritic and Terminal Field Dopamine Release
Authors: Kalivas Peter W. , Duffy Patricia
Published: October 2006

Abstract: Daily injections of cocaine or morphine into rodents produces behavioral sensitization such that the last daily injection results in a greater motor stimulant effect than the first injection. To evaluate a role for brain dopamine in behavioral sensitization to cocaine and morphine, tissue slices from the ventromedial mesencephalon (containing dopamine cell bodies), the nucleus accumbens, and striatum (dopamine terminal fields) were obtained from rats pretreated with daily cocaine, morphine, or saline 2–3 weeks earlier. When the tissue slices were depolarized by increasing potassium concentration in the superfusate, the release of endogenous dopamine from the ventromedial mesencephalon of cocaine‐ and morphine‐pretreated rats was significantly decreased. In contrast, the release of dopamine from the nucleus accumbens and striatum was either unaltered or slightly enhanced in rats pretreated with cocaine and morphine. When dopamine was released by amphetamine, a significant decrease in dopamine release from the ventromedial mesencephalon of cocaine‐pretreated rats was measured. No other significant changes were measured after amphetamine‐induced release. It is postulated that the decrease in dopamine release from the ventromedial mesencephalon of cocaine‐ and morphinesensitized rats results in less somatodendritic autoreceptor stimulation, and thereby produces an increase in dopamine neuronal activity.
Rat Brain Apotransketolase: Activation and Inactivation
Authors: Jeyasingham Marina D. , Pratt Oliver E.
Published: October 2006

Abstract: Kinetic analysis of the combination of rat brain apotransketolase with thiamine diphosphate suggested that the enzyme exists in more than one form. One part of the apoenzyme reacted rapidly with thiamine diphosphate to reconstitute the holoenzyme, but another part appeared to combine only relatively slowly. In addition, an apparently irreversible further change took place, the apoenzyme being converted progressively to a form which apparently could not be activated by thiamine diphosphate. The relative proportions of the three forms i.e., that reacting rapidly, slowly, or not at all with thiamine diphosphate, were a function of the duration and conditions of storage, with the proportion of the apoenzyme form which reacted rapidly with thiamine diphosphate decreasing progressively. The findings reported here provide a possible explanation for problems various workers have encountered in attempting to evaluate Michaelis constants for the reaction of thiamine diphosphate with apotransketolase. Key Words: Transketolase—Brain—Wernicke‐Korsakoff syndrome—Thiamine. Jeyasingham M. D. and Pratt O. E. Rat brain apotransketolase: Activation and inactivation.
Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly
Authors: Yamamoto Hideyuki , Saitoh Yoshiki , Fukunaga Kohji , Nishimura Hiroshi , Miyamoto Eishichi
Published: October 2006

Abstract: Protein phosphatase C was purified 140‐fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP‐dependent protein kinase (cyclic AMP‐kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor‐2. Protein phosphatase C dephosphorylated microtubule‐associated protein 2 (MAP2), τ factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin‐dependent protein kinase (calmodulin‐kinase) and the catalytic subunit of cyclic AMP‐kinase. The properties of dephosphorylation of MAP2, τ factor, and tubulin were compared. The Km values were in the ranges of 1.6–2.7 μM for MAP2 and τ factor. The Km value for tubulin decreased from 25 to 10–12.5 μM in the presence of 1.0 μM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, τ factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and τ factor by calmodulin‐kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch‐off mechanism of both Ca2+/calmodulin‐dependent and cyclic AMP‐dependent regulation of microtubule formation.
Phorbol Esters Attenuate Glutamate‐Stimulated Inositol Phospholipid Hydrolysis in Neuronal Cultures
Authors: Canonico P. L. , Favit A. , Catania M. V. , Nicoletti F.
Published: October 2006

Abstract: The phorbol diesters 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and phorbol‐12,13‐dibutyrate, but not 4–α‐phorbol‐didecanoate, inhibited the stimulation of inositol phospholipid hydrolysis by excitatory amino acids and carbamylcholine in primary cultures of cerebellar neurons. This inhibition was mimicked by the synthetic diacylglycerol 1,2‐dioleoyl‐rac‐glycerol (DOG) and was selective for a specific glutamate‐phosphoinositide receptor subtype (GP2 receptor) activated by glutamate and quis‐qualate. TPA was nearly inactive in inhibiting the stimulation of inositol phospholipid hydrolysis by N‐methyl‐d‐aspartate, a selective agonist of the GP1 receptor. Phorbol diesters and DOG attenuated the stimulation of inositol phospholipid hydrolysis by glutamate and quisqualate also in cerebellar slices from 9–15‐day‐old rats; however, using this preparation, their action was weak and required high concentrations (> 1 μM). The inhibition of signal transduc‐tion by phorbol diesters was not consequent to a reduced binding of glutamate to its membrane recognition sites. In fact, TPA induced only a small increase in the KD but no change in the Bmax of [3H]glutamate binding in cerebellar membranes. Phorbol diesters may act to inhibit specific GTP‐binding proteins or particular molecular forms of phosphoinositidase C associated with GP2 or muscarinic cholinergic receptors.
Treatment with GM1 Ganglioside Restores Striatal Dopamine in the 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Treated Mouse
Authors: Hadjiconstantinou M. , Neff N. H.
Published: October 2006

Abstract: 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), 30 mg/kg i.p. daily for 7 days, was administered to mice. This dosage regimen resulted in an ∼50% reduction of striatal dopamine (DA) level. Chronic administration of GM1 ganglioside (II3NeuAc‐GgOse Cer), beginning between 1 to 4 days after terminating MPTP dosing, resulted in partial restoration of the striatal DA level. From dose‐ and time‐response studies, it appeared that 30 mg/kg i.p. of GM1 administered daily for ∼23 days resulted in an ∼80% restoration of the DA level and complete restoration of the 3,4‐dihydroxyphenylacetic acid (DOPAC) content. This dosage of GM1 also restored the turnover rate of DA in the striatum to near normal. Discontinuing GM1 treatment resulted in a fall of DA and DOPAC levels to values found in mice treated with MPTP alone. There was no evidence for regeneration of nerve terminal amine reuptake in the GM1‐treated mice as evaluated by DA uptake into synaptosomes. Our biochemical findings in animals suggest that early GM1 ganglioside treatment of individuals with degenerative diseases of dopaminergic nigrostriatal neurons might be fruitful.
Alteration in Fatty Acid Composition of Adult Rat Brain Capillaries and Choroid Plexus Induced by a Diet Deficient in n‐3 Fatty Acids: Slow Recovery After Substitution with a Nondeficient Diet
Authors: Homayoun P. , Durand G. , Pascal G. , Bourre J. M.
Published: October 2006

Wistar rats were fed for three generations with a semisynthetic diet containing either 1.5% sunflower oil (940 mg% of C18:2n‐6, 6 mg% of C18:3n‐3) or 1.9% soya oil (940 mg% of C18:2n‐6, 130 mg% of C18:3n‐3). At 60 days of age, the male offspring of the third generation were killed. The fatty acyl composition of isolated capillaries and choroid plexus was determined. The major changes noted in the fatty acid profile of isolated capillaries were a reduction (threefold) in the level of docosahexaenoic acid and, consequently, a fourfold increase in docosapentaenoic acid in sunflower oil‐fed animals. The total percentage of poly‐unsaturated fatty acids was close to that in the soya oil‐fed rats, but the ratio of n‐3/n‐6 fatty acids was reduced by threefold. In the choroid plexus, the C22:6n‐3 content was also reduced, but by 2.6‐fold, whereas the C22:5n‐6 content was increased by 2.3‐fold and the ratio of n‐3/n‐6 fatty acids was reduced by 2.4‐fold. When the diet of sunflower oil‐fed rats was replaced with a diet containing soya oil at 60 days of age, the recovery in content of n‐6 and n‐3 fatty acids started immediately after diet substitution; it progressed slowly to reach normal values after 2 months for C22:6n‐5 and 2.5 months for C22:6n‐3. The recovery in altered fatty acids of choroid plexus was also immediate and very fast. Recovery in content of C22:5n‐6 and C22:6n‐3 was complete by 46 days after diet substitution.
Dose‐Dependent, K+‐Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+‐Dependent
Authors: Philibert Robert A. , Rogers Keith L. , Allen Albert J. , Dutton Gary R.
Published: October 2006

The K+‐stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7–9‐day‐old rats was studied at 16–18 days in vitro using HPLC analysis. Taurine efflux was dose‐dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K.+‐stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3–5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0–5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.
Possible Involvement of Pertussis Toxin Substrates (Gi, Go) in Desipramine‐Induced Refractoriness of Adenylate Cyclase in Cerebral Cortices of Rats
Authors: Okada Fumihiko , Tokumitsu Yukiko , Ui Michio
Published: October 2006

To evaluate the efficiency of coupling between β‐receptor and adenylate cyclase catalyst via a GTP‐binding protein, Gs, in the brain membrane two parameters were employed: a β‐agonist‐induced increase in the membrane GTP‐dependent adenylate cyclase activity and a β‐agonist‐induced shortening of the lag time preceding the onset of the steady‐state activation by guanyl‐5′‐yl‐β‐γ‐imidodiphosphate [Gpp(NH)p] of the membrane cyclase. Both parameters showed lower values in membranes from desipramine‐treated rats compared with untreated rats. Thus, coupling of β‐adrenergic receptors to adenylate cyclase in the brain membrane was impaired by the desipramine treatment. Rats once injected intraventricularly with isletactivating protein (IAP), pertussis toxin, were subjected to desipramine treatment, for the purpose of studying effects of another kind of the GTP‐binding protein (Gi), which loses its function as a signal transducer on being ADP‐ribosylated selectively by the toxin. IAP treatment did not impair the β‐receptor coupling by itself, since neither of the above two parameters for the coupling were reduced by IAP treatment. Moreover, the first parameter was normalized. though the second one was not, by superimposition of the IAP treatment upon the desipramine‐treated rats. It seems likely, therefore, that Gi interacts with a Gs‐adenylate cyclase coupling in an inhibitory fashion in brain membranes. The desensitization might be overcome when the inhibitory interaction of Gi on the subsequent process is attenuated by IAP treatment.
Three Distinct Types of Monoclonal Antibodies After Long‐Term Immunization of Rats with Mouse Nerve Growth Factor
Authors: Cattaneo Antonino , Rapposelli Bruna , Calissano Pietro
Published: October 2006

Abstract: This article reports the results of a systematic investigation of the different types of antibodies produced in the course of a long‐term immunization of rats with mouse nerve growth factor (NGF). We have characterized three types of monoclonal antibodies, namely: (1) antibodies that bind to NGF and inhibit its binding to target cells and its biological activity in culture (type A); (2) antibodies that bind to and precipitate NGF but do not inhibit its binding to target cells or its biological activity (type B); (3) antibodies that fail to recognize NGF itself, but inhibit nonetheless its binding to target cells (type C). These antibodies bind to an antigen present on NGF target cells and not on rat fibroblasts lacking NGF receptor. They appear thus to be antiidiotypic antibodies directed against the NGF receptor, developed as a consequence of the long‐term immunization with NGF.
Increases in Striatal and Hippocampal Impedance and Extracellular Levels of Amino Acids by Cardiac Arrest in Freely Moving Rats
Authors: Korf J. , Klein H. C. , Venema K. , Postema F.
Published: October 2006

Abstract: The time course of changes in the tissue impedance and the levels of extracellular transmitter and non‐transmitter amino acids was studied in the striatum and hippocampus of the unanesthetized rat after cardiac arrest. Electrodes were implanted for the continuous measurement of tissue impedance so that a measure of the volume of extracellular space was provided. Alternatively, bilateral dialysis probes were used for monitoring levels of extracellular amino acids in subsequent 30‐s samples using an automated precolumn derivatization technique for reversed‐phase HPLC analysis and fluorimetric detection. The impedance started to rise ∼1.2 min following cardiac arrest, increased rapidly during the first 5 min, and increased almost linearly thereafter. After 15 min, a decrease of ∼50% in the extracellular space was calculated. The impedance rose more steeply in the striatum than in the hippocampus. The extracellular levels of taurine, which increased >300% within 5 min after cardiac arrest, most closely resembled the time course of the change in impedance. Glutamate and aspartate levels did not increase until 5 min after circulatory arrest, and at 15 min they had risen to a level of 465 and 265% for the striatum and 298 and 140% for the hippocampus of the resting release, respectively. The release of γ‐aminobutyric acid (GABA) was multiphasic and did not resemble that of any of the other—putative—transmitter amino acids. Fifteen minutes after cardiac arrest, the levels of GABA were 617 and 774% of the resting release in the striatum and hippocampus, respectively. Glycine and ala‐nine efflux substantially increased (232 and 151% in striatum and 141 and 154% in hippocampus, respectively) 15 min postmortem, whereas the glutamine level was slightly increased and levels of asparagine, histidine, threonine, ethanolamine, serine, arginine, and tyrosine were inconsistently higher in the two brain regions. At this time, the extracellular levels of glutamate, GABA, and aspartate were only slightly lower, as expected from the tissue levels and from levels of the other amino acids, an observation indicating that all the amino acids may diffuse through postmortem brain tissue to a nearly similar extent. This study provides evidence that extracellular levels of taurine reflect changes in distribution of electrolytes (and in membrane potentials), that the postmortem release of transmitter amino acids is multiphasic with a delay of at least 1 min, that postmortem shrinkage in extracellular volume cannot account for the increase in the content of transmitter amino acids in the dialysate, and that the massive overflow of glutamate, aspartate, GABA, and taurine seen during ischemia is the result of both release and the failure of uptake. Possible implications of the present findings for excitotoxic damage of the brain are discussed.
Specificity and Reversibility of the Inhibition by HgCl2 of Glutamate Transport in Astrocyte Cultures
Authors: Brookes N.
Published: October 2006

Abstract: Inhibition of glutamate transport is a potential indirect cause of excitotoxic damage by glutamate in the CNS. The mercuric ion, the form in which metallic mercury vapor is believed to exert its neurotoxic action, is a known inhibitor of amino acid transport. This study examines the specificity with which HgCl2 inhibits glutamate transport in mouse cerebral astrocytes by means of comparative measurements of 2‐deoxyglucose uptake. Uptake of 2‐deoxyglucose is an index of glucose utilization that reflects the function of Na+,K+‐ATPase and hexokinase, and is sensitive to Na+ entry. The kinetic parameters, ionic dependence, and substrate specificity of glutamate transport in these astrocyte cultures were consistent with the commonly occurring system designated XAg. Acute exposure to 0.5 μM HgCl2 inhibited by 50% the initial rate of glutamate transport but did not affect 2‐deoxyglucose uptake. Glutamate transport was not detectably inhibited by Al2+, Pb2+, Co2+, Sr2+, Cd2+, or Zn2+ (10 μM as chlorides). The inhibitory action of 0.5 μM HgCl2 on glutamate transport was rapidly reversible. The action of 1–2 μM HgCl2 was progressive when exposures were extended to 1–3 h, and was more slowly reversible. These results suggest that Hg2+ can impair glial glutamate transport reversibly at exposure levels that do not compromise some other vital cell functions.
Stereoselective Nicotine‐Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation
Authors: Rapier Catherine , Lunt George G. , Wonnacott Susan
Published: October 2006

Abstract: Using a sensitive perfusion system we have studied the nicotine‐induced release of [3H]dopamine ([3H]DA) from striatal synaptosomes. Nicotine‐evoked release was concentration dependent with an EC50 of 3.8 μM. The response to 1 μM nicotine was comparable to that to 16 mM K+ 10 μM veratridine evoked a larger response. All three samuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 μM (–)‐nicotine (100 μl) at 30‐min intervals resulted in similar levels of [3H]DA release; higher concentrations of (–)‐nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100‐fold higher concentration of (+)‐nicotine was necessary to evoke the same level of response as 1 μM (–)‐nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high‐affinity nicotine binding sites described in mammalian brain.
Lysosulfatide (Galactosylsphingosine‐3‐O‐Sulfate) from Metachromatic Leukodystrophy and Normal Human Brain
Authors: Rosengren Birgitta , Fredman Pam , Mansson Jan‐Eric , Svennerholm Lars
Published: October 2006

Abstract The glycosphingolipid pattern was examined in three cases of late infantile metachromatic leukodystrophy (MLD): one with a relatively short (2.5 years), one with a long (7.8 years), and one with a very long (13.2 years) survival time. All values were compared with those of age‐matched normal controls. The cerebroside concentration was reduced to 25, 12, and 4%, respectively, in the MLD white matter, whereas the sulfatide concentration was increased up to 200% of the control value. The yield of myelin was reduced to <15% in the early case and to <3 and 1%, respectively, in the two later cases. There was no sign of increased sulfatide proportion in the myelin. The ganglioside pattern was normal in cerebral gray matter, but in the white matter, contents of gangliosides of the lacto series were significantly increased, in particular, the ganglioside suggested by us as being characteristic of reactive astrocytosis. For the first time, lysosulfatide was identified in MLD and normal human brains by mass spectrometry and radioimmunoaffinity TLC using specific monoclonal antibody. Its quantity was found to be similar in normal and MLD brains. These findings support our postulation that the lysoglycosphingolipids are synthesized de novo from sphingosine and that they do not play a key role in pathogenetic mechanisms.
Further Physicochemical Characterization of the Novel Human Brain Protein h3
Authors: Bollengier F. , Beeckmans S. , Mahler A. , Kanarek L.
Published: October 2006

Abstract Recently we reported the isolation and partial biochemical characterization of the novel polypeptide h3 from human brain and liver. In this report, the physicochemical characterization is further established by the use of several analytical methods. The following results were obtained: the ultraviolet absorption spectrum is not influenced by pH, and the circular dichroism (CD) spectrum reveals that this protein has no a‐helices, whereas ± 25% of the polypeptide chain is found to be folded as a β‐pleated sheet structure. Neither the conformation of h3 as assessed by CD nor the titration kinetics of sulfhydryl groups with Ellman's reagent are affected by the presence of the ions K+, Na+, Ca2+, and Mg2+. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in a β‐mercaptoethanol gradient and Cleveland sequential SDS‐PAGE showed that the frequent formation of h3 polymers and doublets, as observed earlier, is almost exclusively due to disulfide bonding.
Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2‐Oxoglutarate
Authors: Kihara Minoru , Kubo Takao
Published: October 2006

Abstract The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2‐oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+‐dependent stimulus‐evoked release of endogenous glutamate, γ‐aminobutyric acid (GABA), and β‐alanine in all regions examined. AOAA (10‐4 and 10‐3M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. l‐Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2‐Oxoglutarate (10‐3M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10‐4M) almost abolished the activities of AAT in all regions studied. AOAA (10‐4 and 10‐3M) increased the stimulus‐evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus‐evoked release of β‐alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.