bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanolamine Base‐Exchange Reaction in Rat Brain Microsomal Subfractions
Authors: Corazzi Lanfranco , Porcellati Giuseppe , Freysz Louis , Binaglia Luciano , Roberti Rita , Arienti Giuseppe
Published: December 2006

Abstract: Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subtractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base‐exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury‐dextran produced inactivation of the ethanolamine base‐exchange reaction in the three subfractions. These findings suggest that the active site of base‐exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base‐exchange reaction has similar properties in all subfractions.
Enhancement of ATPase Activity by a Lipid Peroxide of Arachidonic Acid in Rat Brain Microvessels
Authors: Koide Tohru , Asano Takao , Matsushita Hiroshi , Takakura Kintomo
Published: December 2006

Abstract: The effects of 15‐hydroperoxyarachidonic acid (15‐hpaa) on Na+, K+‐ and Mg + ‐ATPase activities in the blood‐brain barrier (BBB) were examined using rat brain microvessels (MV). 15‐HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+ ‐ATPase activity was inhibited in a dose‐dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15‐HPAA in MV was not due to a simple detergent‐like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti‐edema drugs as 1,2‐bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered.
Extra‐ and Intracellular pH During Near‐Complete Forebrain Ischemia in the Rat
Authors: von Hanwehr Roger , Smith Maj‐Lis , Siesjö Bo K.
Published: December 2006

Abstract: The objective of the present study was to estimate extracellular pH (pHe) and intracellular pH (pHi) during near‐complete forebrain ischemia in the rat, and to evaluate the relative importance of lactic acidosis and rise in tissue Pco2, (Ptco2) in causing pHe and pHi to fall. The animals, which were ventilated, normoxic, normocapnic, and normothermic, were subjected to 15 min of ischemia, either without or with 30–60 min of recirculation. Ptco, was measured with a tissue electrode, pH, with a double‐barrel liquid ion‐exchanger microelectrode, changes in extracellular fluid (ECF) volume by impedance measurements, tissue CO, content by a microdiffusion technique, and labile tissue metabolites by enzymatic fluorometric methods. Ischemia caused Ptco2 to rise to between 95 and 190 mm Hg (mean 149 mm Hg), and pH, to fall by 0.45–1.05 units (mean 0.70 units). During recovery, Ptco, normalized within 5 min and pHe after 15–30 min. During ischemia, high‐energy phosphates were depleted and tissue lactate content increased to 15 μmol · g−1. The total CO2 content (Tco2) was minimally or moderately reduced (normal, 11.9 μmol · g−1; range of ischemic values, 7.9–12.1 μmol · g0‐, this range probably reflecting variable amounts of remaining blood flow. Impedance measurements demonstrated that ECF volume during ischemia was reduced to 55% of control, with gradual normalization during the first 15–30 min of recirculation. From values for Ptco2, Tco2, [HCO3−]e, and ECF volume, [HCO3−]i, and pH, could be calculated. These values pertain to an idealized homogeneous intracellular compartment, and the methods used cannot detect whether different intracellular compartments diverge in their acid‐base responses. Ischemia caused pH, to fall from 7.05 to a mean of 6.15 (range 5.9–6.4). Previous data, and those obtained at present, suggest that pH, normalizes after 15 min of recirculation, with a subsequent, secondary alkalosis. Calculations indicate that about half of the pH, change was due to the hypercapnia. In intracellular fluid, though, the hypercapnia must play a minor role in reducing pH, the predominate cause of the acidosis being lactic acid production.
Schwann Cell Marker Defined by a Monoclonal Antibody (224–58) with Species Cross‐Reactivity. I. Cellular Localization
Authors: Guerci A. , Monge M. , Evercooren A. Baron‐Van , Lubetzki C. , Dancea S. , Boutry J. M. , Goujet‐Zalc C. , Zalc B.
Published: December 2006

Abstract: We have demonstrated by indirect immunoflu‐orescence the cellular localization of a monoclonal antibody (mAb 224–58), produced after immunization of a mouse with human central nervous system (CNS) myelin. Serologically, mAb 224‐58 was found to be specific for 3′‐sulfomonogalactosylglycolipids, namely 3′‐sulfogalac‐tosylceramide (SGC) and 3′‐sulfogalactosyl 1‐O‐alkyl ether 2‐O‐acylglycerol (seminolipid). This mAb did not bind to SGC‐containing tissues such as kidney, liver, spleen, or brain, nor to muscle. However mAb 224–58 did stain positively mouse, rat, and human peripheral nerve sections. In these latter sections, mAb 224–58 was bound to Schwann cell bodies and processes. The specificity of mAb 224–58 for Schwann cells was ascertained on teased rat sciatic nerves and rat Schwann cell cultures. Cells positive for mAb 224–58 were also positive for laminin, and negative for Thy 1–1 antigens both in teased fibers and Schwann cell cultures. In addition, in teased nerve preparations, mAb 224–58‐positive cells were also galactosylceramide (GalC)‐ and SGC‐positive. Isolated Schwann cells also expressed 224–58 antigen, even after prolonged time in culture. On testis sections, which contain both SGC and seminolipid, the SGC‐positive cells, i.e., the spermatogonia, were always 224–58‐negative. But the other germinal cells were 224–58‐positive. This suggests that although 224–58 does not discriminate between SGC and seminolipid in serological tests, these lipids in their naturally occurring membrane acquire a spatial configuration that renders them distinguishable to their respective antibody.
Schwann Cell Marker Defined by a Monoclonal Antibody (224–58) with Species Cross‐Reactivity. II. Molecular Characterization of the Epitope
Authors: Goujet‐Zalc C. , Guerci A. , Dubois G. , Zalc B.
Published: December 2006

Abstract: A monoclonal antibody (mAb) designated 224–58 (IgM‐K) has been raised by fusion of Sp2/0‐Ag14 mouse hybridoma cell line with spleen cells of a mouse immunized with human brain myelin. This mAb binds specifically to mouse, rat, and human Schwann cells membrane. Serological tests showed that mAb 224–58 reacted with total lipid extract, but not with total protein extract of human myelin. Combination of ELISA, complement fixation, and immunoautoradiographic detection on silica gel TLC allowed us to determine that mAb 224–58 reacted with sulfomonogalactolipids, namely 3′‐sulfogalac‐tosylceramide (SGC) and 3′‐sulfogalactosyl 1‐O‐alkylether 2–O‐acylglycerol (seminolipid). The fine molecular structure of the epitope recognized by mAb 224–58 was established by studying the cross‐reactivity of this mAb toward closely related sulfolipids and by comparing its reactivity after submitting either purified sulfolipids or total lipid extracts to various chemical and enzymatic treatments. The lipidic hapten‐binding site to mAb 224–58 is dependent on (1) the sulfoester on carbon 3 of the galactose molecule, (2) the osidic bond, and (3) the carbonyl group of the fatty acid. Interestingly enough, neither the amide bond and the long‐chain base nor the OH group of the fatty acid belongs to the antigenic determinant recognized by mAb 224–58.
Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture
Authors: Yanagihara Nobuyuki , Tank A. William , Langan Thomas A. , Weiner Norman
Published: December 2006

Abstract: Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P‐peptides can be identified after paper electro‐phoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+‐induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP‐dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.
Purification and Characterization of a Soluble Cyclic Nucleotide‐Independent Ca2+‐Calmodulin‐Sensitive Protein Kinase from Rat Brain
Authors: Alderson Ralph F. , Sze Paul Y.
Published: December 2006

Abstract: Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl‐cellulose, ATP‐aga‐rose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442‐fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory sub‐unit from the type II cyclic AMP‐dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G‐200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 μM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.
Arginine‐Vasopressin Stimulates Inositol Phospholipid Metabolism in Rat Hippocampus
Authors: Stephens L. R. , Logan S. D.
Published: December 2006

Abstract: The hippocampal vasopressin receptors have been characterised by measuring the stimulated accumulation of inositol monophosphate in the presence of 10 mM LiCl after hippocampal slices were prelabelled with [3H]inositol. Arginine‐vasopressin caused a dose‐dependent increase in inositol monophosphate accumulation (ED50=7.1 nM). The response was unchanged in the absence of Ca2+ and significantly reduced in the presence of a V1,‐receptor antagonist. Equimolar oxytocin was ineffective as a stimulus. This suggests that the hippocampal receptors are of the V1 type.
Chloroquine Reduces Neuronal and Glial Iron Uptake
Authors: Swaiman Kenneth F. , Machen Valynda L.
Published: December 2006

Abstract: The effect of chloroquine, a lysosomotropic agent, on iron uptake into neuronal and glial cell cultures is reported. Chloroquine significantly inhibited iron uptake in both neuronal and glial cells. These findings suggest that iron transport into both neuronal and glial cells is mediated by the transferrin‐iron complex.
Release of Monoamines from Striatum of Rat and Mouse Evoked by Local Application of Potassium: Evaluation of a New In Vivo Electrochemical Technique
Authors: Gerhardt Greg A. , Rose Greg M. , Hoffer Barry J.
Published: December 2006

Abstract: Local application of K+ via micropressure‐ejection, coupled with in vivo electrochemical detection, was used to study stimulated release from monoaminergic nerve terminals in the striatum of anesthetized rats and mice. K − ‐evoked releases were reversible, reproducible, and dose‐dependent. In contrast, releases of electroactive species could not be evoked by local ejection of Na+. The magnitudes and time courses of K+‐evoked releases recorded from the caudate nucleus of mice were greater than those seen in rats. Local application of nomifensine, a putative catecholamine reuptake blocker, augmented the magnitudes and time courses of K+‐evoked releases. Releases were also recorded from brain regions adjacentthe striatum; these signals were always smaller than those seen in the caudate nucleus and had amplitudes that showed good correspondence to the relative degree of dopaminergic input to these areas. These data, taken together with other information in the literature, suggest that this new technique is well suited for in situ studies of monoamine release and reuptake in intact animals.
Immunochemical and Immunohistochemical Localization of Parvalbumin in Rat Nervous Tissues
Authors: Endo Toyoshi , Takazawa Kazunaga , Kobayashi Shigeru , Onaya Toshimasa
Published: December 2006

Abstract: The contents of parvalbumin in various nervous tissues of the rat were measured by radioimmunoassay (RIA) and its cellular distribution was immuno‐histochemically examined by peroxidase‐antiperoxidase methods. The antibody, raised in rabbits using rat skeletal muscle parvalbumin, did not cross‐react with other Ca2+ ‐binding proteins such as calmodulin or S‐100 proteins. The RIA demonstrated the wide distribution of the antigen, with very high levels in the cerebellum (3.217 ± 519 ng/mg protein). The immunohistochemical description by Celio and Heizmann [Nature 293, 300‐302 (1981)] was confirmed concerning the existence of the antigen in Purkinje cells of the cerebellum; nonpyramidal neurons of the cerebral cortex; and medium‐sized cells of the olfactory bulb, hippocampus, and reticular nucleus of the thalamus. In addition to these neurons, we found the parvalbumin‐like immunoreactivity in the large neurons of the superior vestibular nucleus and the neurons of the medial superior olive nucleus. In the γ‐aminobutyric acid (GABA)‐containing nuclei such as substantia nigra, caudatoputamen, and globus pallidus. parvalbumin‐positive cells and fibers were rare. In the medial lemniscus of the midbrain which contains no GABA, parvalbumin‐immunoreactive fibers were prominent. The possibility was discussed that parvalbumin exists in a specific population of neurons that differ from those containing GABA.
Studies on a Possible Functional Coupling Between Presynaptic Acetylcholinesterase and High‐Affinity Choline Uptake in the Rat Brain
Authors: Raiteri Maurizio , Marchi Mario , Caviglia Anna Maria
Published: December 2006

Abstract: The relationships between presynaptic acetylcho‐linesterase (AChE) and high‐affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: (a) determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; (b) determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; (c) evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and (d) comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 μM [N‐methyl‐3H] ACh was two‐thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was of the amount passing through the synapto‐somal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.
Distribution of Glial Fibrillary Acidic Protein in Gliosed Human White Matter
Authors: Newcombe J. , Woodroofe M. N. , Cuzner M. L.
Published: December 2006

Abstract: Glial fibrillary acidic protein (GFAP) in gliosed white matter from multiple sclerosis plaques and cerebral infarcts was examined by polyacrylamide gel electrophoresis and immunoblotting. Using a monoclonal antibody raised against human GFAP, up to 11 GFAP polypeptide bands of molecular weight 37–49 kilodaltons were identified in paniculate and supernatant fractions of CNS tissue homogenates. Soluble GFAP constituted about one‐quarter of the total GFAP in normal cerebral white matter. In brain lesions in which reactive astrocytes were observed microscopically, the proportion of soluble GFAP was increased, with a greater representation of the lower‐molecular‐weight forms. In brain chronic sclerotic plaques, almost all of the GFAP was in the paniculate form. Purified paniculate GFAP was susceptible to proteolysis at acid but not at neutral pH in the presence of CNS homogenates. In tissue autolysis studies, GFAP was stable in situ for periods well in excess of average CNS postmortem times.
Subcellular Localization of Angiotensin‐Converting Enzyme in Cultured Neuroblastoma Cells
Authors: Clemens D. L. , Okamura T. , Inagami T.
Published: December 2006

Abstract: Angiotensin‐converting enzyme (ACE) activity of Neuro‐2A mouse neurobiastoma cells was found predominantly in particulate fractions. Density gradient centrifugation of the particulate fractions showed ACE activity in light fractions of the gradient, a result suggesting a plasma membrane localization. This was confirmed using the aqueous two‐phase polymer system of plasma membrane isolation. The rapid and energy‐independent hydrolysis of exogenous substrate by ACE of intact cells and the sensitivity of the enzyme of intact cells to proteases indicate further that the active site of ACE is oriented extracellularly.
Ethanolamine Kinase Activity in Purified Myelin of Rat Brain
Authors: Kunishita Tatsuhide , Vaswani Kuldeep K. , Morrow Charles R. , Novak Gerald P. , Ledeen Robert W.
Published: December 2006

Abstract: Highly purified rat brain myelin showed a significant level of ethanolamine kinase, amounting to 17% of the specific activity of whole brain homogenate. This kinase level in myelin was an order of magnitude higher than that of lactate dehydrogenase, a marker for cytosol. Subcellular distribution studies revealed that in addition to myelin, this kinase was present in the P1, P2, P3, and cytosolic fractions with highest relative specific activity in the latter. The possibility that myelin activity resulted from adsorption of the soluble enzyme was unlikely since activity was retained in myelin that had been washed with buffered sodium chloride or taurocholate. Mixing experiments and repeated purification further indicated that the enzyme is intrinsic to myelin. Kinetic studies indicated similar Km values for ethanolamine in the microsomal, cytosolic, and myelin fractions but a significantly lower apparent Km for ATP in myelin. This and other differences suggested the possible existence of isozymes. Establishment of the presence of this kinase completes the list of phospholipid synthesizing enzymes needed to synthesize phosphatidylethanolamine from diacylglycerol within the myelin membrane.
Kinetic Modeling of Receptor‐Ligand Binding Applied to Positron Emission Tomographic Studies with Neuroleptic Tracers
Authors: Logan J. , Wolf A. P. , Shiue C.‐Y. , Fowler J. S.
Published: December 2006

Abstract: Positron emission tomography (PET) with labeled neuroleptics has made possible the study of neurotransmitter‐receptor systems in vivo. In this study we investigate the kinetics of the 3, 4‐dihydroxyphenylethylamine (dopamine) receptor‐ligand binding using PET data from a series of experiments in the baboon with the 18F‐labeled drugs spiperone, haloperidol, and benperidol. Models used to describe these systems are based on first‐order kinetics which applies at high specific activity (low receptor occupancy). The parameters governing the uptake and loss of drug from the brain were found by fitting PET data from regions with little or no receptor concentration (cerebellum) and from experiments in which specific binding was blocked by pretreatment with the drug (+)‐butaclamol. Receptor constants were determined by fitting data from receptor‐containing structures. Correcting the arterial plasma activities (the model driving function) for the presence of drug metabolites was found to be important in the modeling of these systems.
N1‐Methyl‐2‐125I‐Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors
Authors: Hoffman Beth J. , Scheffel Ursula , Lever John R. , Karpa Michael D. , Hartig Paul R.
Published: December 2006

Abstract: Methylation of 2‐125I‐lysergic acid diethylamide (125I‐LSD) at the N1 position produces a new derivative, N1‐methyl‐2‐125I‐lysergic acid diethylamide (125I‐MIL), with improved selectivity and higher affinity for serotonin 5‐HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I‐MIL represents 80–90% of total binding, and the apparent dissociation constant (KD) for serotonin 5‐HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I‐MIL also displays a high affinity for serotonin 5‐HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I‐MIL exhibits at least 60‐fold higher affinity for serotonin 5‐HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I‐MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0°C. Autoradiographic experiments confirm the improved specificity of 125I‐MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I‐MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I‐MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.
Ontogenesis of Adenosine Deaminase Activity in Rat Brain
Authors: Geiger J. D. , Nagy J. I.
Published: December 2006

Abstract: The activity of adenosine deaminase (ADA) was determined in whole brain of rats at the embryonic age of 15 days through to adulthood and in nine brain regions in rats 1 day old through to adulthood. In 1‐day‐old rats, the highest activity was seen in olfactory bulbs (550 ± 15 nmol/mg protein/30 min) and this was 4.5‐fold higher than that in the pons, which was the lowest. In adult animals, olfactory bulb still contained the greatest activity, which was about eightfold higher than hippocampus, which had the lowest. Except for hypothalamus, where ADA activity increased nearly twofold in rats between the ages of 1 and 50 days, significant decreases of as much as fivefold were found in whole brain, superior colliculus, cortex, hippocampus, cerebellum, olfactory bulbs, and olfactory nucleus. In contrast, ADA activity in pons and subcortex remained relatively constant throughout the developmental period. The Km values for ADA in whole brain at 18 days gestation (48 ± 5 μM) were not significantly different from that observed in adult rats (38 ± 7 μM), whereas the Vmax values decreased significantly from 339 ± 9 to 108 ± 8 nmol/mg protein/30 min. Taken together, the developmental patterns observed in the various brain regions appear not to correspond to any one particular process such as periods of rapid cell proliferation, cell death, synaptogenesis, or myelination. Nor do they correspond to known developmental profiles of transmitters, their receptors, or their metabolic enzymes. The complex changes in ADA activity during ontogenesis suggest an important role of ADA at very early stages of development as well as in specific regions of the adult rat brain.
Lactate and pH in the Brain: Association and Dissociation in Different Pathophysiological States
Authors: Paschen W. , Djuricic B. , Mies G. , Schmidt‐Kastner R. , Linn F.
Published: December 2006

Abstract: Brain tissue pH and lactate content were measured in rats under three different experimental conditions, namely: (1) during complete global cerebral ischemia; (2) after reversible near‐complete cerebral ischemia; and (3) in experimental brain tumors. At the end of the experiments brains were frozen with liquid nitrogen. A series of 20‐μm thick coronal sections was prepared in a cryostat and then used for the regional determination of tissue pH (umbelliferone technique) and tissue lactate (bioluminescent technique). In addition, tissue samples were taken for the quantitative measurement of brain lactate (enzymatic fluorometric technique). The relationship between lactate content and tissue pH was different for each of the three experimental models studied: only after short‐term global cerebral ischemia did an increase in the lactate content correlate with a decrease in tissue pH (r= 0.94; p < 0.001). A highly significant increase in the lactate content (p < 0.001) was accompanied by physiological pH values (6.96 ± 0.08 in comparison to 6.97 ± 0.04 in controls) during recirculation after transient cerebral ischemia and in brain tumors even by an alkaline pH shift. In view of these observations the term “lactacidosis” should not be used without measuring both the lactate content and the pH. The observed dissociation between pH and lactate is due to the fact that both parameters are regulated independently. During anaerobiosis the main source of proton production is ATP hydrolysis rather than glycolysis. It is, therefore, suggested that the terms “acidosis” and “lactosis” should be used instead of “lactacidosis.”
Endogenous Basic Protein Phosphatases in the Brain Myelin
Authors: Yang Shiaw‐Der , Liu Jen‐Sing , Fong Yiu‐Lian , Yu Jau‐Song , Tzen Tse‐Cheng
Published: December 2006

Abstract: Direct treatment of brain myelin with freezing/thawing in 0.2 M2‐mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P‐la‐beled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X‐100, this endogenous latent phosphatase activity was further stimulated 2.5‐fold. Diethylaminoethyl‐cellulose and Sephadex G‐200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M2‐mercaptoethanol and with a molecular weight of 350, 000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP. Mg‐dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high‐molecular‐weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.
Neurochemistry of GABAergic System in Cerebral Cortex Chronically Exposed to Bromide In Vivo
Authors: Balcar Vladimir J. , Erdö Sandor L. , Joó Ferenc , Kása Péter , Wolff Joachim R.
Published: December 2006

Abstract: Neurochemical correlates of GABAergic synaptic transmission [binding, uptake, metabolism, and tissue content of γ‐aminobutyric acid (GABA)] were investigated in the cortex of rats that had been given 27 mM bromide in drinking water for periods of time ranging from 1 day to 1 month. No effect of bromide on any of the parameters was found and it is concluded that chronic administration of bromide has no profound effect on GABAergic inhibitory system in the rat cortex.
γ‐Aminobutyric Acid‐Activated 36Cl‐ Influx: A Functional In Vitro Assay for CNS γ‐Aminobutyric Acid Receptors of Insects
Authors: Wafford K. A. , Sattelle D. B. , Abalis I. , Eldefrawi A. T. , Eldefrawi M. E.
Published: December 2006

Abstract: The effects of γaminobutyric acid (GABA) on the uptake of 36Cl into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 μM GABA (after 4‐s incubation, then rapid quenching) the influx of 36Cl‐ was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 μM), but was not significantly affected by bicuculline (100 μM)Results of 36Cl‐influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.
Degradation of Luteinizing Hormone‐Releasing Hormone by Neuroblastoma Cells and Their Membrane: Evidence for the Involvement of a Thiol Protease and Angiotensin‐Converting Enzyme
Authors: Yokosawa Hideyoshi , Fujii Yurika , Ishii Shin‐ichi
Published: December 2006

Abstract: Luteinizing hormone‐releasing hormone was degraded by cells of the N‐ I8 line of mouse neuroblastoma and their membrane. Cleavage products were separated by HPLC and identified by amino acid analysis. Fragments (1‐3), (4‐5), and (6‐10) were major cleavage products. All the products increased in level as a function of time except for fragment (1‐9, which increased in amount only during a short incubation time and then decreased. The accumulation of fragment (1‐5) was increased in the presence of captopnl or EDTA, whereas that of fragments (1‐3) and (4‐5) decreased inversely. On the other hand, the generation of either fragment (1‐3) or (4‐5) was stimulated by the presence of C1‐. The results suggest that the conversion of fragment (1‐5) into fragments (1‐3) and (4‐5) is catalyzed by angiotensin‐converting enzyme. p‐Chloromercuribenzoate inhibited the formation of fragment (1‐5), a result suggesting the involvement of a thiol protease in this formation. Thus, the degradation of luteinizing hormone‐releasing hormone by neuroblastoma cells and their membrane seems to take place mainly through the cleavage of the Tyr5‐Gly6 bond by a thiol protease, followed by the release of the dipeptide Ser‐Tyr from the liberated fragment (1‐5) by angotensin‐converting enzyme. It is further suggested that the thiol protease and angiotensin‐converting enzyme are also responsible for the initial minor cleavages of the Gly6‐Leu7 bond and the Trp3‐Ser4 bond, respectively.
Differential Regulation by Butyrate and Dibutyryl Cyclic AMP of δ‐Opioid, α2‐Adrenergic, and Muscarinic Cholinergic Receptors in NCB‐20 Cells
Authors: Zhu Xing‐Zu , Chuang De‐Maw
Published: December 2006

Abstract: Long‐term treatment of NCB‐20 cells with sodium butyrate resulted in a marked increase in the specific binding of [3H]d‐Ala2, d‐Leu5 enkephalin. This increase wm concentration and time dependent, with an EC50 of about 480 μM and a maximal effect detected after 3‐day treatment. At saturating concentration of butyrate (1 mM) the increase was three‐ to fourfold of the untreated control. Scatchard analysis revealed that the butyrate effect was due to an increase in the density of the opioid receptor binding sites. Butyrate also induced a smaller (about twofold) increase in the density of muscarinic cholinergic receptor binding assessed by using [3H]quinuclidinyl benzilate, whereas α2‐adrenergic receptor binding assessed by using [3H]clonidine was not significantly affected. The butyrate‐induced opioid receptor binding could be totally abolished by the presence of cycloheximide, suggesting that the butyrate effect involves synthesis of the receptor protein. Butyrate treatment did not affect basal and prostaglandin E1‐stimulated cyclic AMP levels but caused a three‐ to fourfold decrease in the IC50 of d‐Ala2, d‐Leu5 enkephalin for attenuating these cyclic AMP levels and approximately 25% increase in the maximal extent of attenuation. In contrast to the butyrate effect, long‐term treatment of NCB‐20 cells with 1 mM dibutyryl cyclic AMP induced an 80% decrease in the opioid and α2‐adrenergic receptor bindings and a 57% loss of muscarinic cholinergic receptor binding. This down‐regulation of muscarinic cholinergic receptor binding sites was associated with a 35% decrease of carbachol‐induced phosphoinositide breakdown, whereas the receptor upregulation induced by butyrate was found to increase the carbachol response by about threefold. The differential regulation by butyrate and dibutyryl cyclic AMP suggests that the butyrate effect is mediated by a mechanism independent of intracellular cyclic AMP. The induction by butyrate of opioid receptors and muscarinic cholinergic receptors in NCB‐20 cells may provide a useful system for studying the regulation of gene expression of these receptor proteins.
Blood–Brain Transport of Thiamine Monophosphate in the Rat: A Kinetic Study In Vivo
Authors: Patnni Cesare , Reggiani Carlo , Laforenza Umberto , Rindi Gianguido
Published: December 2006

Abstract: To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood‐brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi·mmol−1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half‐saturation concentration (Km) equaled 27–39 pmol·g−1·min−1 and 2.6–4.8 μM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol·g−1· min−1, thus representing a significant component of thiamine supply to nervous tissue.
A Cyclic AMP Analogue Induces Synthesis of a Myelin‐Specific Glycoprotein by Cultured Schwann Cells
Authors: Shuman Sandra , Hardy Mattie , Sobue Gen , Pleasure David
Published: December 2006

Abstract: Neonatal rat Schwann cells, cultured with agents which increase intracellular cyclic AMP, were prompted to resume synthesis of a 170,000 Mr glycoprotein which is specific to peripheral nervous system myelin and is herein referred to as P170k. We have shown previously that similar treatment induces the synthesis by Schwann cells of the myelin lipid, galactocerebroside. In contrast to P170K and galactocerebroside, syntheses of P0 and myelin basic protein were not induced. Intracellular cyclic AMP is thus likely to be a participant in the complex system regulating myelination.
Changes in the Relative Amounts of Aspartate and Glutamate Released and Retained in Hippocampal Slices During Stimulation
Authors: Szerb John C.
Published: December 2006

Abstract: It has been found previously that the ratio of aspartate to glutamate released and retained by brain slices reversibly changes with changing glucose concentrations in the medium. To find out whether increased neuronal activity also results in changes in the ratio of aspartate to glutamate, in this study electrical‐field stimulation was applied for 10 min to hippocampal slices in the presence of 0.2–5 mM glucose. In 5 mM glucose, the ratio of aspartate to glutamate released did not change during stimulation, but the amount of aspartate retained at the end of stimulation was reduced. In contrast, in 1 mM or less glucose, the ratio of aspartate to glutamate released increased progressively and the rate of increase was inversely proportional to the glucose content of the medium. The evoked release of aspartate and glutamate both in low and high glucose was nearly suppressed in low (0.1 mM) Ca2+ or by tetrodotoxin. In low glucose, the ratio of aspartate to glutamate contained in the slices also increased as a result of stimulation. This increase was reduced only a little in low Ca2+, but was nearly eliminated by tetrodotoxin. Results suggest that increased neuronal activity causes a shift in the ratio of aspartate to glutamate released in the presence of glucose concentrations similar to those found in the brain in normoglycemic rats. This shift, due to an increased energy demand, probably originates from terminals which release aspartate and glutamate in different proportions.
In Vivo Substitution of Choline for Sodium Evokes a Selective Osmoinsensitive Increase of Extracellular Taurine in the Rat Hippocampus
Authors: Lehmann Anders , Sandberg Mats
Published: December 2006

Abstract: Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis‐perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl‐ with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane‐anesthetized rats was dialyzed with Krebs‐Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium‐containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl‐ with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl‐ substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl‐ causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.
Hyaluronate‐Binding Proteins of Murine Brain
Authors: Marks Michelle S. , Chi‐Rosso Gloria , Toole Bryan P.
Published: December 2006

Abstract: The distribution of hyaluronate‐binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low‐ionic‐strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate‐binding activities were found to be of high affinity (KD= 10‐9M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate‐binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL‐4B and CL‐6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)‐cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate‐binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500‐fold purification of this proteoglycan‐like, hyaluronate‐binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL‐6B, and ion exchange chromatography on DEAE‐cellulose. Another class of hyaluronate‐binding material (25–50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate‐binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.
Marked Amine and Amine Metabolite Changes in Norrie Disease Patients with an X‐Chromosomal Deletion Affecting Monoamine Oxidase
Authors: Murphy D. L. , Sims K. B. , Karoum F. , de la Chapelle A. , Norio R. , Sankila E.‐M. , Breakefield X. O.
Published: December 2006

Abstract: Urinary and plasma amines and amine metabolites were quantified in two individuals with Norrie disease resulting from a deletion in chromosomal region Xp 11.3, recently reported to be associated with absence of the gene encoding monoamine oxidase (MAO)‐A and nondetectable MAO‐A activity in fibroblasts and MAO‐B activity in platelets. Marked (four‐ to 100‐fold) elevations in levels of urinary phenylethylamine, o‐tyramine, and m‐tyramine (which are preferential substrates for MAO‐B) and marked reductions (90%) in levels of 3‐methoxy‐4‐hydroxyphenylglycol (a deaminated metabolite of norepinephrine, a preferential substrate for MAO‐A) in urine and plasma confirmed the presence of a systemic, functionally significant reduction in the activities of both MAO isozymes. The magnitude of these changes, which are equivalent to those found in subjects taking MAO‐inhibiting antidepressants, suggests that early initiation of dietary and drug restrictions may be clinically important in these and other patients with X‐chromosomal mutations involving MAO. These findings further support the proposition that the MAOA and MAOB genes are located in close proximity on the X chromosome. Negligible changes in the metabolites of dopamine and serotonin raise the possibility that other metabolic pathways are of importance for their production, that dietary or intestinal bacterial sources contribute substantially to the presence of these amine metabolites in urine, or both.
Protein Kinase C and Its 80‐Kilodalton Substrate Protein in Neuroblastoma Cell Neurite Outgrowth
Authors: Girard Peggy R. , Kuo J. F.
Published: December 2006

Abstract: A potential role of the protein kinase C (PKC) system in differentiation of human neuroblastoma cell line LA‐N‐5 was investigated. It was found that neurite outgrowth induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA, 81 nM) was associated with a down‐regulation of PKC as determined independently by immunocytochemistry, immunoblot, and enzyme activity assay. Down‐regulation of PKC in cells induced to differentiate by retinoic acid (1 μM) was less pronounced, whereas it was undetected in cells induced to differentiate by nerve growth factor (100 ng/ml). The in vitro phosphorylation of an 80‐kilodalton protein present in control LA‐N‐5 cells or in cells treated with TPA, retinoic acid, or nerve growth factor for 1 day decreased to various extents at days 4 or 7 concomitant with neuritogenesis. Pretreatment of LA‐N‐5 cells with a high concentration (1 μM) of TPA to deplete cellular PKC rendered the cells unresponsive to the differentiating effect of the agents. It was observed that CHP‐100 cells, another human neuroblastoma line shown to be resistant to differentiation induced by the agents, had a reduced PKC level and the amount of in vitro phosphorylation of the 80‐kilodalton protein was greatly reduced in control cells and remained relatively unchanged when the cells were treated with the agents for up to 7 days. The present studies suggested that PKC and its 80‐kilodalton substrate protein were likely involved in initiation and/or progression of LA‐N‐5 cell differentiation induced by TPA and that separate PKC‐independent pathways might also be involved in the differentiating effect of retinoic acid or nerve growth factor.
Stress Preferentially Increases Extraneuronal Levels of Excitatory Amino Acids in the Prefrontal Cortex: Comparison to Hippocampus and Basal Ganglia
Authors: Moghaddam Bita
Published: December 2006

Abstract: The technique of intracerebral microdialysis was used to assess the effect of stress on the extracellular concentrations of excitatory amino acids, glutamate and aspartate, in the rat medial prefrontal cortex, hippocampus, striatum, and nucleus accumbens. A 20‐min restraint procedure led to an increase in extracellular glutamate in all regions tested. The increase in glutamate levels was significantly higher in the prefrontal cortex than that observed in other regions. With the exception of the striatum, extracellular levels of aspartate were increased in all regions. Furthermore, the increase in aspartate levels was significantly higher in prefrontal cortex compared to hippocampus and nucleus accumbens. Local perfusion of tetrodotoxin during the restraint procedure significantly decreased the stress‐induced increase in extracellular excitatory amino acids. In order to ensure that the above results were not an artifact of restraint not associated with stress (e.g., decreased mobility), we also examined the effect of swimming stress on the extracellular levels of excitatory amino acids in selected regions, i.e., striatum and medial prefrontal cortex. Both regions displayed a significant increase in extracellular levels of aspartate and glutamate following 20 min of swimming in room temperature water. This study provides direct evidence that stress increases the neuronal release of excitatory amino acids in a regionally selective manner. The implications of the present findings for stress‐induced catecholamine release and/or hippocampal degeneration are discussed.