bluuebunny/crossref_metadata_2025 huggingface dataset:
Variability in the Structural Requirements for Binding of Human Monoclonal Anti‐Myelin‐Associated Glycoprotein Immunoglobulin M Antibodies and HNK‐1 to Sphingoglycolipid Antigens
Authors: Ilyas Amjad A. , Chou Denise K. H. , Jungalwala Firoze B. , Costello Catherine , Quarles Richard H.
Published: October 2006

A high proportion of patients with neuropathy have immunoglobulin M (IgM) paraproteins that react with carbohydrate determinants on the myelin‐associated glycoprotein (MAG) and two sphingoglycolipids, 3‐sulfoglucuronyl paragloboside (SGPG) and 3‐sulfoglucuronyl lactosaminyl paragloboside. In order to characterize the fine specificities of these human antibodies further, the binding of 10 anti‐MAG paraproteins to several chemically modified derivatives of SGPG was compared with the binding to intact SGPG by both TLC‐overlay and enzyme‐linked immunosorbent assay. The following derivatives were tested: the desulfated lipid, glucuronyl paragloboside (GPG); the methyl ester of GPG (MeGPG); the methyl ester of SGPG, 3‐sulfomethylglucuronyl paragloboside (SMeGPG); and 3‐sulfoglucosyl paragloboside (SGlcPG) produced by reduction of the carboxyl group of the glucuronic acid with sodium borohydride. All 10 IgM paraproteins and the related mouse IgM antibody, HNK‐1, reacted most strongly with intact SGPG, but variations in the reactivity with the derivatives revealed striking differences in the structural requirements for binding between the antibodies. Five distinct patterns of reactivity were observed: (a) three of the human antibodies and HNK‐1 exhibited partial reactivity with the sulfated derivatives, SMeGPG and SGlcPG, but not with GPG or MeGPG, indicating an absolute requirement for the sulfate group; (b) two of the human antibodies reacted only with GPG, demonstrating a requirement for the free carboxyl group on the glucuronic acid; (c) three of the antibodies bound to SMeGPG, SGlcPG, and GPG, but not to MeGPG, suggesting that at least one negative charge was needed for binding; (d) one antibody bound to SMeGPG and GPG, but not to SGlcPG or MeGPG, suggesting that both the carbonyl group and at least one negative charge were required; and (e) another antibody bound to MeGPG, SMeGPG, and SGlcPG, but not to GPG, a pattern that is difficult to explain simply based on the chemical structures. Interestingly, only those antibodies that exhibited reactivity with GPG bound to a third, minor, unidentified glycolipid of normal peripheral nerve that chromatographs faster than SGPG. The results clearly demonstrate heterogeneity in the fine specificities of the anti‐MAG antibodies that may affect their pathogenic properties.
Characterization of Opioid Receptors in Cultured Neurons
Authors: Vaysse Pierre J.‐J. , Zukin R. Suzanne , Fields Kay L. , Kessler John A.
Published: October 2006

The appearance of μ‐, δ‐, and k‐opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high‐affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (‐)‐[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax= 210 ± 40 fmol/mg protein); this density is approximately two‐thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the μ‐specific ligand [3H]D‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 ± 0.1 nM and a density (Bmax) of 160 ± 20 fmol/mg of protein. Specific binding of (‐)‐[3H]bremazocine under conditions in which μ‐ and δ‐receptor binding was suppressed (k‐receptor labeling conditions) occurred to an apparent single class of sites (KD= 2 ± 1 nM;Bmax= 40 ± 15 fmol/mg of protein). There was no detectable binding of the selective δ‐ligand [3H]D‐Pen2,d‐Pen5‐enkephalin. Thus, cultured striatal neurons expressed μ‐ and k‐receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not δ‐receptors.
Inositol Metabolism During Neuroblastoma B50 Cell Differentiation: Effects of Differentiating Agents on Inositol Uptake
Authors: Reboulleau Christian P.
Published: October 2006

Inositol uptake was studied in the rat CNS neuroblastoma B50 cell line. Eadie‐Hofstee analysis of the uptake pattern reveals two defined modes of inositol entry into the cell. The high‐affinity uptake component requires the presence of extracellular sodium and is inhibited by phloridzin. Analysis of the uptake velocities of the high‐affinity uptake component provided the following apparent kinetic parameters: Km= 13.7 μM and Vmax= 14.7 pmol/mg of protein/min (without correcting for residual diffusion) and Km= 12.9 μM and Vmax= 12.3 pmol/mg of protein/min (with correction). At physiological concentrations, the high‐affinity . transport process contributes approximately 70% to total uptake; the remainder is due to a low‐affinity diffusion‐like process. Uptake inhibition studies reveal that the uptake process is sensitive to ouabain, amiloride, and dichlorobenzamil inhibition but relatively insensitive to cytochalasin B or phloretin. When neuroblastoma B50 cells are induced to differentiate morphologically with high extracellular calcium or with dibutyryl cyclic AMP, a significant decrease in inositol uptake is observed. The dibutyrl cyclic AMP‐mediated inhibition of uptake affects only the high‐affinity uptake component and is noncompetitive in nature. The high extracellular calcium‐mediated inhibition is less specific; it involves “disappearance” of the high‐affinity process, some inhibition of the low‐affinity process, and an increase of inositol efflux. The significance of these observations is discussed in the context of neuroblastoma B50 cell differentiation.
Regional Central Nervous System Oligosaccharide Storage in Caprine β‐Mannosidosis
Authors: Boyer Philip J. , Jones Margaret Z. , Rathke Eileen J. S. , Truscott Nancy K. , Lovell Kathryn L.
Published: October 2006

Goats affected with β‐mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme β‐mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(β1–4)GlcNAc(β1–4)GlcNAc] and a disaccharide [Man(β1–4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of β‐mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional β‐mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.
Role of Endogenous Taurine on the Glutamate Analogue‐Induced Neurotoxicity in the Rat Hippocampus In Vivo
Authors: Menéndez Nieves , Solís Jose María , Herreras Oscar , Herranz Antonio Sánchez , Martín del Río Rafael
Published: October 2006

The glutamate analogues N‐methyl‐d‐aspartate (NMDA), kainic acid (KA), and quisqualic acid (QA), prepared in different hypertonic media, were perfused in vivo in the hippocampal CA1 field of rats using a microdialysis technique. Extracellular taurine levels, estimated after analysis of the taurine content of dialysates, increased during perfusion of all three agonists but varied according to the osmolarity of the medium. The NMDA‐induced increase in extracellular taurine content was only slightly inhibited by perfusion of 150 and 300 mM sucrose. The KA‐evoked increase was partially dependent on extracellular osmolarity, because addition of 50 and 150 mM sucrose caused a dose‐dependent inhibition that was not augmented using higher sucrose concentrations. QA caused a taurine increase that was totally abolished by addition of 50 mM sucrose. These results indicate that the rise in extracellular taurine level elicited by QA and part of the increase elicited by KA are probably due to a release caused by the cellular swelling that these substances evoke, a finding substantiating the previously proposed osmoregulatory role of taurine. However, almost all the increase in extracellular taurine content caused by NMDA and all the osmotically insensitive part of the KA‐evoked rise cannot be explained as release triggered by cell swelling and may reflect a function of taurine other than osmore‐gulation.
Developmental Profile of Striatal Preprotachykinin Gene Expression
Authors: Haverstick Doris M. , Jeziorski Michael , Bannon Michael J.
Published: October 2006

Abstract: The normal developmental profile of preprotachykinin (PPT) gene expression was determined in rat striatum from embryonic day 20 to adulthood (>45 days of age). At embryonic day 20, the amount of striatal PPT mRNA was ∼8% of adult levels, and this specific mRNA approached adult levels by postnatal days 12–15. The absolute amount of PPT mRNA, determined by comparison with PPT mRNA synthesized in vitro, ranged from 0.2 pg/μg of total RNA at embryonic day 22 to 5–6 pg/μg of total RNA in adult striata. In addition, the proportion of the various PPT mRNAs derived from the same primary transcript by alternate splicing was determined in the developing animal. At those ages at which PPT mRNA levels were significantly less than those in the adult, there was a slight (10%) but statistically significant increase in the relative amount of γ‐PPT mRNA with respect to the amount of β‐PPT mRNA. Because these mRNA species encode different combinations of tachykinin peptides, these data suggest that the proportion of substance P versus various neurokinin A‐related peptides may be developmen‐tally regulated.
N‐Cadherin Gene Maps to Human Chromosome 18 and Is Not Linked to the E‐Cadherin Gene
Authors: Walsh Frank S. , Barton C. Howard , Putt Wendy , Moore Stephen E. , Kelsell David , Spurr Nigel , Goodfellow Peter N.
Published: October 2006

Abstract: cDNA clones encoding the human N‐cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe λN2. Comparison of the predicted protein sequences revealed >91% homology between chick brain, mouse brain, and human muscle N‐cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3’of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3’untranslated sequence followed. Northern analysis identified a number of major N‐cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N‐cadherin gene to be mapped to chromosome 18. This is distinct from the E‐cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.
Analysis of Acidic Metabolites of Biogenic Amines in Bovine Retina and Vitreous and Aqueous Humour by Gas Chromatography‐Negative Ion Chemical Ionisation Mass Spectrometry
Authors: Midgley J. M. , Watson D. G. , Macfarlane R. G. , Macfarlane S. C. , McGhee C. N. J.
Published: October 2006

Abstract: Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (>60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical—limits of detection were established at ∼200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine. dopamine, and adrenaline/noradrenaline (4‐hydroxyphenylacetic acid, 3,4‐dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p‐Hydroxymandelic acid, the acidic metabolite of p‐octopamine‐p‐synephrine, was identified in vitreous and in aqueous humour.
A Novel and Neurotoxic Tetrahydroisoquinoline Derivative In Vivo: Formation of 1,3‐Dimethyl‐1,2,3,4‐Tetrahydroisoquinoline, a Condensation Product of Amphetamines, in Brains of Rats Under Chronic Ethanol Treatment
Authors: Makino Yukiko , Ohta Shigeru , Tasaki Yoshikazu , Tachikawa Osamu , Kashiwasake Masaki , Hirobe Masaaki
Published: October 2006

Abstract: Repeated amphetamine administration to rats under chronic ethanol intoxication resulted in the formation of 1,3‐dimethyl‐1,2,3,4‐tetrahydroisoquinoline (1,3‐DiMeTIQ), a novel metabolite of amphetamines. 1,3‐DiMeTIQ was quantified with a sensitive, specific assay using gas chromatography‐mass spectrometry. It was not found in the brains of rats given repeated amphetamine administration but no ethanol. The chronic ethanol‐intoxicated rats subjected to repeated amphetamine administration exhibited behavioral abnormalities, such as repeated convulsions and curving of the back. 1,3‐DiMeTIQ contents were markedly higher in the brain or plasma of rats manifesting abnormal behavior in comparison with those in rats behaving normally. Thus, the 1,3‐DiMeTIQ content in the rat brain seems to have some relationship with behavioral abnormalities. This study also confirmed that 1,3‐DiMeTIQ can cross the blood‐brain barrier in the rat. Intraperitoneal 1,3‐DiMeTIQ injections to rats caused behavioral symptoms similar to those observed in rats with chronic ethanol intoxication and repeated amphetamine administration. The effect of toxic doses of 1,3‐DiMeTIQ on dopaminergic and serotonergic metabolism in the whole rat brain was also investigated.
Phosphoinositide Breakdown in Rat Hippocampal Slices: Sensitivity to Glutamate Induced by In Vitro Anoxia
Authors: Ninomiya Haruaki , Taniguchi Takashi , Fujiwara Motohatsu
Published: October 2006

Abstract: We examined the effects of in vitro anoxia on phosphoinositide (PI) breakdown in rat hippocampal slices stimulated by glutamate and quisqualate. In addition to assays of accumulations of 3H‐inositol phosphates (3H‐IPs) degraded from prelabeled PI, we adopted direct assay procedures of inositol 1,4,5‐triphosphate (1,4,5‐IP3) using l,4,5‐IP3‐specific binding protein to determine the formation of 1,4,5‐IP3. The first effect, observed with anoxic incubation by itself, was the diminished quisqualate (10‐‐5M)‐stimulated accumulation of 3H‐IPs degraded from prelabeled PI under prolonged anoxia. Quisqualate caused a transient increase in 1,4,5‐IP3 formation in the early phase of anoxia, similar to that under oxygenated conditions. Glutamate (10‐‐5M), under normal conditions, influenced neither the accumulation of 3H‐IPs nor the formation of 1,4,5‐IP3. Also, the accumulation of 3H‐IPs under prolonged anoxia was unaffected. The same concentration of glutamate, however, gave rise to a transient increase in 1,4,5‐IP3 content in the early phase of anoxia, similar to that caused by quisqualate. The second effect, observed by oxygenation following anoxia, was the induction of glutamate‐stimulated accumulation of 3H‐IPs. When the hippocampal slices were oxygenated following a sufficiently long (>30‐min) exposure to anoxia, glutamate (10‐‐5M) caused a significant increase in accumulation of 3H‐IPs degraded from prelabeled PI. Quisqualate‐stimulated accumulation of 3H‐IPs under oxygenated incubations was also increased by prior exposure of slices to anoxia. These results support the hypothesis that an exposure of hippocampal slices to anoxia induces a sensitivity of the PI breakdown pathway to glutamate and that, given an oxygen supply following sufficiently long exposure to anoxia, the slices maintain their sensitivity to glutamate with an apparent increase in the accumulation of 3H‐IPs.
In Vivo Stimulation of D1 Receptors Increases the Phosphorylation of Proteins in the Striatum
Authors: Lewis Robert M. , Levari Isaac , Ihrig Bryan , Zigmond Michael J.
Published: October 2006

Abstract: In vivo changes in levels of DARPP‐32 [dopamine (DA)‐and cyclic AMP‐regulated phosphoprotein, Mr= 32,000] protein phosphorylation in response to DA agonists in the rat striatum were measured using a novel assay that combines the benefits of rapid quenching of enzyme activity by focused microwave irradiation with a back‐phosphorylation assay. The basal level of phospho‐DARPP‐32 was 5.6% of total DARPP‐32. Injections of l‐3,4‐dihydroxyphe‐nylalanine (100 mg/kg) increased this level to 44.4%. This effect was not as great if focused microwave irradiation was not used. The D1‐specific agonist SKF 38393 (10 mg/kg) increased the level of phospho‐DARPP‐32 to 36.4%. A further modification of the back‐phosphorylation assay was used to detect other phosphoproteins that appear to be regulated by DA. These results establish an assay for in vivo studies of postsynaptic responses involving second messengers in the DA system and provide direct in vivo evidence for the hypothesis that stimulation of D1 receptors increases the phosphorylation of DARPP‐32, as well as several other proteins.
Chronic Cocaine Treatment Decreases Levels of the G Protein Subunits Giα and Goα in Discrete Regions of Rat Brain
Authors: Nestler Eric J. , Terwilliger Rose Z. , Walker John R. , Sevarino Kevin A. , Duman Ronald S.
Published: October 2006

Abstract: A possible role for G proteins in contributing to the chronic actions of cocaine was investigated in three rat brain regions known to exhibit electrophysiological responses to chronic cocaine: the ventral tegmental area, nucleus accumbens, and locus coeruleus. It was found that chronic, but not acute, treatment of rats with cocaine produced a small (∼ 15%), but statistically significant, decrease in levels of pertussis toxin‐mediated ADP‐ribosylation of Giα and Goα in each of these three brain regions. The decreased ADP‐ribosylation levels of the G protein subunits were shown to be associated with 20–30% decreases in levels of their immunoreactivity. In contrast, chronic cocaine had no effect on levels of G protein ADP‐ribosylation or immunoreactivity in other brain regions studied for comparison. Chronic cocaine also had no effect on levels of GSα or Gβ immunoreactivity in the ventral tegmental area and nucleus accumbens. Specific decreases in Giα and Goα levels observed in response to chronic cocaine in the ventral tegmental area, nucleus accumbens, and locus coeruleus are consistent with the known electrophysiological actions of chronic cocaine on these neurons, raising the possibility that regulation of G proteins represents part of the biochemical changes that underlie chronic cocaine action in these brain regions.
Poly‐ and Monoclonal Antibodies Against Recombinant Rat Brain Calbindin D‐28K Were Produced to Map Its Selective Distribution in the Central Nervous System
Authors: Pinol M. Rosa , Kägi Urs , Heizmann Claus W. , Vogel Brigitte , Séquier Jean‐Marc , Haas Werner , Hunziker Willi
Published: October 2006

Abstract: Many processes in the CNS depend on calcium. The calcium signal is transduced into an intracellular response via Ca2+‐binding proteins, including calbindin D‐28K. In many laboratories, polyclonal antibodies against chicken in testinal calbindin D‐28K have been used to study its localization in the brain (normal and degenerated) of various species, including humans, but some of these antisera cross‐reacted with other proteins, including calretinin. We purified recombinant rat brain calbindin D‐28K to raise antisera in rabbits and purified a recombinant rat–chicken calbindin D28K hybrid protein to immunize mice for the generation of monoclonal antibodies. These antisera were highly specific for calbindin D‐28K, as demonstrated by two‐dimensional Western blotting analysis. Immunohistochemical analyses combined with in situ hybridization studies demonstrated that calbindin D‐28K in the Purkinje cells of the cerebellum is independent of vitamin D. The antibodies described here will be important tools for studying the regulation of expression of calbindin D‐28K and its biological function in the brain and in the PNS.
In Vivo Mechanisms Underlying Dopamine Release from Rat Nigrostriatal Terminals: II. Studies Using Potassium and Tyramine
Authors: Fairbrother Iain S. , Arbuthnott Gordon W. , Kelly John S. , Butcher Steven P.
Published: October 2006

Abstract: The brain microdialysis technique has been used to examine the in vivo effects of potassium and tyramine on dopamine (DA) release and metabolism in the striatum of halothane‐anaesthetised rats. Increasing the concentration of potassium perfusing the dialysis probe (30–120 mM) induced a dose‐related efflux of DA. A dose‐related release of DA was also observed following addition of tyramine (1–100 μM) to the perfusing buffer. High concentrations of potassium were found to reduce the dialysate content of the DA metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid and the serotonin metabolite 5‐hydroxyindoleacetic acid. No such effect was observed even when using the highest concentration of tyramine tested. Potassium‐evoked DA release was facilitated by pretreatment with the DA uptake inhibitor nomifensine, was inhibited by depletion of extracellular calcium, and was not significantly affected by tetrodotoxin (TTX). The effect of tyramine on DA efflux was inhibited by nomifensine and was insensitive to both TTX and calcium depletion. These data suggest that potassium and tyramine induce release of DA via different mechanisms. Potassium‐induced DA release involves a carrier‐independent process and may utilise an exocytotic release mechanism. On the other hand, tyramine‐induced DA release would appear to involve a carrier‐dependent process. Depletion of vesicular stores of DA by pretreatment with reserpine did not significantly affect potassium‐induced DA release, whereas a marked inhibition of the effects of tyramine was noted. However, in reserpinised animals the potassium‐induced release of DA was inhibited by nomifensine, a result suggesting that a carrier‐dependent release mechanism operates in the absence of vesicular DA. Inhibition of DA synthesis by pretreatment with α‐methyl‐p‐tyrosine (α‐MPT) reduced potassium‐evoked DA release. However, this treatment only impaired the effect of tyramine when α‐MPT was given 120 min before tyramine, when vesicular DA stores are also depleted. These data suggest that tyramine induces release of DA from vesicular stores, whereas the effect of potassium involves newly synthesised DA. Because DA metabolite levels are reduced by potassium but not by tyramine, these data suggest that extracellular DOPAC is derived from a newly synthesised pool(s) of DA.
Capillary Depletion Method for Quantification of Blood–Brain Barrier Transport of Circulating Peptides and Plasma Proteins
Authors: Triguero Domingo , Buciak Jody , Pardridge William M.
Published: October 2006

Abstract: Recent studies indicate that circulating peptides or plasma proteins, such as insulin or transferrin, or modified proteins, such as cationized albumin, undergo receptor‐mediated or absorptive‐mediated transport through the brain capillary wall, i.e., the blood‐brain barrier (BBB). Although morphologic studies such as autoradiography or immunoperoxidase labeling can demonstrate transport of blood‐borne protein into brain, there is a need for a rapid, sensitive, and quantifiable physiology‐based technique for comparing the relative rates of transport of several different blood‐borne peptides or proteins into brain. Therefore, the present investigations describe a carotid arterial infusion technique coupled with a capillary depletion method for quantifying transport of blood‐borne cationized albumin, cationized IgG, and acetylated low‐density lipoprotein (LDL). Because differentiation of true transcytosis into the postcapillary compartment of brain parenchyma from binding and/or endocytosis to the brain microvasculature is important, the present studies use a dextran density centrifugation step to deplete brain homogenate of the vasculature. In addition, 3H‐labeled native albumin is used as a vascular space marker to account for release of capillary contents into the postcapillary supernatant following homogenization of brain. This study demonstrates rapid transport of cationized IgG or cationized albumin into brain, as these compounds achieve a volume of distribution of 20–30 μl/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the postcapillary compartment of brain parenchyma. These studies provide the basis for a sensitive, quantifiable technique for studying transport of radiolabeled blood‐borne peptides and proteins across the BBB of anesthetized animals.
Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate‐Early Genes c‐fos and c‐jun in Rat Cerebral Cortex
Authors: Winston Sheryl M. , Hayward Michael D. , Nestler Eric J. , Duman Ronald S.
Published: October 2006

Abstract: Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate‐early genes c‐fos and c‐jun, also referred to as nuclear proto‐on‐cogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c‐fos and c‐jun to an acute, “test” seizure was examined. Four hours after a single ECS, the induction of c‐fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c‐fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to “reset” the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time‐dependent decrease in the expression of c‐fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8–10 days of treatment, at which time the induction of c‐fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c‐jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8–10 days) ECS block the induction of c‐fos may differ.
Diazepam Sensitivity of the Binding of an Imidazobenzodiazepine, [3H]Ro 15‐4513, in Cerebellar Membranes from Two Rat Lines Developed for High and Low Alcohol Sensitivity
Authors: Uusi‐Oukari M. , Korpi E. R.
Published: October 2006

Abstract: Ro 15‐4513 (ethyl‐8‐azido‐5,6‐dihydro‐5‐methyl‐6‐oxo‐4H‐imidazo[1,5‐a][1,4]benzodiazepine‐3‐carboxylate), a partial inverse agonist of brain benzodiazepine receptors, has been shown to antagonize some actions of ethanol. In addition to conventional benzodiazepine binding sites, Ro 15‐4513 binds to a specific cerebellar protein, the binding of which has been shown to be insensitive to diazepam. The binding of [3H]Ro 15‐4513 was studied in washed membranes of the cerebellum, hippocampus, and cerebral cortex of two rat lines developed for differences in their sensitivity to ethanol‐induced motor impairment. Only minor differences were found in the estimated parameters (KD and Bmax) for the total specific binding between the rat lines. The main difference between the rat lines was, however, observed in the characteristics of the cerebellar binding, all of which was displaced by diazepam in most of the alcohol‐sensitive [alcohol‐nontolerant (ANT)] rats, in contrast to only 75% displacement in most of the alcohol‐insensitive [alcohol‐tolerant (AT)] ones. The following cerebellar results were obtained with the major subgroups of both lines, i.e., with the AT rats chosen for the presence of the diazepam‐insensitive binding and with the ANT rats chosen for its absence. The KD for the total specific [3H]Ro 15‐4513 binding in the ANT animals was about half of that in the AT animals. No line difference was found in the Bmax of the binding in these rats. Photolabeling with [3H]Ro 15‐4513 showed that the diazepam‐insensitive binding was in a protein with a molecular weight of 55,000. In the absence of diazepam, this band was labeled in both rat lines, but micromolar diazepam abolished the labeling more thoroughly in the ANT rats. There were only minor line differences in the potency of the inverse agonists ethyl‐β‐carboline‐3‐carboxylate, methyl‐6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate, and FG 7142 (N‐methyl‐β‐carboline‐3‐carboxamide) to displace the cerebellar [3H]Ro 15‐4513 binding. This result suggests that the line difference has developed specifically in the affinity of the protein with a molecular weight of 55,000 to micromolar diazepam and possibly to other benzodiazepine agonists as well. Because the rat lines differ in their sensitivity to benzodiazepines in a similar way as to ethanol, the diazepam‐insensitive binding sites of [3H]Ro 15‐4513 might be involved in the mechanisms causing the impairment of motor performance after sedative drugs.
Sulfoglucuronyl Glycolipids Bind Laminin
Authors: Mohan Panaiyur S. , Chou Denise K. H. , Jungalwala Firoze B.
Published: October 2006

Abstract: Previous studies have shown that HNK‐1 antibody reactive glycoconjugates, including the glycolipids 3‐sulfoglucuronylneolactotetraosylceramide (SGGL‐1) and 3‐sulfoglucuronylneolactohexaosylceramide (SGGL‐2), are temporally and spatially regulated antigens in the developing mammalian cortex. Extracellular matrix glycoprotein laminin is involved in cell adhesion by interacting with cell surface components and also promotes neurite outgrowth. Laminin has been shown to bind sulfatide. The interaction of sulfated glycolipids SGGL‐1 and SGGL‐2 with laminin was studied by employing a solid‐phase radioimmunoassay and by HPTLC‐immunoblotting. Laminin binding was detected with anti‐laminin antibodies followed by 125I‐labelled Protein A and autoradiography. Laminin binds SGGL‐1 and SGGL‐2, besides sulfatide, but does not bind significantly gangliosides and neutral glycolipids. The binding of SGGLs to laminin was two to three times less compared to sulfatide when compared on a molar basis. Desulfation of SGGLs and sulfatide by mild acid treatent resulted in abolition of laminin binding. On the other hand, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect. This showed that the sulfate group was essential for laminin binding. Of the various glycosaminoglycans tested, only heparin inhibited the binding of laminin to SGGLs and sulfatide in a dose‐dependent manner. This indicated that SGGLs and sulfatide bind to the heparin binding site present in the laminin molecule. The availability of HNK‐1 reactive glycolipids and glycoproteins such as SGGLs and several neural cell adhesion molecules to bind laminin at critical stages of neural development may serve as important physiological signals.
In Vivo Monitoring of Catecholaminergic Metabolism in the C1 Region of Rat Medulla Oblongata: A Comparative Study by Voltammetry and Intracerebral Microdialysis
Authors: Lambás‐Señas Laura , Gillon Jean‐Yves , Bouilloux Jean‐Pierre , Seccia Michèle , Buda Michel , Renaud Bernard
Published: October 2006

Abstract: In vivo voltammetry or microdialysis was used to monitor catecholaminergic metabolism in the C1 region of the ventrolateral medulla oblongata of anesthetized rats. In vivo voltammetry allowed the recording of a catechol oxidation current (CA.OC) peak in this region. This CA.OC was suppressed after inhibition of monoamine oxidase by pargyline or after inhibition of tyrosine hydroxylase by α‐methyl‐p‐tyrosine and was markedly increased after blockade of dopamine‐β‐hydroxylase by FLA 63. Similar results were found when intracerebral microdialysis coupled with HPLC and electrochemical detection was used to measure the concentration of 3,4‐dihydroxyphenylacetic acid (DOPAC) in the dialysates obtained from the C1 region: The changes in CA.OC and DOPAC concentration in the dialysates exhibited very similar kinetic characteristics in the three pharmacological experiments. These results support the involvement of DOPAC as a major component of the electrochemical signal recorded by voltammetry in the C1 group of adrenergic neurons.
Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes
Authors: Erecińska Maria , Zaleska Malgorzata M. , Nelson David , Nissim Itzhak , Yudkoff Marc
Published: October 2006

Abstract: The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2‐15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mMKCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na+‐amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine‐induced increase in intrasyn‐aptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.
Dopamine Release via Protein Kinase C Activation in the Fish Retina
Authors: Kato S. , Ishita S. , Mawatari K , Matsukawa T. , Negishi K.
Published: October 2006

Abstract: Calcium‐dependent phospholipid‐sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) activated PKC in the nanomolar range. A major 38‐kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (>15 min). Rod‐bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (α/β‐subtype). The PKC antibody recognized a 78‐kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium‐ and dose‐dependent [3H]DA release during a short period (2 min), with the minimal effective dose being 1 nM. Other phorbols having no tumor‐promoting activity, such as 4β‐phorbol and 4α‐phorbol 12, 13‐didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1‐oleoyl‐2‐acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG‐ or TPA‐induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7) and staurosporine. In contrast, release of [3H]glycine, γ‐[3H]aminobutyric acid, and D‐[3H]aspartic acid was never induced by TPA. Taken together, the present data indicate that TPA or OAG triggers a release of DA synaptically and that the induction is due to a PKC activation specifically in the membrane of DA cells in the fish retina. The functional role of PKC in rod‐bipolar cells is unknown at present.
Incorporation into Phospholipid Classes and Metabolism via Desaturation and Elongation of Various 14C‐Labelled (n‐3) and (n‐6) Polyunsaturated Fatty Acids in Trout Astrocytes in Primary Culture
Authors: Tocher Douglas R. , Sargent John R.
Published: October 2006

Abstract: The incorporation and metabolism of [1‐14C]18: 3(n‐3), [1‐14C]20:5(n‐3), [1‐14C]18:2(n‐6), and [1‐14C]20:4(n‐ 6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22°C. with >90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n‐6), 18:3(n‐3), and 20:5(n‐3) in individual phospholipid classes at 22°C were very similar, with 57–63 and 18–24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n‐6), ∼30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n–3) fatty acids to longer‐chain and more unsaturated species was significantly greater than that of(n 6) acids, but Δ4‐desaturase activity was very low. A culture temperature of 10°C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10°C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidyicholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.
Effect of Differentiation and Cell Density on Glycosphingolipid Class and Molecular Species Composition of Mouse Neuroblastoma NB2a Cells
Authors: Kadowaki Hiroko , Evans James E. , Rys‐Sikora Krystyna E. , Koff Raymond S.
Published: October 2006

Abstract: The effects of cell density and retinoic acid‐induced differentiation on the class and molecular species composition of mouse neuroblastoma NB2a cell glycosphingolipids were examined under conditions where the period of culture was controlled. The total amount of neutral glycosphingolipids per cell decreased both with differentiation and as the cells became confluent. The relative amount of the neutral glycosphingolipid classes was not affected by differentiation, whereas there were small but significant changes in the relative amount of the neutral glycosphingolipid classes as the cells became confluent. The total amount of the gangliosides was unaffected by either differentiation or cell density, but there were significant changes in the ganglioside class composition as a result of both cell density and differentiation, and the effects were additive. The molecular species of all the major neutral glycosphingolipid and ganglioside classes were essentially identical, and were altered only slightly by either differentiation or cell density.
Reversible Inhibition of Acetylcholine Synthesis and Behavioural Effects Caused by 3‐Bromopyruvate
Authors: Arendt T. , Schugens M. M. , Marchbanks R. M.
Published: October 2006

Abstract: 3‐Bromopyruvate inhibits pyruvate decarboxylase in brain homogenates and causes a 90% drop in acetylcholine tissue content at a concentration of 2 mM. Stereotaxic injection of 3‐bromopyruvate into the basal forebrain causes after 7 days a 40% drop of acetylcholine concentration and pyruvate decarboxylase activity in the cortex and hippocampus, and greater decreases at the site of injection. However, values return to normal 18 days after injection. Choline ace‐tyltransferase is partially inhibited only at the site of injection after 7 days. Choline transport and choline concentration are not affected at either 7 or 18 days after injection. Impairments in spontaneous alternation and in retention of passive avoidance were seen only 7 days after the injection. The results suggest that stereotaxic injection of bromopyruvate can induce discrete reversible cholinergic lesions on a time scale useful for behavioural experiments and for comparison with neurodegeneration.
Effect of In Vivo Modulation of Membrane Docosahexaenoic Acid Levels on the Dopamine‐Dependent Adenylate Cyclase Activity in the Rat Retina
Authors: Malnoe Armand , Milon Hubert , Reme Charlotte
Published: October 2006

Abstract: We have studied the effect of a dietary deprivation of n‐3 fatty acids on the activity of the dopamine (DA)‐de‐pendent adenylate cyclase in the rat retina. Experiments were conducted in 6‐month‐old rats raised on semipurified diets containing either safflower oil (n‐3 deficient diet) or soybean oil (control diet). The levels of docosahexaenoic acid [22:6 (n‐3)] in retinal phospholipids were significantly decreased in n‐3 deficient rats (35–42% of control levels). This was compensated by a rise in 22:5 (n‐6), the total content of poly‐unsaturated fatty acids (PUFA) remaining approximately constant. Adenylate cyclase activity was measured in retinal membrane preparations from dark‐adapted or light‐exposed rats. The enzyme activity was stimulated by DA and SKF 38393 in a light‐dependent fashion. The activation was lower in rats exposed to light than in dark‐adapted animals, suggesting a down‐regulation of the DI DA receptors by light. The activation by guanine nucleotides and forskolin was also decreased in light‐exposed rats. There was no significant effect of the dietary regimen on the various adenylate cyclase activities and their response to light. Furthermore, the guanine nucleotide‐ and DA‐dependent adenylate cyclase activities of retinal membranes were found to be relatively resistant to changes in membrane fluidity induced in vitro by benzyl alcohol. The results indicate that in the absence of changes in total PUFA content, a decreased ratio of n‐3 to n‐6 fatty acids in membrane phospholipids does not significantly affect the properties of adenylate cyclase in the rat retina.
Different Ceramide Compositions of Gangliosides Between Human Motor and Sensory Nerves
Authors: Ogawa‐Goto K. , Funamoto N. , Abe T. , Nagashima K.
Published: October 2006

Abstract: Ganglioside analysis of human motor and sensory nerves revealed that ceramide compositions of sensory nerve GD1a, GD1b, and GMl differed apparently from those in the motor nerve. These gangliosides from sensory nerve contained a large amount of long‐chain fatty acids and d18:1 as a major long chain base. On the contrary, the motor nerve gangliosides contained C16–18 fatty acids and a large amount of d20:1 besides d 18:1. Furthermore, these gangliosides were enriched more in the axon fraction than in the myelin fraction. LM1, which was a major ganglioside in myelin from human peripheral nerve, was composed of similar ceramide compositions in the two nerves. The present findings suggest that the characteristic ceramide species of nerve gangliosides may reflect in part properties of their own neurons.
Evidence that Somatostatin Enhances Endogenous Acetylcholine Release in the Rat Hippocampus
Authors: Araujo D. M. , Lapchak P. A. , Collier B. , Quirion R.
Published: October 2006

Abstract: The present experiments show that somatostatin (SS)‐like immunoreactive material is present in the hippocampus and that its release can be increased by K+ stimulation of rat hippocampal slices, suggesting that SS‐like peptides may be of significance to neurotransmission in the hippocampus. Exogenous SS‐28 and SS‐14 enhanced the K+‐evoked release of endogenous acetylcholine (ACh) from rat hippocampal slices, whereas amino‐terminal fragments of SS‐28 did not. The increased ACh release in the presence of either peptide appeared to be mediated by an interaction with SS receptors because cyclo‐SS, a putative SS antagonist, abolished the effects of both SS‐28 and SS‐14. In addition, the increase in ACh release induced by SS‐14 or SS‐28 was antagonized by the calcium channel antagonists ω‐conotoxin GVIA, nifedipine, and cinnarizine, implicating voltage‐sensitive calcium channels in this effect. Moreover, the effect was sensitive to tetrodotoxin, suggesting an indirect action of the peptides at a site distal to cholinergic nerve terminals. Cysteaminc, which has been reported to deplete SS content and to increase SS release in brain, augmented the basal and evoked release of ACh from hippocampal slices, without affecting SS‐like content and release. Finally, neuropeptide Y, which is colocalized with SS in many neurons of the hippocampal formation, did not alter ACh release, nor did it facilitate the SS‐induced increase. The results suggest that in the rat hippocampus, both SS‐28 and SS‐14 interact with SS receptors to regulate ACh release indirectly by a mechanism that involves alterations of calcium influx during depolarization.