bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulated Galactolipid Synthesis and Cell Surface Expression in Schwann Cell Line D6P2T
Authors: Bansal Rashmi , Pfeiffer S. E.
Published: October 2006

Clonal cell line D6P2T, subcloned from an ethylnitrosourea‐induced tumor line D6 of the rat peripheral nervous system, has been characterized with particular attention to galactolipid metabolism. Galactosylcerebroside and sulfatide synthesis and expression on the cell surface are highly regulated in D6P2T cells by mechanisms involving serum‐and cyclic AMP‐mediated pathways. These cells also express 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (Wolfgram protein Wla) and laminin. In contrast, myelin basic protein and antigen HNK‐1 were not detected. Line D6P2T appears to be a semi‐differentiated Schwann cell model, which offers interesting possibilities for studies of galactolipid synthesis, transport, and sorting.
Chronic Low‐Level Lead Exposure Impairs Embryonic to Adult Conversion of the Neural Cell Adhesion Molecule
Authors: Cookman Gillian R. , King William , Regan Ciaran M.
Published: October 2006

The neural cell adhesion molecule (N‐CAM) is a complex of surface glycoproteins that are developmentally regulated and believed to be intimately involved in the orderly structuring of the CNS. Here the effect of chronic low‐level lead exposure on their expression in the postnatally developing cerebellum is described. Rat pups were chronically exposed to lead via their dam's drinking water which contained either 200 or 400 mg PbCl2/L from time of birth. Pup postnatal blood lead levels ranged between 10 and 20 μg/dl until day 16 after which they became elevated to 40 μg/dl on day 20. During this period the developmental sialylation state of N‐CAM, which is believed to regulate cell‐cell interaction, fibre outgrowth, and synapse formation, was monitored by rocket immunoelectrophoresis. In control animals the expected desialylation of N‐CAM was found to occur at times coincident with postnatal synapto‐genesis. In contrast, desialylation in animals chronically exposed to lead was found to be significantly impaired when blood lead levels exceeded 20 μg/dl. This could not be attributed to lead‐induced undernutrition or alterations in immu‐noprecipitate formation. These observations could account for the neurobehavioural deficits that are known to be induced at similar blood lead levels, and the potential contribution of impaired N‐CAM desialylation to synaptic elaboration is discussed.
Noninvasive Demonstration of In Vivo 3‐Fluoro‐3‐Deoxy‐D‐Glucose Metabolism in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy: Suitable Probe for Monitoring Cerebral Aldose Reductase Activities
Authors: Kwee Ingrid L. , Nakada Tsutomu , Card Peter J.
Published: October 2006

The metabolism of 3‐fluoro‐3‐deoxy‐D‐glucose (3‐FDG) in rat brain in vivo was investigated noninvasively using 19F nuclear magnetic resonance (NMR) Spectroscopy. Following an intravenous infusion of 3‐FDG, 400 mg/kg, four resonances assigned to the α and β anomers of 3‐FDG, 3‐fluoro‐3‐deoxy‐D‐sorbitol, and 3‐fluoro‐3‐deoxy‐D‐fructose were clearly resolved in brain, a result indicating that 3‐FDG is metabolized primarily into the aldose reductase sorbitol (ARS) pathway. An orally administered aldose reductase inhibitor, sorbinil, caused reduction of the flux of 3‐FDG into the ARS, an observation suggesting that the method can be applied in quantitative studies of ARS path way activities. Studies of 24‐h urine specimens showed that in addition to the two metabolites observed in brain, F‐was excreted into the urine. 3‐FDG appears to be a suitable metabolic probe for assessing glucose metabolism in the ARS pathway by in vivo 19F NMR Spectroscopy.
Turnover of Brain Monoamine Oxidase Measured In Vivo by Positron Emission Tomography Using l‐[11C]Deprenyl
Authors: Arnett Carroll D. , Fowler Joanna S. , MacGregor Robert R. , Schlyer David J. , Wolf Alfred P. , Långström Bengt , Halldin Christer
Published: October 2006

The distribution of carbon‐11‐labeled L‐deprenyl, an irreversible inhibitor of monoamine oxidase type B (MAO‐B), was determined in the baboon brain by positron emission tomography. The irreversible blood‐to‐brain transfer constant (influx constant, Ki) was measured using a complete metabolite‐corrected arterial plasma concentration curve. This influx constant was used as a measure of functional enzyme activity for sequential determinations of MAO‐B recovery following a single high dose of unlabeled l‐deprenyl. The half‐life for turnover of MAO‐B was thus determined to be 30 days. Using appropriate irreversible inhibitors, this procedure should be generally useful for determining enzyme turnover rates in any organ in vivo and can be applied to some human studies as well.
Effects of Methylprednisolone and the Combination of α‐Tocopherol and Selenium on Arachidonic Acid Metabolism and Lipid Peroxidation in Traumatized Spinal Cord Tissue
Authors: Saunders Royal D. , Dugan Laura L. , Demediuk Paul , Means Eugene D. , Horrocks Lloyd A. , Anderson Douglas K.
Published: October 2006

Abstract: Traumatic injury of the spinal cord leads to a series of pathological events that result in tissue necrosis and paralysis. Among the earliest biochemical reactions are hydrolysis of fatty acids from membrane phospholipids, production of biologically active eicosanoids, and peroxidation of lipids. This study examines the effect of agents purported to improve recovery following spinal cord trauma, methyl‐prednisolone sodium succinate (MPSS) and the combination of α‐tocopherol and selenium (Se), on the posttraumatic alterations of membrane lipid metabolism. Pretreatment with either MPSS or a‐tocopherol and Se reduced the trauma‐induced release of total FFA including arachidonate in the injured spinal cord tissue. In addition, these agents decreased the postinjury levels of prostanoids. Pre‐treatment with either MPSS or a‐tocopherol and Se also completely prevented the trauma‐induced loss of cholesterol while inhibiting the increase of a cholesterol peroxidation product, 25‐hydroxycholesterol. These data suggest that: (a) perturbation of membrane lipid metabolism may contribute to the tissue necrosis and functional deficit of spinal cord injury and (b) MPSS or the combination of a‐tocopherol and Se may protect injured spinal cord tissue, at least in part, by limiting these posttraumatic membrane lipid changes.
Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus
Authors: Staatz‐Benson C. , Potashner S. J.
Published: October 2006

Abstract: This study attempts to determine if the cochlear nucleus (CN) contains glycinergic synaptic endings. The uptake and release of exogenous radiolabeled glycine were measured in vitro in the three major subdivisions of the guinea pig CN: anteroventral, posteroventral, and dorsal. A kinetic analysis of [3H]glycine uptake revealed the presence in each CN subdivision of a high‐and a low‐affinity uptake mechanism. The high‐affinity mechanism had a Km of 25.2–30.5 μM and a Vmax of 3.8–4.8 nmol/10 mg of cell water/5min, whereas the low‐affinity mechanism had a Km of 633–718 μM and a Vmax of 26.6–37.1 nmol/10 mg of cell water/5 min. At steady state, the high‐affinity mechanism accumulated 10 μM [3H]glycine from the medium, achieving tissue concentrations that were 13–24 times that in the medium. The high‐affinity uptake was dependent on the temperature and on the concentrations of NaCl and glucose in the incubation medium. It exhibited a high degree of substrate specificity, as determined by the effects of structural analogues of glycine on the uptake of [3H]glycine. Each CN subdivision also contained two mechanisms mediating [14C]glycine release. One was activated by depolarizing electrical stimuli, produced a rapid transient release of [14C]glycine, and was dependent on the presence of extracellular Ca2+. The other was continuous, producing a slow spontaneous efflux of [14C]glycine. Released glycine could be removed primarily by uptake, because during release measurements, the amount of [14C]glycine detected in the medium decreased when glycine uptake activity was optimized. The electrically evoked, Ca2+‐dependent release and the high‐affinity uptake of glycine may mediate the synaptic release and inactivation of glycine, respectively. These findings, therefore, support the presence of glycinergic synaptic endings in each CN subdivision.
Quantitative Development and Molecular Forms of Acetyl‐and Butyrylcholinesterase During Morphogenesis and Synaptogenesis of Chick Brain and Retina
Authors: Layer Paul G. , Alber Regina , Sporns Olaf
Published: October 2006

Abstract: The embryonic development of total specific activities as well as of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and of butyrylcholinesterase (BChE, EC 3.1.1.8) have been studied in the chick brain. A comparison of the development in different brain parts shows that cholinesterases first develop in diencephalon, then in tectum and telencephalon; cholinesterase development in retina is delayed by about 2–3 days; and the development in rhomb‐encephalon [not studied until embryonic day 6 (E6)] and cerebellum is last. Both enzymes show complex and independent developmental patterns. During the early period (E3‐E7) first BChE expresses high specific activities that decline rapidly, but in contrast AChE increases more or less constantly with a short temporal delay. Thereafter the developmental courses approach a late phase (E14‐E20), during which AChE reaches very high specific activities and BChE follows at much lower but about parallel levels. By extraction of tissues from brain and retina in high salt plus 1% Triton X‐100, we find that both cholinesterases are present in two major molecular forms, AChE sedimenting at 5.9S and 11.68 (corresponding to G2 and G4 globular forms) and BChE at 2.9S and 10.3S (G1 and G4, globular). During development there is a continuous increase of G4 over G2 AChE, the G4 form reaching 80% in brain but only 30% in retina. The proportion of G1 BChE in brain remains almost constant at 55%, but in retina there is a drastic shift from 65% G1 before E5 to 70% G4 form at E7. Our results show that the light‐molecular‐weight forms of both enzymes are prevalent during the morphogenetic period, whereas the G4 forms correlate with final differentiation processes, e.g., synaptogenesis.
Isolation of a Human Brain cDNA for Glutamate Dehydrogenase
Authors: Banner Carl , Silverman Sanford , Thomas John W. , Lampel Keith A. , Vitkovic Ljubisa , Huie Diana , Wenthold Robert J.
Published: October 2006

Abstract: A cDNA has been isolated from a human brain expression library using anti‐bovine glutamate dehydrogenase (GDH) antibodies. The cDNA has an open reading frame of 774 nucleotides, which codes for 258 amino acids. The 258‐amino‐acid sequence is 95% homologous to the carboxy terminus of human liver GDH. This high degree of homology indicates that the cDNA codes for brain GDH. Fourteen differences between the amino acid sequence deduced from this cDNA and the sequence reported for human liver GDH suggest that there may be two active human GDH genes. A cRNA probe synthesized from the cDNA detects a 3.7‐kilobase (kb) mRNA from human brain. Rat liver and kidney each contain two GDH mRNAs, 3.5 and 2.8 kb, respectively. The 3.5‐kb transcript is prominent in rat brain, whereas the 2.8‐kb transcript is barely detectable, a result suggesting that GDH gene expression is differentially controlled in rat brain.
An In Vivo Kinetic Model with L‐[35S]Methionine for the Determination of Local Cerebral Rates for Methionine Incorporation into Protein in the Rat
Authors: Lestage P. , Gonon M. , Lepetit P. , Vitte P. A. , Debilly G. , Rossatto C. , Lecestre D. , Bobillier P.
Published: October 2006

Abstract: A method has been developed for the simultaneous in vivo measurement of local rates for methionine incorporation into cerebral protein in the rat. It is based on the use of l‐[35S]methionine as a tracer for reflecting the bidirectional exchange of methionine between plasma and brain and its incorporation into cerebral protein, using a dynamic three‐compartment model. An operational equation based on this model has been derived in terms of deter‐minable variables. The method has been applied to the normal freely moving rat and to the rat under chloral hydrate anesthesia. In the freely moving rat, the values of methionine incorporation into cerebral protein in the gray matter vary widely from structure to structure (50–300 nmo1/100 g/min), with the highest values in structures related to neurosecretory functions, e.g., supraoptic and para ventricular nuclei. The values for white matter are more uniform (24–28 nmol/100 g/min) at levels approximately six‐ to sevenfold lower than for gray matter. Chloral hydrate anesthesia depresses the rate of methionine incorporation in all the structures examined. Anesthesia did not reduce the heterogeneity normally present within gray matter.
The Metabolism of [18F]6‐Fluoro‐l‐3,4‐Dihydroxyphenylalanine in the Hooded Rat
Authors: Dimming P. , Boyes B. E. , Martin W. R. W. , Adam M. , Grierson J. , Ruth T. , McGeer E. G.
Published: October 2006

Abstract: The metabolism of the positron‐emitting compound [18F]6‐fluoro‐l‐3,4‐dihydroxyphenylalanine (*F‐DOPA) was studied in carbidopa‐pretreated male hooded rats. Thirty minutes following carbidopa administration (5 mg/kg i.p.), animals received *F‐DOPA (500 μg/kg; specific activity, 175–230 Ci/mol) as an intrajugular bolus. Blood samples were taken at various times between 5 and 90 min, and the plasma was analyzed by HPLC with gamma counting of fractions. *F‐DOPA disappeared rapidly from plasma in concert with the formation of the 3‐O‐methylated metabolite, Me‐*F‐DOPA. Animals were killed from 5 to 120 min after injection, and the brains were rapidly dissected. The disappearance of *F‐DOPA from both vermis and striatal samples was rapid. Me‐*F‐DOPA, the sole metabolite observed in the vermis, was the major labeled material in the striatum at ≥20 min after injection. Fluorodopamine was an important metabolite in the striatum, making up 25% of total radioactivity at early intervals. Striatal samples also contained fluoro‐3,4‐dihydroxyphenylacetic acid, which constituted ∼10% of the total radioactivity, and traces of two radiolabeled compounds, tentatively identified as fluorohomovanillic acid and fluoro‐3‐methoxytyramine.
Regional Distribution of High‐Affinity γ‐[3H] Hydroxybutyrate Binding Sites as Determined by Quantitative Autoradiography
Authors: Hechler Viviane , Weissmann Dinah , March Evelyne , Pujol Jean‐François , Maitre Michel
Published: October 2006

Abstract: The distribution of high‐affinity binding sites for γ‐[3H]hydroxybutyrate in coronal sections of rat brain was studied by quantitative autoradiographic techniques. Binding sites for this naturally occumng substance, which may possibly have a neurotransmitter role, are concentrated in some restricted areas of the brain, particularly in the limbic system. The hippocampus (especially field CAI of Ammon's horn, at 292 fmol/mg of tissue), septum (72 fmol/mg of tissue), and cortex (frontal, 113 fmol/mg of tissue; parietal, 103 fmol/mg of tissue; cingulate, 114 fmol/mg of tissue; and entorhinal, 134 fmol/mg of tissue) show pronounced labeling with γ‐[3H]hydroxybutyrate. Binding is much lower in caudatus‐putamen (50 fmol/mg of tissue), thalamus, and hypothalamus. Caudal parts of the brain (cerebellum, pons, and medulla) are practically devoid of binding sites. These results strongly support a functional role of endogenous y‐hydroxybutyrate in particularly restricted areas of the rat brain.
In Vitro Protein Phosphorylation in Head Preparations from Normal and Mutant Drosophila melanogaster
Authors: Buxbaum Joseph D. , Dudai Yadin
Published: October 2006

Abstract: We have characterized protein phosphorylation in vitro in subcellular fractions from Drosophila melanogaster heads. Optimal conditions for the incorporation of 32P into proteins, and its dependence on ATP, divalent cations, and cyclic nucleotides have been determined, as well as the effect of inhibitors of ATPase, protein phospha‐tase, and protein kinase on protein phosphorylation. Among these inhibitors, Zn2+ was found to affect the incorporation of 32P into specific bands and p‐hydroxymercuri‐benzoate was found to be most suited for freezing the activity of both kinases and phosphatases. Cyclic AMP‐depen‐dent protein kinase (cAMP‐dPK) activity was present in both supernatant (S2) and paniculate (P2) fractions, with the majority (60–85%, depending on the homogenization medium) being associated with S2, as determined by phosphorylation of exogenous synapsin I. cAMP‐dPK catalyzed the phosphorylation of at least 18 endogenous polypeptides in S2 and at least 10 endogenous polypeptides in P2. These proteins could be classified on the basis of the extent of stimulation of phosphorylation by cyclic nucleotides, dependence on cyclic nucleotide concentration, and rate of phosphorylation. A phosphoprotein of 51 kilodaltons (pp51) was a major component of the S2 and P2 fractions and displayed properties expected from the regulatory sub‐unit of the cAMP‐dPK, R‐II. A phosphoprotein doublet of approximately 37 kilodaltons (pp37) was stimulated to the largest extent by cAMP in the P2 and S2 fractions. The phosphorylation of several proteins in both fractions was significantly lowered by the mammalian Walsh inhibitor of cAMP‐dPK, whereas in some cases the stimulation of phosphorylation of the same proteins by exogeneous cAMP was relatively small. Phosphoproteins from two learning mutants known to be deficient in cAMP metabolism, dnc and rut, were analyzed for their extent of phosphorylation in the presence of a stable cAMP analogue; no significant differences from normal were detected, suggesting that the genetic defect in cAMP metabolism is not accompanied by constituent abnormalities in phosphorylated substrates in the adult fly, and that the physiological defects in these mutants result from aberrations in the interaction of the cAMP cascade with normal substrates. The majority of Ca2+/calmodulin kinase activity (80–90%, depending on the homogenization procedure) was associated with S2 as revealed by phosphorylation of exogenous synapsin I. Two endogenous substrates for this kinase in P2 had molecular masses of approximately 45 and 87 kilodaltons. At least 11 substrates for the Ca2+/calmodulin‐dependent kinase were detected in S2. The 45‐kilodalton protein was a major substrate in this fraction too, as was pp37.
Phosphorylation of Purified Rat Striatal Tyrosine Hydroxylase by Ca2+/Calmodulin‐Dependent Protein Kinase II: Effect of an Activator Protein
Authors: Atkinson Janet , Richtand Neil , Schworer Charles , Kuczenski Ronald , Soldering Thomas
Published: October 2006

Abstract: The phosphorylation of tyrosine hydroxylase, purified from rat striatum, was investigated using purified Ca2+/calmodulin (CaM)‐dependent protein kinase II. This kinase catalyzed the Ca2+‐dependent incorporation of up to 0.8 mol 32PO4/mol tyrosine hydroxylase subunit (62 kilo‐daltons). Reverse‐phase high‐performance liquid chroma‐tography mapping of tryptic 32P‐peptides established that the Ca2+/CaM‐dependent protein kinase II phosphorylated a different serine residue than was phosphorylated by the cyclic AMP‐dependent protein kinase. Limited proteolysis sequentially reduced the subunit Mr from 62 to 59 kilodaltons and finally to 57 kilodaltons, resulting in loss of the site phosphorylated by the Ca2+/CaM‐dependent protein kinase II, but not the site phosphorylated by the cyclic AMP‐dependent protein kinase. Phosphorylation by the Ca2+/ CaM‐dependent protein kinase II had little direct effect on the kinetic properties of tyrosine hydroxylase, but did convert it to a form that could be activated twofold by addition of an activator protein. This heat‐labile activator protein increased the Vmax without affecting the Km for the pterin cofactor. This effect was specific in that the activator protein was without effect on nonphosphorylated tyrosine hydroxylase or on tyrosine hydroxylase phosphorylated by the cyclic AMP‐dependent protein kinase. These results are consistent with the hypothesis that the „Vmax‐type” activation of tyrosine hydroxylase observed upon depolarization of neural and adrenal tissues may be mediated by the Ca2+/ CaM‐dependent protein kinase II.
Myelin Basic Protein‐Specific RNA Levels in Interleukin‐2‐Stimulated Oligodendrocytes
Authors: Benveniste Etty N. , Herman Paula K. , Whitaker John N.
Published: October 2006

Abstract: In the immune system, T‐and B‐cell proliferation, as well as B‐cell immunoglobulin secretion, is induced by interleukin‐2 (IL‐2), a T‐cell‐derived lymphokine. IL‐2 also influences the growth of glial cells, specifically, the proliferation and maturation of oligodendrocytes. Studies were conducted to investigate further IL‐2‐induced maturation of oligodendrocytes through its effect on the regulation of the myelin basic protein (MBP) gene. A cDNA probe specific for rat MBP and a double‐antibody radioimmuno‐assay for MBP were used to quantitate MBP mRNA and protein levels in oligodendrocytes under different experimental conditions. We demonstrate that both MBP mRNA and protein levels are increased in IL‐2‐stimulated oligodendrocytes. MBP mRNA levels increase within 8 h after IL‐2 stimulation, peak between 24 and 48 h, and then deline slightly. MBP protein levels increase 24 h after stimulation and peak at 72 h. MBP mRNA transcripts in the range of 2.0–2.4 kilobases are present in cultured rat oligodendrocytes, which are similar to the MBP mRNA transcripts detected in whole rat and mouse brain. These mRNA transcripts are specifically increased in quantity after oligodendrocyte stimulation with IL‐2. These results suggest that one component of oligodendrocyte differentiation/maturation—MBP mRNA and protein expression—can be regulated in part by IL‐2.
Effects of the Binding of Imipramine to Erythrocytes and Plasma Proteins on Its Transport Through the Rat Blood‐Brain Barrier
Authors: Riant P. , Urien S. , Albengres E. , Renouard A. , Tillement J. P.
Published: October 2006

Abstract: Brain extraction of a tricyclic antidepressant, imipramine, was investigated using the carotid injection technique in the rat. The extent to which drug binding to plasma proteins and erythrocytes could inhibit the brain extraction was measured. Equilibrium dialysis showed that imipramine is highly bound to human serum albumin (HSA), α1‐acid glycoprotein (AAG), lipoproteins, and erythrocytes. The free dialyzable drug fraction was inversely related to the protein concentration. Despite this degree of binding, no significant reduction in the brain extraction of the drug was observed in the presence of HSA, lipoprotein, or erythrocytes. Only AAG reduced the brain transport of this drug in a ratio related to the protein concentration. However, the rat brain extraction was higher than expected from the in vitro measurement of the dialyzable fraction. These data indicate that the amount of circulating imipramine available for penetration in brain exceeds widely the dialyzable fraction of the drug as measured in vitro.
Regional Differences in the Capacity for Ammonia Removal by Brain Following Portocaval Anastomosis
Authors: Butterworth Roger F. , Girard Guylaine , Giguère Jean‐Francois
Published: October 2006

Abstract: Portocaval anastomosis (PCA) in the rat leads, within 4 weeks, to severe liver atrophy, sustained hyperam‐monemia, and increased brain ammonia. Because brain is not equipped with an effective urea cycle, removal of ammonia involves glutamine synthesis and PCA results in significantly increased brain glutamine. Glutamine synthetase activities, however, are decreased by 15% in cerebral cortex and are unchanged in brainstem of shunted rats. Administration of ammonium acetate to rats following PCA results in severe encephalopathy (loss of righting reflex and, ultimately, coma). Glutamine concentrations in brainstem of comatose rats are increased a further two‐fold, whereas those of cerebral cortex are unchanged. Consequently, ammonia levels in cerebral cortex reach disproportionately high levels (of the order of 5 mM). These findings suggest a limitation in the capacity of cerebral cortex to remove additional blood‐borne ammonia by glutamine formation following PCA. Such mechanisms may explain the hypersensitivity of rats with PCA and of patients with portal‐systemic shunting to small increases of blood ammonia. Disproportionately high levels of brain ammonia in certain regions, such as cerebral cortex, may then result in alterations of inhibitory neurotransmission and, ultimately, loss of cellular (astrocytic) integrity.
Intracellular pH, Lactate, and Energy Metabolism in Neonatal Brain During Partial Ischemia Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy
Authors: Corbett R. J. T. , Laptook A. R. , Nunnally R. L. , Hassan A. , Jackson J.
Published: October 2006

Abstract: Sequential 31P and 1H nuclear magnetic resonance spectra were measured for neonatal piglets (n = 7) to determine the relationship between brain intracellular pH (pHi), lactate, and phosphorylated energy metabolites during partial ischemia. Simultaneous determinations of arterial and cerebral venous blood gases, pH, O2 content, and plasma concentrations of glucose and lactate were also made. Ischemia, induced by bilateral carotid artery ligation plus hemorrhagic hypotension for 35 min, resulted in variable reductions in ATP, phosphocreatine, and increases in Pi, H+, and lactate relative to control levels. In four piglets, whose arterial blood glucose rose above control, brain lactate exceeded 20 μmol g−1 with corresponding decreases in pHi of >0.7 units compared to control levels. The extents of brain acidosis and lactosis showed a strong linear correlation with each other (r= 0.94). Maximal changes in brain lactate, pHi, and ATP at the end of ischemia showed significant positive linear correlations with the control levels of arterial blood glucose, but did not correlate with arterial glucose or arterial cerebral‐venous glucose difference values during ischemia. The relationship between pHi and buffer base deficit was comparable to results reported for adult animals up to 20 μmol ml−1. However, in contrast to models proposed for adult brain, the continued linear relationship between pH and higher buffer base levels is most consistent with a theoretical model that assumes the presence of weak acid buffers with pKa values from 6.7 to 5.2.
Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain
Authors: Montpied Pascale , Martin Brian M. , Cottingham Sandra L. , Stubblefield Barbara K. , Ginns Edward I. , Paul Steven M.
Published: October 2006

Abstract: A human cDNA clone containing the 5’coding region of the GABAA/benzodiazepine receptor α subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP‐labelled antisense RNA probe (860 bases) prepared from the α subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus ≫ pons = striatum = medulla. In situ hybridization revealed high levels of α subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor α subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.
Glucose Transporter of the Blood‐Brain Barrier and Brain in Chronic Hyperglycemia
Authors: Harik Sami I. , Gravina Stephen A. , Kalaria Rajesh N.
Published: October 2006

Abstract: The effect of chronic hyperglycemia on the glucose transporter moiety of the blood‐brain barrier and cerebral cortex was studied in rats 3 weeks after the administration of a single intravenous dose of streptozotocin (60 mg/kg), using specific [3H]cytochalasin B binding methods. Streptozotocin‐treated rats developed hyperglycemia, as well as polydipsia and polyuria, and failed to gain weight. The density of d‐glucose‐displaceable cytochalasin B binding sites in the brain microvessels of streptozotocin‐treated hyperglycemic rats was increased by about 30% compared with those of control rats, without change in the affinity of binding. Chronic hyperglycemia had no effect on the density or affinity of specific binding of cytochalasin B to cerebral cortical membranes. These findings do not support the hypothesis that glucose transporters in brain microvessels comprising the blood‐brain barrier are “down‐regulated” in chronic hyperglycemia.
Excitatory Amino Acid Release from Rat Hippocampal Slices as a Consequence of Free‐Radical Formation
Authors: Pellegrini‐Giampietro Domenico E. , Cherici Giovanna , Alesiani Marina , Carlà Vincenzo , Moroni Flavio
Published: October 2006

Abstract: The release of D‐[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hy‐droxyl‐radical scavenger D‐mannitol, or the superoxide‐radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.
Inhibition of DNA Synthesis in C6 Glioma Cells Following Cellular Incorporation of GM1 Ganglioside and Choleragenoid Exposure
Authors: Skaper Stephen D. , Facci Laura , Favaron Marco , Leon Alberta
Published: October 2006

Abstract: The B subunit of cholera toxin, which is multiva‐lent and binds specifically to GM1 ganglioside on the cell surface, has previously been used as a ganglioside‐specific probe to regulate DNA synthesis in thymocytes and fibro‐blasts. To explore in more detail this growth‐regulatory action of gangliosides, C6 glioma cells (which are GM1 ganglioside deficient) were used as a model system. When cultures of C6 cells were first treated with GM1, followed by exposure to the B subunit, proliferation was inhibited, as measured by 3H‐labeled thymidine incorporation into DNA. Pretreatment of the cells with 50 μM GM1 for 15 min (followed by washing with fetal calf serum) and incubation with 1 μ/ml of B subunit for 21 h was sufficient to reduce DNA synthesis to 15% of control values (and confirmed by autoradiographic analysis), although maximal inhibition could be achieved with as little as 30 min exposure to B, followed by washing. Furthermore, the B subunit inhibited the response of the C6 cells to basic fibroblast growth factor only following GM1 pretreatment. The B subunit‐induced inhibition of DNA synthesis was specific for the ganglioside GM 1, and was unrelated to increases of cyclic AMP. These results demonstrate that cell‐incorporated GM1 ganglioside may act as a receptor capable of undergoing a specific ligand interaction, subsequently affecting molecular processes at the nuclear level.
Distribution of Elements in Rat Peripheral Axons and Nerve Cell Bodies Determined by X‐Ray Microprobe Analysis
Authors: LoPachin Richard M. , Lowery Joan , Eichberg Joseph , Kirkpatrick Joel B. , Cartwright Joiner , Saubermann Albert J.
Published: October 2006

Abstract: X‐ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, my‐elin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K. contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellu‐lar distribution of elements was clearly shown by color‐coded images generated by computer‐directed digital x‐ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.
Identification and Characterization of Gangliosides Reacting with IgM Paraproteins in Three Patients with Neuropathy Associated with Biclonal Gammopathy
Authors: Ilyas Amjad A. , Willison Hugh J. , Dalakas Marinos C. , Whitaker John N. , Quarles Richard H.
Published: October 2006

Abstract: IgM monoclonal antibodies from three patients with polyneuropathy associated with biclonal gammopathy reacted with monosialoganglioside GM1 on thin‐layer chromatograms. An IgM paraprotein in one of the patients with a predominantly motor neuropathy also reacted strongly with the ganglioside GD1b and asialo‐GM1. All three of these antigenic lipids have a Gal(β1–3)GalNAc moiety in common which would appear to be the antigenic determinant. However, this IgM also cross‐reacted weakly with paragloboside which has an N‐acetyllactosaminyl [Gal(β1–4)GlcNAc] terminal structure. The specificity of the other paraprotein in this patient is not known. The IgM paraproteins reacting with GM1 in both of the other patients exhibited different specificity because they did not react with GD1 b and asialo‐GM1, but reacted strongly with GM2 ganglioside. The data suggest that the epitope for both of these IgMs is in the GalNAc(β31–4)(NeuAcα2–3)Gal(β1–4)Glc region of the gangliosides that is common to both GM2 and GM1. The second IgM paraproteins in both of these latter patients react with the myelin‐associated glycoprotein (MAG) and two 3‐sulfoglucuronyl glycolipids that share antigenic determinants with MAG.
Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate
Authors: Erecińska Maria , Zaleska Malgorzata M. , Nissim Itzhak , Nelson David , Dagani Fiorenzo , Yudkoff Marc
Published: October 2006

Abstract: The metabolism of [15N]glutamate was studied with gas chromatography‐mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]‐Glutamate was taken up rapidly by the preparation, reaching a steady‐state level in <5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into γ‐aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2‐oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transami‐nation to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate de‐hydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factors), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.
Characterization of [3H]Paroxetine Binding in Rat Brain
Authors: Marcusson Jan O. , Bergstrom Maria , Eriksson Kristina , Ross Svante B.
Published: October 2006

Abstract: The binding of the 5‐hydroxytryptamine (5‐HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 μl, the binding was optimized with an apparent dissociation constant (KD) of 0.03–0.05 nM. Competition experiments with 5‐HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single‐site binding model. Fluoxetine and imipramine revealed, in addition to a high‐affinity (nanomolar) site, also a low‐affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5‐HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5‐HT‐ and fluoxetine‐sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5‐HT (100 μM) sensitive [3H]paroxetine binding were also consistent with a single‐site binding model, and the binding was competitively inhibited by 5‐HT and imipramine. The number of binding sites (Bmax) for 5‐HT‐sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p‐chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a >60% reduction in the striatum and hypothalamus, indicating a selective localization on 5‐HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5‐HT uptake in rat brain.
Cold Blockade of Axonal Transport Activates Premitotic Activity of Schwann Cells and Wallerian Degeneration
Authors: Oaklander Anne Louise , Spencer Peter S.
Published: October 2006

Between 3 and 4 days after transection of cat sciatic nerve, Schwann cell‐associated premitotic activity spreads anterogradely along degenerating distal nerve stumps at a rate of approximately 200 mm/day. We investigated whether fast anterograde axonal transport contributes to the initiation of this component of Wallerian degeneration. Axonal transport was blocked in intact and transected cat sciatic nerves by focally chilling a proximal segment to temperatures below 11°C for 24 hr. Incorporation of [3H]thymidine (a marker of premitotic DNA synthesis) was then measured 3 and 4 days posttransection in cold blocked‐ and control‐degenerating nerves. Effects of cold block prior to and concomitant with nerve transection were studied. Results failed to support the hypothesis that Schwann‐cell premitotic activity after axotomy is associated with entry into the axon of mitogenic substances and their anterograde fast transport along the distal stump. Instead, data suggested that progressive anterograde failure of fast anterograde transport distal to transection serves to effect the Schwann‐cell premitotic response to axotomy.
Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1
Authors: Sadoul Karin , Sadoul Rémy , Faissner Andreas , Schachner Melitta
Published: October 2006

The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane‐attached, phosphorylated carboxy terminal L1–30. This site is cleaved during treatment of live cultured cells with broad‐spectrum, protease‐free phospholipase C (but not phosphatidylinositol‐specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1–180 and L1–30, suggesting a particular cell‐intrinsic cleavage mechanism. L1–180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane‐integrated phosphorylated L1–30. Phase separation experiments with Triton X‐114 show that the released forms of L1–180 and L1–140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane‐anchored L1–30, L1–140 is released into the supernatant together with L1–50. A strong affinity of L1–200, L1–140, and L1–80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L1‐containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin‐associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.
Opiate‐Inhibited Adenylate Cyclase in Rat Brain Membranes Depleted of Gs‐Stimulated Adenylate Cyclase
Authors: Childers Steven R.
Published: October 2006

Opiate agonists inhibit adenylate cyclase in brain membranes, but under normal conditions the maximal inhibition is small (10–15%). When rat brain membranes were preincubated at pH 4.5, washed, and then assayed for adenylate cyclase at pH 7.4, stimulation of activity by agents (fluoride, guanylyl‐5′‐imidodiphosphate, cholera toxin) that act through the stimulatory GTP‐binding coupling protein (Gs) protein was lost. At the same time, inhibition of basal adenylate cyclase by opiate agonists was increased to a maximum of 30–40%. Opiate inhibition was maximal at low magnesium concentrations (<5 mM), required guanine nucleotides, and decreased the Vmax, not Km, of the enzyme. Incubation of membranes with per tussis toxin lowered the apparent affinity for agonists in inhibiting activity. The δ opioid agonists were more potent than μ agonists, and the Ke values for naloxone in blocking agonist inhibition were similar for both μ and δ agonists (50–90 nM). These results suggest that inhibition of adenylate cyclase in brain is not mediated by μ opiate receptors, but whether classic high‐affinity δ and k receptors are involved with this enzyme cannot be confirmed by these experiments.
Identification and Function of Octopamine and Tyramine Conjugates in the Limulus Visual System
Authors: Battelle B.‐A. , Edwards S. C. , Kass L. , Maresch H. M. , Pierce †S. K. , Wishart A. C.
Published: October 2006

Abstract: Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as γ‐glutamyl octopamine and γ‐glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine‐rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of γ‐glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that γ‐glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that γ‐glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.
High‐Resolution Proton Magnetic Resonance Spectroscopy of Rabbit Brain: Regional Metabolite Levels and Postmortem Changes
Authors: Petroff Ognen A. C. , Ogino Takashi , Alger Jeffry R.
Published: October 2006

The changes in 16 cerebral metabolites produced by cardiac arrest and subsequent room temperature auto‐lysis were studied using high‐resolution proton nuclear magnetic resonance spectroscopy. Biopsies of rabbit cerebral cortex, cerebral white matter, and cerebellum were quantitatively analyzed for acetate, alanine, γ‐aminobu‐tyric acid, creatine, glutamate, glycine, inositol, lactate, N‐acetylaspartate, phosphocreatine, succinate, taurine, and threonine. Of these, N‐acetylaspartate and the total creatine pool are the best candidates for use as concentration reference standards linking in vitro to in vivo 1H nuclear magnetic resonance measurements. Both changed little immediately after death, and they varied in a distinctive way among cortex, white matter, and cerebellum.
ADP‐Ribosylation of Highly Purified Rat Brain Mitochondria
Authors: Masmoudi A. , Islam F. , Mandel P.
Published: October 2006

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP‐ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP‐ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3‐aminobenzamide or other adenine nucleotides, such as ATP or ADP‐ribose. The inhibitory effect of nicotinamide or 3‐aminobenzamide on ADP‐ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP‐ribose and ATP appeared more effective in inhibiting ADP‐ribosyl transferase. Our results provide evidence for the existence of ADP‐ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP‐ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP‐ribose to mitochondrial proteins is oligomeric.