bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Cycle‐Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures
Authors: Langan Thomas J. , Volpe Joseph J.
Published: October 2006

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/ vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose‐dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein‐poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low‐density lipoprotein (LDL) to raise the cholesterol concentration 80‐fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more. This temporal aspect of the requirement for mevalonate was unaffected by the presence of a 90‐fold excess of cholesterol, provided as LDL. Thus, these data establish a requirement for mevalonate or for a presumably nonsterol derivative of mevalonate for the occurrence of DNA synthesis in synchronized glial primary cultures, and demonstrate that this requirement is expressed at a specific time in the cell cycle.
Feeding‐Related Circadian Variation in tele‐Methylhistamine Levels of Mouse and Rat Brains
Authors: Oishi Ryozo , Itoh Yoshinori , Nishibori Masahiro , Saeki Kiyomi
Published: October 2006

Circadian changes in the brain histamine (HA) and tele‐methylhistamine (t‐MH) levels were studied in mice and rats after adaptation to an alternating 12‐h light/dark cycle (lights on at 0600). Although there was no significant circadian fluctuation of the brain HA levels, the levels of t‐MH, a major metabolite of brain HA, showed a marked circadian variation. In mice, the t‐MH levels were about 80 ng/g from 1200 to 1800 but about two times higher values were obtained from 2400 to 0600 of the next morning. In rats, the t‐MH levels ranged from 24 to 28 ng/g at 0600 and 1200, slightly increased at 1800, and reached at 2400 a peak twice as high as the levels seen during the light period. The t‐MH levels again rapidly decreased during the subsequent 3 h. In mice fasted from 1200, the t‐MH levels did not increase during the period of darkness. When mice were fed at 1200 after a 24‐h fast, a significant increase in the t‐MH levels was observed at 1800. There was no significant circadian variation of the HA and t‐MH levels in the plasma of mice and rats. These results suggest that circadian variation in brain t‐MH levels is related to feeding and possible subsequent changes in elimination of t‐MH from the brain and/or turnover of HA in the brain. This phenomenon should be given due attention when HA dynamics in the brain are being assessed.
Changes in Extracellular Amino Acids During Soman‐and Kainic Acid‐Induced Seizures
Authors: Wade John V. , Samson Fred E. , Nelson Stanley R. , Pazdernik Thomas L.
Published: October 2006

Extracellular amino acid levels in the rat piriform cortex, an area highly susceptible to seizure‐induced neuropathology, were determined by means of intracranial microdialysis. Seizures were induced by systemic administration of either soman (O‐1,2,2‐trimethylpropyl methylphosphonofluoridate), a potent inhibitor of acetylcholinesterase, or the excitotoxin kainic acid. Extracellular glutamate levels increased in animals with seizures shortly after administration of either convulsant, but this change was statistically significant only in the case of soman‐treated animals. Extracellular taurine levels increased markedly, reaching two‐and fourfold baseline levels during the second hour of soman‐and kainic acid‐induced seizures, respectively. Taurine levels did not increase in the subpopulation of soman‐treated animals without seizures, a finding indicating that elevation of extracellular taurine level is seizure related. Thus, we propose that taurine efflux may be a physiological cellular response to neuronal changes produced by excito‐toxic chemicals, either directly or as a consequence of seizures.
Delayed Cerebroventricular Metabolism of [125I]Angiotensins in the Spontaneously Hypertensive Rat
Authors: Wright John W. , Sullivan Margaret J. , Bredl Charles R. , Hanesworth Jodie M. , Cushing Laurie L. , Harding Joseph W.
Published: October 2006

This study was designed to evaluate the hypothesis that impaired brain angiotensin signal termination contributes to the sustained blood pressure elevations noted in the genetically hypertensive rat model of human essential hypertension. A technique that combined the intracerebroventricular injection of [125I]angiotensins, followed by focused microwave fixation to stop all peptidase activity and subsequent HPLC analyses, was used for determining half‐lives of [I25I]angiotensin II and [125I]angiotensin III in the ventricular space. The results indicate that the spontaneously hypertensive rat evidenced significantly longer half‐lives for intracerebroventricularly injected [125I]angiotensin II over those measured for the Wistar‐Kyoto and Sprague‐Dawley normotensive rat strains: 45.0, 27.2, and 25.0 s, respectively. This was also true for intracerebroventricularly administered [125I]angiotensin III: 19.5, 11.4, and 9.0 s, respectively. These results support the notion that a dysfunction in central aminopeptidase activity in the spontaneously hypertensive rat may result in prolonged half‐lives of endogenously synthesized angiotensins II and III, which are known to serve as ligands at central angiotensin receptors responsible for the control of cardiovascular function. The extended half‐lives of these ligands may contribute to the sustained elevations in blood pressure observed in this animal model.
Tetrameric Detergent‐Soluble Acetylcholinesterase from Human Caudate Nucleus: Subunit Composition and Number of Active Sites
Authors: Gennari K. , Brunner J. , Brodbeck U.
Published: October 2006

Abstract: Purified tetrameric detergent‐soluble acetylcho‐linesterase (DS‐AChE) from human caudate nucleus was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the absence as well as in presence of a reducing agent. Staining for protein revealed a main band at 66,000 daltons (light monomer) with additional bands at 78,000 daltons (heavy monomer) as well as 130,000 and 150,000 daltons (light and heavy dimers). The same four polypeptides were also detected by Western blotting and by autoradiography of [3H]diisopropylphosphoryl enzyme. Labeling of the enzyme with 3‐trifluoromethyl‐3‐(m‐[125I]‐iodophenyl)diazirine showed that the heavy monomer contained the hydrophobic anchor of the enzyme, whereas the light monomer was practically not labeled. The hydrophobic anchor was susceptible to proteolytic degradation by proteinase K. The functional molarity of DS‐AChE was determined by two independent methods. Four active sites for the tetrameric enzyme were estimated. The turnover number per site was 1.7 ± 107 mol of acetylthiocholine iodide hydrolyzed ± h−1.
Calcium‐Dependent and‐Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry
Authors: Nicholls David G. , Sihra Talvinder S. , Sanchez‐Prieto Jose
Published: October 2006

Abstract: An enzyme‐linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea‐pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35–0.39 nmol min−1 mg of protein−1 and is not significantly affected by external Ca2+. KC1 depolarization (30 mM KCI) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein−1. The final extent of Ca2+‐dependent release amounts to 1.9 nmol mg of protein−1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30°C for 2 h before depolarization leads to a decrease in Ca2+‐independent release and an increase in Ca2+‐dependent release, consistent with an intrasynaptosomal relocation of the amino acid.
Characterization of the Exocytotic Release of Glutamate from Guinea‐Pig Cerebral Cortical Synaptosomes
Authors: Sanchez‐Prieto Jose , Sihra Talvinder S. , Nicholls David G.
Published: October 2006

Abstract: A continuous enzyme‐linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea‐pig cerebrocortical synaptosomes. Ca2+‐dependent release can be induced not only by K+, but also by the Na+ channel activator veratridine and the Ca2+ ionophore ionomycin. K+‐induced release can be inhibited by the Ca2+ channel inhibitor verapamil. Sr2+ and Ba2+ substitute for Ca2+ in promoting K+‐induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy‐anide p‐trifluoromethoxyphenylhydrazone causes a time‐dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca2+‐independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca2+‐dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.
Accumulation of Galactosylsphingosine (Psychosine) in the Twitcher Mouse: Determination by HPLC
Authors: Shinoda Hisaharu , Kobayashi Takuro , Katayama Mono , Goto Ikuo , Nagara Hitoshi
Published: October 2006

Abstract: We developed a sensitive and simple procedure for determination of galactosylsphingosine (psychosine), using HPLC. The method involved extraction of lipids, separation by cation‐exchange and C18 reverse‐phase columns, and derivatization with o‐phthalaldehyde. The fluorescent galactosylsphingosine was detected by HPLC. The amount of galactosylsphingosine was accurately assayed by simultaneous determination of glucosylsphingosine, as the internal standard. The detection limit was 0.5 ng/assay tube, and the quantitative range of the method was up to 750 ng. This procedure was applied to tissue from the twitcher mouse, an animal model of human globoid cell leukodystrophy, as well as tissue from normal and carrier mice. In the latter mice, a small amount of galactosylsphingosine was detected in the spinal cord (21.6–37.2 ng/100 mg wet weight) but not in the cerebrum and sciatic nerve. Marked accumulation of galactosylsphingosine was noted in the nervous tissues of the twitcher strain, even on postnatal day 4. The concentration of galactosylsphingosine was greater in the peripheral than in central nervous tissues. The spinal cord and brainstem contained more galactosylsphingosine than did the cerebrum and cerebellum. The concentration increased with age from 764 ng/100 mg in the sciatic nerve at 4 days to 5,910 ng/100 mg at 37 days. These data correlate well with the pathological changes; tissues containing higher concentrations of galactosylsphingosine show earlier and more severe pathological changes than those containing lower concentrations, thereby indicating the close link of galactosylsphingosine to the pathogenesis of the twitcher mouse.
Effects of Antidepressant Drug Treatments on α2‐Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes
Authors: William Janet R. Me , Campbell Iain C.
Published: October 2006

Abstract: : KC1 (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+‐dependent manner; this release was attenuated by clonidine (0.01–100 μM). Changes in the release of [3H]NA and the functional status of α2‐adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 μM) or the potentiating effects of yohimbine (1 μM) on K+‐evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous 4‐K+‐evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+‐evoked release from the DMI‐treated tissue. Attenuation of K+‐evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI‐treated tissue remained significantly reduced although total percent and K+‐evoked [3H]NA release were unchanged. The K+‐evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+‐evoked [3H]NA release by clonidine in both chronic antidepressant‐treated tissues was not significantly changed. It is concluded that the functional sensitivity of α2‐adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+‐evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants. This reduction in the potentiation induced by yohimbine may be a result of a decrease in the biophase concentration of NA at α2‐adrenoceptors in the hypothalamus of the chronic antidepressant drug‐treated samples.
Effects of Injury on the Indoleamines in Cerebral Cortex
Authors: Pappius Hanna M. , Dadoun Ralph
Published: October 2006

Abstract: : It was shown previously that focal cortical freezing lesions in rats cause widespread depression of local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere. This was interpreted as reflecting functional depression. The underlying mechanisms were postulated to involve alterations of bioorganic amine systems. Accordingly, levels of serotonin (5‐HT), its metabolite 5‐hydroxyin‐dole acetic acid (5‐HIAA), and its precursor tryptophan were determined by an HPLC method with electrochemical detection in frontoparietal cortical areas of both hemispheres at 4 h and 1, 3, 6, 8, and 10 days after a unilateral cortical freezing lesion. The 5‐HT content was significantly lower than normal in the lesioned hemisphere only at 24 h, whereas the 5‐HIAA level peaked at 24 h but was significantly elevated above normal values between 4 h and 6 days after lesioning. No changes were noted in 5‐HT and 5‐HIAA contents in the hemisphere contralateral to the lesion. These results indicate that cortical 5‐HT metabolism is increased throughout the lesioned hemisphere of a focally injured brain. The increase in tryptophan content of the lesioned brain appeared to have a time course more closely related to previously demonstrated changes in cortical LCGU than to the increase in 5‐HIAA content.
Effects of Cerebral Ischemia on [3H]Inositol Lipids and [3H]Inositol Phosphates of Gerbil Brain and Subcellular Fractions
Authors: Strosznajder Joanna , Wikiel Hanna , Sun Grace Y.
Published: October 2006

Abstract: Intracerebral injection of [3H]inositoi into gerbil brain resulted in labeling of phosphoinositides and inositolphosphates in various subcellular membrane fractions. Phosphatidylinositol (PI) comprised >90% of the radioactivity of inositol lipids. However, the level of labeled poly‐PI (with respect to PI) was higher in synaptosomes than in other membrane fractions. Ischemia induced in gerbils by ligation of the common carotid arteries resulted in a 30% decrease in labeled poly‐PI in brain homogenates and this decrease was largely attributed to the poly‐PI in synaptosomes (50% decrease). Among the inositol phosphates, the ischemia induction resulted in a decrease in labeling of inositol trisphosphate (63%) and inositol bisphosphate (38%), but labeling of inositol phosphate (IP) was increased by 59%. The results suggested a rapid turnover of the inositol phosphates in the gerbil brain. In general, changes in inositol lipids and inositol phosphates due to ischemia were attenuated after pretreatment with lithium (3 meq/kg) injected intraperitoneally 5 h prior to ligation. Surprisingly, lithium treatment alone did not cause an increase in IP labeling in the gerbil brain.
Inactivation and Reactivation of the Multifunctional Calmodulin‐Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain
Authors: Saitoh Yoshiki , Yamamoto Hideyuki , Fukunaga Kohji , Matsukado Yasuhiko , Miyamoto Eishichi
Published: October 2006

Abstract: The multifunctional calmodulin‐dependent protein kinase (calmodulin‐kinase) from rat brain was autophosphorylated in a Ca2+‐and calmodulin‐dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin‐kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin‐kinase were universally dephosphorylated by protein phosphatase C. Calmodulin‐kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin‐kinase by autophosphorylation and dephosphorylation was observed using calmodulin‐kinase from canine heart. These results suggest that the activity of calmodulin‐kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin‐kinases in various tissues.
Enzymes of Fatty Acid β‐Oxidation in Developing Brain
Authors: Reichmann Heinz , Maltese William A. , DeVivo Darryl C.
Published: October 2006

Abstract: Developmental profiles were determined for the activities of eight enzymes involved in fatty acid β‐oxida‐tion in rat brain. The enzymes studied were the palmitoyl‐CoA, octanoyl‐CoA, butyryl‐CoA, glutaryl‐CoA, and 3‐hy‐droxyacyl‐CoA dehydrogenases, the enoyl‐CoA hydratase (crotonase), and the C4‐ and C10‐thiolases. With the exception of the thiolases, all of the activities (expressed on the basis of brain weight) increased during the postnatal period of brain maturation. The activity of octanoyl‐CoA dehy‐drogenase was elevated markedly compared to that of pal‐mitoyl‐CoA dehydrogenase at all developmental stages and in all brain regions in the rat. A similar relationship between these enzymes was observed in various regions of adult human brain. Comparisons of the activities of the β‐oxidation enzymes in human brain versus human skeletal muscle and in cultured neural cell lines (neuroblastoma and glioma) versus cultured skin fibroblasts revealed that the elevated activity of octanoyl‐CoA dehydrogenase relative to palmitoyl‐CoA dehydrogenase was specific to the neural tissues. This relationship was particularly evident when the enzyme activities were normalized to the activity of crotonase. The data support previous findings with ra‐diochemical tracers, indicating that the brain is capable of utilizing fatty acids as substrates for oxidative energy metabolism. The relatively high activity of the medium‐chain fatty acyl‐CoA dehydrogenase in neural tissue may represent an adaptive mechanism to protect the brain from the known encephalopathic effects of octanoate and other medium‐chain fatty acids that readily cross the blood‐brain barrier.
Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain
Authors: Habermann Ernst , Müller Helmut , Hudel Martina
Published: October 2006

Abstract: Primary nerve cell cultures from the brainstem of embryonic mice take up [3H]noradrenaline. Release can be evoked by high K+ or sea anemone toxin II and depends on Ca2+. The cultures allow neurochemical studies on the long‐term actions of clostridial neurotoxins. Tetanus and botulinum A and C neurotoxins partially inhibit the absolute and fractional release evoked by high K+, as well as the fractional basal release. The detection limit for the toxins is below 5 pM. Total radioactivity is higher in the poisoned cultures, although the initial velocity of uptake is not measurably influenced by tetanus or botulinum A toxin. Pretreatment with neuraminidase prevents the effects of botulinum A toxin and diminishes those of botulinum C and tetanus toxins. Within 6 days, the cultures partially recover from tetanus toxin poisoning. Antitoxin prevents the actions of the toxin, but only slightly promotes recovery. The data indicate close pharmacological analogies between the clostridial neurotoxins.
α‐[3H]Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions
Authors: Errami Mohammed , Nieoullon André
Published: October 2006

Abstract: The binding of α‐[3H]amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl‐/Ca2+, saturation‐curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 ± 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several otner excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, l‐G1u, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N‐Methyl‐D‐aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intra‐striatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2–3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopainergic pathway (using nigral injection of 6‐hydroxydopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA‐preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.
Effect of High Doses of Dietary Vitamin E on the Concentrations of Vitamin E in Several Brain Regions, Plasma, Liver, and Adipose Tissue of Rats
Authors: Vatassery G. T. , Brin M. F. , Fahn S. , Kayden H. J. , Traber M. G.
Published: October 2006

Abstract: The object of this study was to assess the influence of high levels of dietary vitamin E on vitamin E concentrations in specific areas of the brain. Four‐week‐old male rats were fed vitamin E‐deficient, control, and high‐vitamin E (1,000 IU/kg) diets for 4 months. Concentrations of α‐tocopherol in serum, adipose tissue, liver, cerebrum, cerebellum, and striatum were determined by liquid chromatography with fluorescence detection. In the high‐vitamin E group, α‐tocopherol concentrations in cerebrum, cerebellum, and striatum increased uniformly to 1.4‐fold of values in controls; serum, adipose tissue, and liver attained even higher concentrations: 2.2‐, 2.2‐, and 4.6‐fold, respectively, of control values. As observed before, brain levels of α‐tocopherol were somewhat resistant to vitamin E deficiency, in contrast to the peripheral tissues.
Reduced Axonal Transport of the G4 Molecular Form of Acetylcholinesterase in the Rat Sciatic Nerve During Aging
Authors: Goemaere‐Vanneste J. , Couraud J.‐Y. , Hassig R. , Di Giamberardino L. , van den Bosch de Aguilar P.
Published: October 2006

Abstract: Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40–60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.
Systemic Lipopolysaccharide and Pokeweed Mitogen Increase Quinolinic Acid Content of Mouse Cerebral Cortex
Authors: Heyes Melvyn P. , Kim Phillip , Markey Sanford P.
Published: October 2006

Abstract: The effects of lipopolysaccharide and pokeweed mitogen on brain l‐tryptophan and quinolinic acid (QUIN) concentrations were investigated in C57BL/6NCR mice. Twenty‐four hours after an intraperitoneal injection of iipopolysaccharide (5 μg from Salmonella abortus equii) or pokeweed mitogen (500 μg), cortical QUIN concentrations were increased by 81 ± 6% and 182 ± 15%, respectively. Plasma QUIN was increased 175 ± 7% of control in pokeweed‐mitogen treated mice only. Brain l‐tryptophan concentrations were increased, whereas plasma l‐tryptophan concentrations were decreased. The consequences of increased QUIN concentrations during endotoxin and mitogen exposure remain to be determined.
Methylation Deficiency Causes Vitamin B12‐Associated Neuropathy in the Pig
Authors: Weir D. G. , Keating S. , Molloy A. , McPartlin J. , Kennedy S. , Blanchflower J. , Kennedy D. G. , Rice D. , Scott J. M.
Published: October 2006

Abstract: Pigs were treated with N2O which is known to impair vitamin B12 function in vivo. Such pigs demonstrated an inability to gain weight, progressive ataxia, and spinal neuropathy. The ataxia was totally and the neuropathy partially preventable by dietary methionine supplementation. Methionine synthase activity was inhibited in both the liver and brain. There was a marked elevation of S‐adenosylhomocysteine in the neural tissues and a concomitant failure of S‐adenosylmethionine to rise and thus maintain the methylation ratio, except when supplementary dietary methionine was added. In contrast, the methylation ratio in the rat was affected to a lesser extent. The neuropathy, it is suggested, is caused by raised S‐adenosylhomocysteine levels in neural tissue; as a result, the methylation ratio is inverted and S‐adenosylmethionine‐dependent methylation reactions are inhibited.
Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain
Authors: Ientile Riccardo , De Luca Grazia , Di Giorgio Rosa Maria , Macaione Salvatore
Published: October 2006

Abstract: The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putres‐cine, spermidine, and spermine levels as well as ornithine decarboxylase, S‐adenosylmethionine decarboxylase, and N1‐acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adre‐nalectomized rats. At 6 h after corticosterone or dexameth‐asone administration, the specific activities of ornithine decarboxylase and N1‐acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S‐adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1‐acetylspermidine transferase activities in a dose‐dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses >5 mg/kg. The great increases in ornithine decarboxylase and N1‐acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1‐acetylspermidine transferase induction.
ADP‐Ribosylation by Cholera Toxin of Membranes Derived from Brain Modifies the Interaction of Adenylate Cyclase with Guanine Nucleotides and NaF
Authors: Tamir Amichai , Gill D. Michael
Published: October 2006

Abstract: We have developed a method to ADP‐ribosylate the stimulatory guanine nucleotide‐binding protein of adenylate cyclase (Gs) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3‐acetylpyridine adenine dinucleotide to inhibit the potent NAD‐glycohydrolase activity of brain membranes, and we used the detergent Triton X‐100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP‐ribosylation. This method reveals that Gs is a very abundant protein in membranes derived from calf brain (∼30 pmol/mg of protein). In brain, Gs exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of Gs with an ADP‐ribosyl residue (a) elicits a four‐ to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5′‐(β,γ‐imido)triphosphate, or the sensitivity of adenylate cyclase to heat‐induced denaturation. A correlation between ADP‐ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.
Bovine P2 Myelin Basic Protein Crystallizes in Three Different Forms
Authors: Sedzik Jan , Bergfors Terese , Jones T. Alwyn , Weise Michael
Published: October 2006

Abstract: P2 protein is a minor component of the myelin membrane. We have crystallized this protein for high‐resolution crystallographic study. Three crystal morphologies are available. Two of them are from ammonium sulfate, and one is from polyethyleneglycol (PEG). The unit cell of the most suitable crystals from PEG 4000 has the dimensions a= 91.3 Å, b= 99.8 Å, c= 56.0 Å; is of space group P212121; and contains up to four molecules per asymmetric unit. The limit of resolution is 2.7 Å.
Choline Acetyltransferase Activity in the Rat Brain Cortex Homogenate, Synaptosomes, and Capillaries After Lesioning the Nucleus Basalis Magnocellularis
Authors: Santos‐Benito F. F. , González J. L. , de La Torre F.
Published: October 2006

Stereotaxic lesions of the nucleus basalis magnocellularis were made unilaterally in male Wistar rats with either kainic or ibotenic acid, using the contralateral side as control. Differences in behavior, body weight, and survival were observed between the kainic and ibotenic acid‐treated rats. One week after surgery, the rats were sacrificed and the effect of the lesions on choline acetyltransferase activity was measured in brain cortex homogenate, synaptosomes, and capillaries. In kainic acid‐lesioned rats, choline acetyltransferase activity decreased in homogenate and synaptosomes of the ipsilateral side with respect to that of the contralateral side; but the ibotenic acid lesion, which also reduced the ipsilateral choline acetyltransferase activity in homogenate, showed a rather different effect on the enzymatic activity of the synaptosomes. There were also differences between the effect of kainic and ibotenic acid lesions on choline acetyltransferase activity in the capillaries of the ipsilateral side with respect to that of the contralateral one. However, capillary choline acetyltransferase activity of the treated rats was in both sides three times higher than that of unoperated rats.
Energy Metabolism and Quantal Acetylcholine Release: Effects of Botulinum Toxin, l‐Fluoro‐2,4‐Dinitrobenzene, and Diamide in the Torpedo Electric Organ
Authors: Dunant Yves , Loctin Francoise , Marsal Jordi , Muller Dominique , Parducz Arpad , Rabasseda Xavier
Published: October 2006

In the Torpedo electric organ, a modified nervemuscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous. At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK). Thus, we compared this pattern of changes with those provoked by l‐fluoro‐2,4‐dinitrobenzene (FDNB), an efficient inhibitor of CK. As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved. In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion. This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminiature potentials was markedly increased. Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials. Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited. The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure. Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release. These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication. On the other hand, similarities between the effect of the clostridial toxin and those of diamide may suggest that the effects of botulinum toxin in nerve terminals result from a general oxidation of thiols. in parallel damaging energy‐providing enzymes (including creatine kinase) and components responsible for the quantal mode of ACh release.
Synthesis and Transport of Cerebrosides and Sulfatides in Rat Brain During Development
Authors: Koul Omanand , Singh Indrapal , Jungalwala Firoze B.
Published: October 2006

Synthesis and transport of nonhydroxy fatty acid (NFA)‐ and hydroxy fatty acid (HFA)‐containing ceramides, cerebrosides, and sulfatides were studied in vivo in rat brain during development. After an intracerebral injection of [3H]serine, incorporation into these lipids of microsomal and myelin membranes was analyzed after HPLC. Distribution of amounts and incorporation of radioactivity were also determined in individual molecular species of these lipids. The results showed that HFA‐ceramides and long‐chain NFA‐ceramides have small pool sizes and rapid turnover rates in the microsomal membranes and are preferentially utilized for the synthesis of long‐chain (≥20:0) HFA‐and NFA‐galactocerebrosides of both microsomal and myelin membranes. Glucocerebrosides are not expressed in myelin and their synthesis in microsomal membranes is predominant before the onset of myelination. With development, synthesis and accumulation of HFA‐cerebrosides increase over NFA‐cerebrosides in both microsomal and myelin membranes. In myelin, incorporation of radioactivity into HFA‐cerebrosides is even higher than that expected by transport alone from microsomal membranes and it is possible that part of the HFA‐cerebrosides in myelin could be due to de novo synthesis by myelin itself. The amount of NFA‐ and HFA‐sulfatides is about equal, both in myelin and microsomal membranes, and this relative proportion does not change with development. Similar relative rates of incorporation of radioactivity into sulfatides of microsomal and myelin membranes are consistent with the notion that both NFA and HFA sulfatides are synthesized in the microsomal (Golgi) membranes and are transported to myelin. In vitro kinetic analysis of both myelin and microsomal galactosyltransferase with different HFA‐ceramide species showed an apparent Km of about 0.12 mM, whereas the Vmax was highest for h24:1 ceramide followed by that for h18:0 and h24:0 ceramides.
Evidence of Glutamatergic Denervation and Possible Abnormal Metabolism in Alzheimer's Disease
Authors: Procter A. W. , Palmer A. M. , Francis P. T. , Lowe S. L. , Neary D. , Murphy E. , Doshi R. , Bowen D. M.
Published: October 2006

Excitatory dicarboxylic amino acids previously have been ascribed several functions in the brain. Here their total concentration and proposed neurochemical markers of neurotransmitter function have been measured in brain from patients with Alzheimer's disease (AD) and controls. Specimens were obtained antemortem (biopsy) approximately 3 years after emergence of symptoms and promptly (<3 h) postmortem some 10 years after onset. Early in the disease a slight elevation in aspartic acid concentration of cerebral cortex was observed in the patients with AD. A reduction in glutamic acid concentration of a similar magnitude was found. It is argued that this, together with a decrease in CSF glutamine content and lack of change in the phosphate‐activated brain glutaminase activity of tissue, reflects an early metabolic abnormality. Later in the disease evidence of glutamatergic neurone loss is provided by the finding that in many regions of the cerebral cortex the Na+‐dependent uptake of D‐[3H]aspartic acid was almost always lowest in AD subjects compared with control when assessed by a method designed to minimise artifacts and epiphenomena. Release of endogenous neuro‐transmitters from human brain tissue postmortem did not appear to have the characteristics of that from human tissue antemortem and rat brain.
Synthesis and Translocation of Gangliosides and Glycoproteins During Urethane Anesthesia
Authors: Domowicz Miriam S. , Kivatinitz Silvia C. , Caputto Beatriz L. , Caputto Ranwel
Published: October 2006

Abstract: In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane‐anesthetized and control chickens after intraocular injection of a labeled N‐acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4‐h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside‐specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.
Differential Regulation of Phosphoinositide Phosphodiesterase Activity in Brain Membranes by Guanine Nucleotides and Calcium
Authors: Gonzales Rueben A. , Crews Fulton T.
Published: October 2006

Abstract: We have shown previously that calcium and guanine nucleotides stimulate the activity of a phosphoinositide (PI) phosphodiesterase in membranes from rat cerebral cortex and that their effects are additive. To understand further guanine nucleotide‐ and calcium‐stimulated PI phosphodiesterase activity, we have investigated the pH sensitivity and effects of inhibitors on the two modes of stimulation. NaF stimulates PI hydrolysis in brain membranes with an EC50 of 2 mM and a maximal effect at 10 mM, suggesting that a guanine nucleotide binding protein can regulate PI phosphodiesterase. Neomycin inhibited guanylylimidodiphosphate (GppNHp)‐stimulated PI phosphodiesterase activity in a concentration‐dependent manner, with 90% inhibition at 0.3 mM. Neomycin was not as effective at inhibiting calcium‐dependent PI hydrolysis (32% inhibition at 0.3 mM). Chloroquine also had a greater inhibitory effect against GppNHp‐stimulated PI phospho diesterase activity compared to calcium‐dependent activity. Guanine nucleotide‐ and NaF‐dependent activations of PI phosphodiesterase were strongly pH‐dependent, with greatest stimulation observed at pH 5–6 and inhibition at more alkaline pH. Calcium‐stimulated PI hydrolysis was not as sensitive to changes in pH and had a peak of activity at pH 9. Our findings of different pH optima and differential sensitivity to inhibitors suggest that calcium and guanine nucleotides may regulate PI phosphodiesterase in rat cortical membranes through independent mechanisms. Key Words: G‐proteins—Calcium—Phosphoinositides—Membranes—Phosphoinositide phosphodiesterase—Brain. Gonzales R. A. and Crews F. T. Differential regulation of phosphoinositide phosphodiesterase activity in brain membranes by guanine nucleotides and calcium. J. Neurochem. 50, 1522–1528 (1988).
Increased Polyphosphoinositide Responsiveness in the Cerebral Cortex Induced by Cholinergic Denervation
Authors: Reed L. J. , de Belleroche J.
Published: October 2006

Abstract: Lesion of the nucleus basalis in the basal fore‐brain of the rat results in the degeneration of the large cholinergic neurones which innervate the cortex. Parameters of cholinergic function, namely, acetylcholinesterase activity, muscarinic acetylcholine receptor number, and the depolarisation‐induced release of acetylcholine, fall in ipsilateral cortex subsequent to lesion. These deficits are likely to reflect the loss of the presynaptic input to the cortex. A reversal in these deficits is seen 1 month after lesion, and a full recovery is seen after 150 days. This is thought to be due to a process of “spared axon sprouting” followed by the reestablishment of synapses. To examine the integrity of the cortical muscarinic receptor response following denervation, an assay of the polyphosphoinositide response was carried out. Cortical tissue slices, prela‐belled with [3H]inositol, were incubated for 40 min with carbachol in the presence of Li+; the accumulation of [3H]‐inositol monophosphate ([3H]IP1) was used as an index of this response. A 92% increase in the carbachol‐stimulated production of [3H]IP1 was seen 5 days after lesion com pared to normal cortex. Sham‐operated animals showed no change in [3H]IP1 accumulation at this time point. Dose‐response experiments showed that this increase was due to an increase in the maximal response to carbachol after lesion with no change in EC50 values. Two weeks after lesion, this increased response was much attenuated; tissue slices from denervated cortex showing a strong acetylcholinesterase decrease (36–66%) showed an increase of just 30% above normal. There was no increase in the polyphosphoinositide response 50 and 118 days after lesion. The transient time course of this response to lesion suggests that it participates in or arises from processes of recovery from denervation and the reestablishment of synapses.
Sulfoglucuronyl Neolactoglycolipids in Adult Cerebellum: Specific Absence in Murine Mutants with Purkinje Cell Abnormality
Authors: Chou Denise K. H. , Jungalwala Firoze B.
Published: October 2006

Abstract: It is shown here that glycolipids of the sulfoglucuronyl neolacto series (SGGLs) are present in the adult rodent cerebellum. SGGLs were not detected in the cerebellar murine mutants lurcher, Purkinje cell degeneration, and staggerer, in which Purkinje cell loss is the primary defect. SGGLs were present, however, in normal amounts in weaver and reeler mutants, in which there is a major and relatively specific loss of granule cells without obvious deficiency in Purkinje cells. In the myelin‐deficient quaking mutant, the expression of SGGLs also was nearly normal. The loss of SGGLs in Purkinje cell‐deficient mutants was specific, since most of the major lipids were not affected significantly and only the percentage composition of other lipids, such as sulfatides and gangliosides, was altered in the mutants. These and other results strongly suggest that SGGLs and other glycolipids of the paragloboside family are localized specifically in Purkinje cells and their arbors in the adult cerebellum. This is the first demonstration of the localization of a specific glycolipid and its analogs in a specific cell type in the nervous system.
Nerve Growth Factor Increases the Intracellular Content of Acetylcholine in Cultured Septal Neurons from Developing Rats
Authors: Takei Nobuyuki , Tsukui Hiroko , Hatanaka Hiroshi
Published: October 2006

Abstract: The effects of nerve growth factor (NGF) on the intracellular content of acetylcholine (ACh) in cultured septal neurons from developing rats have been examined. The content of ACh could be measured by using HPLC and electrochemical detection (HPLC‐ECD), coupled with an immobilized enzyme column. This method of determination is very simple and rapid, and is highly sensitive. The content of ACh and the activity of choline acetyltransferase (ChAT) in cultured postnatal day 1 (PI) septal neurons grown on an astroglial “feeder” layer was increased during the period of cultivation by the addition of NGF. The activities of ChAT and the content of ACh increased in a dose‐dependent manner in direct relationship to the different amounts of NGF employed. These effects of NGF, i.e., elevating the intracellular content of ACh, accompanied by an increase in activity of ChAT, also were confirmed in the PI septal organotypic cultures. Additionally, embryonic day 17 (E17) septal neurons in a serum‐free medium displayed a similar responsiveness to NGF with respect to the elevation in the content of ACh and the increase in activity of ChAT. These results suggest that intracellular levels of ACh are likely to be regulated by NGF in a fashion similar to that of the activity levels of the biosynthetic enzyme.