bluuebunny/crossref_metadata_2025 huggingface dataset:
Lectin Receptors in Central Nervous System Myelin
Authors: Poduslo Joseph F. , Harman Jane L. , McFarlin Dale E.
Published: October 2006

Abstract: Proteins from central nervous system myelin were separated by high‐resolution, sodium dodecyl sulfate‐pore gradient slab gel electrophoresis and the glycoproteins were detected by autoradiography after direct application of radioiodinated lectins. A surprising heterogeneity of lectin binding proteins was found associated with this highly purified membrane fraction. Iodinated wheat germ agglutinin, which has a monosaccharide specificity for N‐acetyl‐D‐glucosamine and N‐acetylneuraminic acid, revealed six major bands and two minor bands. By correlating the molecular weights (Mr) of radioiodinated protein standards with the gel concentration at the position reached by the protein (%T) using the relationship log(Mr) versus log(%T) for gradient gel systems, molecular weight estimates of 128, 300, 109, 800, 75, 300, 48, 800, 26, 100 and 23, 700 were obtained for the major glycoprotein bands and molecular weights of 98, 300 and 86, 600 for the minor bands. When the isolated myelin was extracted with chloroform‐methanol‐a procedure that removes the major myelin proteins, including the proteolipid protein and most of the basic proteins and hence concentrates the minor high molecular weight proteins‐and analyzed after gradient gel electrophoresis, additional glycoproteins of molecular weights 607, 700, 196, 900, 175, 100, 61, 800, 52, 200 and 42, 600 were resolved with this lectin. Radioiodinated soybean agglutinin, which has a specificity for N‐acetyl‐D‐galactosamine and D‐galactose, revealed seven bands, three of which were unique to this lectin (19, 600, 19, 100 and 17,000). Iodinated concanavalin A (d‐mannose, d‐glucose) revealed bands similar to the wheat germ agglutinin as well as additional bands of 40, 300, 37, 300, 35, 700, 21, 800 and 20, 400. The glycoprotein specificity for these lectin binding components was demonstrated by hapten carbohydrate binding inhibition and by organic solvent extraction for removal of glycolipids. Based on these experiments using three lectins with different carbohydrate specificity, 22 lectin‐reactive components were identified; however, six of these bands were removed by chloroform‐methanol extraction. The variations observed in the lectin binding capacity for these different bands suggest possible carbohydrate heterogeneity for these individual glycoproteins. Although many of these bands may be dissociated subunits (monomeric polypeptides) of oligomeric complexes, the observed multiplicity of these quantitatively minor glycoproteins associated with the purified myelin membrane implies a more intricate molecular organization for the myelin sheath complex than previously believed.
Effects of Altered Thyroid States on Myelinogenesis
Authors: Walters Sandra N. , Morell Pierre
Published: October 2006

Abstract: Myelinogenesis was studied in controls and in rats treated since birth with Methimazole (hypothyroid) or thyroxine (hyperthyroid). The amount of myelin in forebrain and its protein composition were determined between 13 and 40 days of age, the period of most rapid myelin accumulation. Hypothyroid rats had reduced body and brain weights relative to controls and the yield of myelin was reduced on both a per brain and a per milligram brain protein basis. Developmental changes in the protein composition of isolated myelin followed the pattern of control animals (the percentage of total myelin protein present as proteolipid protein, large basic protein, and small basic protein increased, as did the ratio of proteolipid/large basic protein) but were delayed temporally by 1–2 days. Hyperthyroid rats also had reduced body and brain weights. At 13 days myelin accumulation was greater than that of controls, corresponding to an earlier initiation of myelination. At later ages myelin yield was reduced on a per brain basis but not on a per milligram brain protein basis. The developmental pattern of myelin protein composition was accelerated temporally by 1–2 days. Myelination in optic nerve, assayed by proteolipid protein content, also was slightly delayed in hypothyroid animals and somewhat accelerated in hyperthyroid animals. The relative synthesis of myelin proteins (determined as incorporation of intracranially injected [3H]glycine into myelin protein relative to incorporation into whole brain protein), as well as distribution of radioactivity among individual myelin proteins, was determined. The results supported the conclusion of the myelin protein accumulation study; hypothyroidism retards the developmental program for myelinogenesis, whereas in the hyperthyroid state myelin synthesis is initiated earlier but is also terminated earlier.
Turnover of Myelin Proteins of Rat Brain, Determined in Fractions Separated by Sedimentation in a Continuous Sucrose Gradient
Authors: Shapira R , Wilhelmi M. R , Kibler R. F
Published: October 2006

Abstract: Rats that Received intracranial injections of [3H]leucine at 14 days of age were killed on days 17, 24, 38, 55, and 89 post‐injection. Brains were homogenized and the myelin membranes separated in a sucrose density gradient. At day 17 sodium dodecylsulfate polyacrylamide gels of water‐shocked, delipidated membrane fractions showed a difference in the specific activity of myelin proteins across the gradient. A decrease in specific activity was found in all of the proteins in the denser fractions, compared with the lighter fractions. As time after injection progressed, the difference became more pronounced; a two‐ to threefold decrease in specific activity was seen across the gradient in the various myelin proteins. The proteins of the lightest membrane fractions retained their high specific activity throughout the experiment in spite of extensive new myelin synthesis. Taking this new myelin into account, the decrease in specific activity in the denser myelin fractions could be explained by isotope dilution. Therefore, proteins present in at least some of the myelin are essentially stable.
The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions
Authors: Linington Christopher , Waehneldt Thomas V
Published: October 2006

Abstract: Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small‐sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M‐sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate‐labelled lectins (WGA, Con A, RCA‐60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.
Reduction of Brain Calcium After Consumption of Diets Deficient in Calcium or Vitamin D
Authors: Harris R. Adron , Carnes David L. , Forte Leonard R.
Published: October 2006

Abstract: Rats fed diets deficient in calcium or vitamin D for 4 weeks displayed hypocalcemia, as indicated by a 50% reduction in serum calcium and a sevenfold elevation of serum parathyroid hormone. These treatments also decreased the calcium content of brain tissue. On a regional basis. this effect was greatest in the brain stem (24% decrease) and least in striatum (10% decrease). Subcellular analysis indicated that the depletion of brain calcium was greatest in the soluble and the microsomal fractions. Infusion of calcium solutions reversed the depletion of brain calcium produced by dietary deficiencies. In control rats. parathyroidectomy or infusion of parathyroid hormone did not alter the calcium content of brain tissue, although these treatments affected the levels of calcium in the serum. In general, these treatments had no effect on the magnesium content of serum or brain tissue. However, vitamin D deficiency did increase the magnesium content of the myelin and synaptosomal fractions. This increase was reversed by parathyroidectomy. These observations demonstrate that long‐term hypocalcemia produces distinct changes in the localization of calcium and magnesium in brain tissue. Furthermore. these studies suggest that though brain calcium levels are influenced by serum concentrations, serum changes must be of large magnitude and long duration for brain calcium levels to be affected.
Leukocyte Sulfatidase for the Reliable Diagnosis of Metachromatic Leukodystrophy
Authors: Raghavan S. S. , Gajewski A. , Kolodny E. H.
Published: October 2006

Abstract: A simple assay technique for the determination of sulfatidase activity in leukocytes has been developed for the reliable diagnosis of metachromatic leukodystrophy (MLD). Sulfatide is tritiated in sphingosine and fatty acid by reduction with [3H]sodium borohydride in alkali in the presence of palladium chloride. This labeled natural substrate for aryl sulfatase A (AsA) is hydrolyzed by normal human leukocytes in 25 mwacetate buffer, pH 5.0, in the presence of 0.3% sodium taurodeoxycholate. The enzyme activity is greatly improved after dialysis, exhibiting better linearity with protein concentration. It is stimulated maximally by 5 mM‐MnCl2 with an apparent Km of 0.17 MM for the substrate. Patients with MLD exhibited virtually no detectable sulfatidase activity although they had residual AsA activity that was measured with the synthetic substrate, p‐nitrocatechol sulfate (NCS). Potential heterozygotes could be identified by the sulfatidase assay in instances where the NCS assay for AsA was inconclusive. Several individuals with levels of AsA activity characteristic of MLD, including a few healthy carriers and certain patients with unknown neurological diseases, were shown not to have MLD by the presence of measurable levels of sulfatidase in their leukocytes.
The Quaternary Structure of Chicken Acetylcholinesterase and Butyrylcholinesterase; Effect of Collagenase and Trypsin
Authors: Allemand Pierre , Bon Suzanne , Massoulié Jean , Vigny Marc
Published: October 2006

Abstract: Acetylcholinesterase (EC 3.1.1.7.; AChE) and butyrylcholinesterase (EC 3.1.1.8.; BuChE) from chicken muscle exist as sets of structurally homologous forms with very similar properties. The collagenase sensitivity and aggregation properties of the‘heavy’forms of both enzymes indicate that they possess a collagen‐like tail, and their stepwise dissociation by trypsin confirms that they correspond to triple (A12) and double (A8) collagen‐tailed tetramers. In addition to this dissociating effect, trypsin digests an important fraction of the catalytic units of AChE, in a progressive manner, removing as much as 30% of the enzyme's mass, without inactivation of the tetramers and of the tailed molecules. The trypsin‐modified AChE forms closely resemble the corresponding mammalian AChE forms in their hydrodynamic properties. It is not known whether the trypsin‐digestible peptides, which do not appear to be involved in the ionic or hydrophobic interactions of the enzymes, are a fragment of the catalytic subunit or whether they constitute distinct polypeptides.
Occurrence and Properties of Phospholipases A1 of Plasma Membranes Prepared from Neuronal‐ and Glial‐Enriched Fractions of the Rabbit Cerebral Cortex
Authors: Woelk H. , Rubly N. , Arienti G. , Gaiti A. , Porcellati G.
Published: October 2006

Abstract: Exogenously added glycerophosphatides, specifically radioactively labelled either in the 1 or in the 2 position, were used to investigate the occurrence and properties of phospholipase A1 in plasma membranes prepared from neuronal‐ and glial‐enriched fractions of rabbit brain. Phospholipase A1 activity was maximal at pH values ranging between 8.0 and 9.0 for the plasma membranes of both cell types. The enzyme activity was most abundant in the microsomal fraction, with a neurondglial ratio of about 2. The plasma membranes displayed about half the enzymic activity of the microsomal fraction, whereas only small amounts of phospholipase A1 were present in the neuronal and glial mitochondria. Investigations on the substrate specificity showed a different pattern for the enzyme of neuronal and glial origin. The release of labelled fatty acids from phosphatidylcholine by the neuronal plasma membrane phospholipase A1 decreased with increasing degree of unsaturation of the fatty acids at the 1 position. The presence of plasmalogens and plasmalogen precursors in the incubation mixture appreciably inhibited the hydrolysis of the corresponding diacyl compounds.
The Human Platelet as a Model for the Glutamatergic Neuron: Platelet Uptake of L‐Glutamate
Authors: Mangano Richard M. , Schwarcz Robert
Published: October 2006

Abstract: L‐Glutamate uptake into human platelets revealed two components: a high‐affinity system (KmH= 3.1 μM), which was sodium‐dependent, and a low‐affinity site (KmL= 88 μM) displaying temperature rather than sodium dependency. These kinetic properties were similar to those found in crude synaptosomal preparations and brain slices. However, Vmax values were far higher in brain(VmaxH= 325 ± 96, VmaxH= 3759 ± 1116 pmol/mg wet weight per min) than in platelets (VmaxH, = 14 ± 6, VmaxL= 313 ± 63 pmol/mg platelet protein per 10 min), indicating a denser population in brain than in platelets of qualitatively similar sites. Pharmacological analysis substantiated the resemblance of nerve endings and platelets: the specific uptake inhibitors threo‐3‐hydroxy‐DL‐aspartate and DL‐aspartate‐β‐hydroxamate as well as D‐and L‐glutamate and L‐aspartate showed similar—though not identical—IC50 values in both preparations; a spectrum of compounds devoid of inhibitory effects in synaptosomes also did not interfere with glutamate uptake in platelets. Uptake parameters were studied in a population of human volunteers to determine the variability of platelet glutamate uptake. Whole blood could be stored up to 6 h after venipuncture without any appreciable change in experimental values. Percentage of variation between 0.09 and 0.28 for three repetitive (weekly) assays in single subjects indicated that glutamate uptake measurements in human platelets are sufficiently suited for future clinical studies.
Effect of Inorganic Lead on Rat Brain Mitochondrial Respiration and Energy Production
Authors: Gmerek Debra E. , McCafferty Mary R. , O'Neill Kevin J. , Melamed Brian R. , O'Neill John J.
Published: October 2006

Abstract: Toxicologically significant amounts of inorganic lead were added to rat brain mitochondrial preparations that did not contain EDTA or Pi. The binding of the lead to the mitochondria was measured by anodic stripping voltometry. In the presence of lead, the respiratory control ratios decreased, implying a decrease in the degree of dependence of respiration on a phosphate acceptor. Nucleotide contents were also measured, and in the presence of inorganic lead the actual amounts of ATP formed from ADP were found to be significantly decreased as well.
Cell‐Free Translation of Free and Membrane‐Bound Polysomes and Polyadenylated mRNA from Rabbit Brain Following Administration of d‐Lysergic Acid Diethylamide In Vivo
Authors: Heikkila John J. , Cosgrove James W. , Brown Ian R.
Published: October 2006

Abstract: Free and membrane‐bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA‐dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell‐free translation products demonstrated that although most of the nascent proteins were common to both free and membrane‐bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell‐free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane‐bound polysomes revealed the appearance of 74K protein following either LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two‐dimensional gel electrophoresis.
Activity and Isoenzyme Pattern of Lactate Dehydrogenase in Neurons and Astroblasts Cultured from Brains of Chick Embryos
Authors: Tholey G. , Roth‐Schechter B. F. , Mandel P.
Published: October 2006

Primary cultures of neurons and glial cells (astroblasts) prepared from brains of 8‐day‐old and 15‐day‐old chick embryos, respectively, were grown for periods between 3 and 19 days. Specific activity of lactate dehydrogenase (LDH) increased in both types of cultures as a function of time and was always significantly higher in glial cells than in neurons. Glial cell extracts were found to contain predominantly the anaerobic isoenzymatic form of LDH (LDH‐H4), and this pattern did not change over a period of 19 days. Cultured neurons contained predominantly the aerobic isoenzymatic form LDH‐H4, and there was a progressive appearance of all other isoenzymes over an 8‐day period. These results support the hypothesis of a different energy metabolism in neurons and glia.
Anticonvulsant Activity of Muscimol and γ‐Aminobutyric Acid Against Pyridoxal Phosphate‐Induced Epileptic Seizures
Authors: Kouyoumdjian Jean Claude , Ebadi Manuchair
Published: October 2006

The intracerebroventricular injection of pyridoxal phosphate (PLP, 0.125‐1.25 μmol/rat) causes epileptic seizures (4 min → 1 min) that are preventable or reversible by GABA (1 μmol/rat), by muscimol (O.025 μmol/rat), or by diazepam (1.75 μmol/rat). At the peak of PLP‐induced convulsions, the activities of GAD and GABA‐T in 14 regions of rat brain remained unaltered, whereas the concentrations of PLP remained elevated. The PLP‐induced convulsion was blocked by DABA (10 μmol/rat) but was not altered by β‐alanine (50 μmol/rat). The previous in vitro studies have shown that PLP increases the uptake of [3H]GABA into synaptosomes and inhibits the binding of [3H]GABA to synaptic membranes. These data suggest that PLP‐induced convulsion is due to reduced availability of GABA to its recognition sites, rather than to alteration in the activity of GABA metabolizing enzymes, or unavailability of PLP as a coenzyme for GAD and GABA‐T. Since the duration of PLP‐induced epileptic seizures is short and can be prevented by GABA agonists, PLP may be used as a tool to study the nature of GABA‐mediated neuroinhibition and the properties of GABA receptor sites.
Blood‐Brain Glucose Transfer in Spreading Depression
Authors: Gjedde Albert , Hansen Anker Jón , Quistorff Bjørn
Published: October 2006

Abstract Spreading depression in rat brain cortex is associated with a twofold increase of cerebral blood flow. It is not known whether this increase is coupled to increases of cerebral metabolic rate and glucose transport from blood to brain. During the passage of a single spreading depression, we measured blood‐brain glucose transport and glucose metabolism in rat cerebral cortex by single intravenous injection of tracer glucose. Blood flow and tissue content of glucose were measured as well. Reduction of tissue glucose and the consequent increase of net transfer of glucose from blood to brain were consistent with a threefold increase of the consumption of glucose before the increase of blood flow. There was no increase of unidirectional blood‐brain transfer.
In Vitro Retina as an Experimental Model of the Central Nervous System
Authors: Ames A. , Nesbett F. B.
Published: October 2006

Abstract Methods are described for isolating adult rabbit retina and maintaining it in a medium designed to resemble CSF. Morphologic, metabolic, and electrophysiologic measurements obtained on the in vitro retinas showed that they remained in a nearly physiological state for at least 8 h, and even after 2 days in vitro they still exhibited a high level of metabolic activity and electrical responsiveness to light. Physiological activity was modified by photic stimulation, and data are presented to document changes in metabolism in response to the changes in function. The isolated retina appears to offer a number of unusual advantages for studying relationships between function and metabolism in organized mammalian central nervous tissue
In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices
Authors: Collingridge G. L. , Thompson P. A. , Davies J. , Mellanby J.
Published: October 2006

Abstract Ca2+‐dependent K+‐stimulated γ‐aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre‐incubation of the slices with 104 mouse LD50/ml tetanus toxin for 3 h at 37°C.
Energy Metabolism in Rat Brain: Inhibition of Pyruvate Decarboxylation by 3‐Hydroxybutyrate in Neonatal Mitochondria
Authors: Booth Robert F. G. , Clark John B.
Published: October 2006

Abstract: The effect of 3‐hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1 ‐14C]pyruvate decarboxylation (1 mm final concentration) by brain synaptosomes derived from 8‐day‐old rats was inhibited by 10% in the presence of 2 mm‐d,l‐3‐hydroxybutyrate and by more than 20% in the presence of 20 mm‐d,l‐3‐hydroxybutyrate. The presence of 2 mm‐l,d‐3‐hydroxybutyrate did not affect the rate of [1‐14T]pyruvate decarboxylation (1 mm final concentration) by brain mitochondria; however, at a concentration of 20 mm‐d,l‐3‐hydroxybutyrate, a marked inhibition was seen in preparations from both 8‐day‐old (35% inhibition) and 21‐day‐old (24% inhibition) but not in those from adult rats. Although the presence of 100 mm‐K+ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in the presence of 1 mm‐K+, the presence of 20 mm‐d,l‐3‐hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mm‐K+). The presence of 20 mm‐d,l‐3‐hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8‐day‐old rats. The concentrations of ATP, ADP, NAD+, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8‐day‐old rats incubated in the presence of 1 mm‐pyruvate alone or 1 mm‐pyruvate plus 20 mm‐d,l‐3‐hydroxybutyrate. Neither the ATP/ADP nor the NADH/NAD+ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3‐hydroxybutyrate. The possible mechanisms and physiological significance of 3‐hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain mitochondria are discussed.
Effects of Long‐Term Low Dietary Tryptophan Intake on Determinants of 5‐Hydroxytryptamine Metabolisn in the Brains of Young Rats
Authors: Ashley David V. M. , Curzon G.
Published: October 2006

Abstract: The relationship between plasma and brain tryptophan (TRP) concentrations and brain 5‐hydroxytryptamine (5‐HT) metabolism was studied in weanling rats fed diets containing either 0.4 g or 1.45 g TRP/ 100 g casein hydrolysate. Both groups gained weight comparably though food intakes were generally higher in the low‐TRP group. Severe depletion of plasma total and free TRP and of brain TRP, 5‐HT, and 5‐hydrox‐yindoleacetic acid (5‐HIAA) occurred within 1 day of feeding the 0.4% TRP diet. Levels became stable after 7 days. The decreased brain TRP concentration of the rats on the 0.4% TRP diet did not cause a compensatory rise of the tryptophan hydroxylase (TRP OHase) activity in vitro. In the low‐TRP group, neither plasma free TRP nor total TRP correlated significantly with brain TRP and although plasma TRP/large neutral amino acid (NAA) ratios (TRP/NAA) correlated significantly (P < 0.05) with the time course of brain TRP, this statistical relationship depended almost completely on the variation of the TRP values alone. In the higher TRP group none of these correlations were significant. A plot of mean plasma free TRP versus brain TRP gave two distinct regression lines with similar slopes and corresponding to values before and after 7 days on the diet. The time course of brain 5‐hydroxyindole concentrations did not parallel those of brain TRP and suggested that changes of TRP OHase activity also had an influence on 5‐HT synthesis.
Energy‐Metabolising Enzymes in Brain Regions of Adult and Aging Rats
Authors: Leong S. F. , Lai J. C. K. , Lim L. , Clark J. B.
Published: October 2006

Abstract: The regional enzyme activities of glucose metabolism in the rat brain were investigated. Hexokinase (EC 2.7.1.1) and pyruvate dehydrogenase (EC 1.2.4.1), key enzymes for glucose metabolism, showed no changes in activity in all the regions studied of the aging brain as compared with the adult brain. However, the activity of d‐3‐hydroxybutyrate dehydrogenase (EC 1.1.1.30) is low throughout the adult brain and, in contrast with hexokinase and pyruvate dehydrogenase, its activity decreases significantly during aging. Other enzymes that showed significant decreases during aging are aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27), citrate synthase (EC 4.1.3.7), and NAD+‐linked isocitrate dehydrogenase (EC 1.1.1.41). The catabolic enzyme in cholinergic metabolism, acetylcholinesterase (EC 3.1.1.7), selected as an example of a non‐energy‐metabolising enzyme, also showed significant decreases in all regions of the brain in aging, although its highest activity remained in the striatum. These results are discussed with respect to the energy metabolism in various brain regions and their status with aging.
Myelin Proteins, Glycoproteins, and Myelin‐Related Enzymes in Experimental Demyelination of the Rabbit Optic Nerve: Sequence of Events
Authors: Reigner Jacqueline , Matthieu Jean‐Marie , Kraus‐Ruppert Rolf , Lassmann Hans , Poduslo Joseph F.
Published: October 2006

Abstract: Wallerian degeneration of the rabbit optic nerve was investigated by the technique of retinal ablation which precludes edema, hemorrhage, or macrophage infiltration. After 8 days of degeneration, marked degradation of axons and some myelin abnormalities appeared in the optic nerve, optic chiasma, and optic tract. Myelin lesions were maximal 32 days after retinal destruction. The amount of material stained with a myelin dye decreased drastically between 32 and 90 days after the operation. Biochemical parameters gave the following sequence of events. The concentration of the major periodic acid‐Schiff staining glycoproteins was decreased after 2 days, and 6 days later the presence of cholesterol esters was detected in the optic tissue. After 16 days of Wallerian degeneration, the specific activity of 2′,3′‐cyclic nucleotide 3′‐phospho‐diesterase not associated with myelin decreased, indicating a possible dedifferentiation of oligodendrocytes. Degradation of myelin basic protein became significant at 32 days and the amount of myelin isolated decreased later. The loss of myelin basic protein coincided with a reduction of myelin periodicity as measured in purified fractions by electron microscopy. These results show that secondary myelin destruction in the absence of edema, hemorrhage, or macrophages is a very slow process, and in this situation myelin undergoes a selective and sequential loss of its constituents.
Evidence That the Synaptic Phosphoprotein B‐50 Is Localized Exclusively in Nerve Tissue
Authors: Kristjansson G. I. , Zwiers H. , Oestreicher A. B. , Gispen W. H.
Published: October 2006

Abstract: The localization of the phosphoprotein B‐50 (molecular weight 48,000, isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B‐50 protein. Two‐dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and an immunochemical detection method employing an anti‐B‐50 antiserum revealed the presence of B‐50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B‐50 in nervous tissue. In addition, the regional distribution of endogenous B‐50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B‐50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1–24 is discussed.
A Novel Inhibitor of Gamma‐Aminobutyrate Aminotransferase with Anorectic Activity
Authors: White H. L. , Howard J. L. , Cooper B. R. , Soroko F. E. , McDermed J. D. , Ingold K. J. , Maxwell R. A.
Published: October 2006

Abstract: 1‐(n‐decyl)‐3‐Pyrazolidinone (BW357U) is a potent, selective inhibitor of gamma‐aminobutyrate aminotransferase (GABA‐T) in vitro and in vivo. After acute or chronic, oral or intraperitoneal administration of BW357U to rats, brain GABA levels were elevated in a dose‐dependent manner. When inhibition of brain GABA‐T exceeded 50%, whole brain GABA levels were elevated approximately threefold, and an anorectic effect was observed in the absence of other symptoms. This compound, because of its potency and selectivity, may be useful in studies relating to the function of GABA‐containing neurons in appetite regulation.
Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin
Authors: Smith Marion Edmonds , Sternberger Nancy H.
Published: October 2006

Abstract: The effect of an inhibitor of N‐glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21‐day‐old Wistar rats. Tunicamycin when incubated with these nerves in the presence of 3H‐labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high‐molecular‐weight proteins, P0, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of 14Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P0 peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with 3H amino acids in the presence of tunicamycin. The new protein, which cross‐reacts with P0 antiserum, was tentatively identified as a nonglycosylated P0 protein that appears to be almost as well incorporated as P0 into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P0 is actually assembled into a site and configuration like that of P0.
The Binding of the GAB A Agonist [3H]THIP to Rat Brain Synaptic Membranes
Authors: Falch E. , Krogsgaard‐Larsen P.
Published: October 2006

Abstract: THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol) is a specific GABA agonist with potent analgesic properties. The binding of radioactive THIP to thoroughly washed, frozen, and thawed membranes isolated from rat brains has been studied at 2°C under sodium ion‐free conditions and compared with the binding of [3H]GABA and [3H]piperidine‐4‐sulphonic acid ([3H]P4S). The best computer fits to the experimental data were in all cases attained with a receptor model based on three independent binding sites, of which only the high‐ and medium‐affinity sites could be characterised satisfactorily. While the KD values were found to be comparable for all three ligands employed, the density of the high‐affinity binding site (BM1) was, with the exception of the membranes from the cerebellum, considerably lower for [3H]THIP than for [3H]GABA and [3 H]P4S. The regional distribution of the GABA receptors, which bind [3H]THIP, was different from those recognizing [3H]GABA and [3H]P4S. A number of analogues, including asymmetric compounds with known configuration, were tested as inhibitors of the binding of [3H]GABA, [3H]muscimol, [3H]THIP, [3H]isoguvacine, and [3H]P4S. The concentrations of the asymmetric compounds required for the inhibition of [3H]P4S binding were much higher than those required for the displacement of [3H]GABA, [3H]muscimol, [3H]THIP, and [3H]isoguvacine. The comparable relative potencies of inhibitors do, however, indicate that all of the ligands bind to the GABA receptors.
Increase in Exogenous Choline Fails to Elevate the Content or Turnover Rate of Cortical, Striatal, or Hippocampal Acetylcholine
Authors: Brunello N. , Cheney D. L. , Costa E.
Published: October 2006

Abstract: The present experiments were designed to test whether increasing the availability of choline to rat brain increases the rate of acetylcholine synthesis in that organ. The content of choline and acetylcholine and the turnover rate of acetylcholine in striatum, hippocampus, and cerebral cortex were measured following changes in dietary choline, intraperitoneal choline, or intravenous infusion of choline. Increasing plasma choline caused some increase in tissue choline but did not increase acetylcholine levels nor acetylcholine turn‐over rate in any of the areas of brain studied. Indeed, in hippocampus, choline decreased the turnover rate of acetylcholine.
Neurotransmitter Metabolism in Rat Brain Synaptosomes: Effect of Anoxia and pH
Authors: Pastuszko Anna , Wilson David F. , Erecińska Maria
Published: October 2006

Abstract: Synaptosomes isolated from the rat cerebral cortex by means of a discontinuous Ficoll gradient carry out net, sodium‐dependent, veratridine‐sensitive accumulation of γ‐aminobutyric acid (GABA), serotonin, norepinephrine, and dopamine. The intrasynaptosomal contents of the four neurotransmitters are: 30.4 nmol/mg protein, 17.4 pmol/mg protein, 13.5 pmol/mg protein, and 21.2 pmol/mg protein, respectively. Anaerobic preincubation of synaptosomes causes an irreversible decrease in the rates of neurotransmitter accumulation but does not affect the rates of their release. The inhibitory effect of anaerobiosis is enhanced by increased concentration of [H+] (decreased pH) in the medium. The most sensitive is the uptake of dopamine, the least that of serotonin. The rates of neurotransmitter efflux are unaffected by anaerobiosis. Synaptosomes leak catecholamines, GABA, and serotonin into the medium when subjected to anaerobiosis, and reintroduction of oxygen is accompanied by a rapid reaccumulation of all four neurotransmitters. It is concluded that: (1) Responses of synaptosomes to anaerobiosis are remarkably similar to the behavior of intact brain in hypoxia and ischemia. (2) Neurotransmitter uptake systems are more sensitive to short periods of anaerobiosis than are either the energy metabolism or ion transport. (3) Some neurotransmitter uptake systems are more easily damaged by anaerobiosis than others.
Serotonin and γ‐Aminobutyric Acid Turnover After Injection into the Median Raphe of Substance P and D‐Ala‐Met‐Enkephalin Amide
Authors: Forchetti Concetta M. , Marco Emilio J. , Meek James L.
Published: October 2006

Abstract: The raphe nuclei [which contain serotonin (5‐HT) cell bodies) are also known to contain axons that store substance P, met‐enkephalin, and γ‐aminobutyric acid (GABA). We have previously shown that GABA has a tonic inhibitory action on 5‐HT turnover. To examine other possible interactions of these neuronal systems, we assessed the effect on 5‐HT turnover of injecting substance P and 2‐n‐ala‐met‐enkephalin into the median raphe nucleus, and the effects of substance P on GABA turnover. Serotonin turnover was increased by 30% in the hippocampus after the injection of substance P (4 μg) into the median raphe, indicating an excitatory effect of substance P on the raphehippocampal system. Local injection of the metabolically stable metenkephalin analog 2‐D‐ala‐met‐enkephalin amide (10 μg) increased the hippocampal steady state content of 5‐hydroxyindoleacetic acid (5‐HIAA) by 60%. The data suggest an excitatory effect of met‐enkephalin within the raphe nucleus. We attempted to estimate GABA turnover from the rate of disappearance of GABA after inhibition of glutamic acid decarboxylase by isoniazid and by the rate of accumulation of GABA after inhibition of GABA transaminase by gabaculine. Isoniazid, which is a competitive inhibitor, had too short and incomplete an action to be of use when injected intranuclearly. Gabaculine, which is an irreversible inhibitor, induced a rapid‐onset increase in GABA content. This accumulation was linear up to 90 min. The injection of gabaculine (80 ng) into the raphe increased GABA content by five times the control values, but hippocampal 5‐HT and 5‐HIAA contents were not significantly changed. Substance P injection increased the GABA turnover by 30%. Gabaculine seems a promising tool for detecting changes in GABA turnover.
Properties of Particulate and Detergent‐Solubilized Phospholipid N‐Methyltransferase Activity from Calf Brain
Authors: Percy A. K. , Moore J. F. , Waechter C. J.
Published: October 2006

Abstract: Calf brain membranes catalyze the enzymatic transfer of [CH3‐3H]methyl groups from S‐adenosyl‐l‐[CH3‐3H]methionine into endogenous phosphatidyl‐N‐methylethanolamine (PME), phosphatidyl‐N,N‐dimethylethanolamine (PDE), and phosphatidylcholine (PC). Phospholipid N‐methylation can be stimulated by the addition of exogenous PME or PDE, added in aqueous dispersions with sodium taurocholate. When membranes are incubated in the presence of exogenous PME, [CH3‐3H]PDE represents 86% of the labeled phospholipid products. When exogenous PME is replaced by PDE, 91% of the label is incorporated into PC. Thus, under these in vitro conditions it is possible to assay PME‐ and PDE‐N‐methyitransferase activity separately. The calf brain phospholipid N‐methyltransferase activity has also been solubilized by treating the membranes ultrasonically in the presence of Triton X‐100 and 10 mM monothioglycerol. When the detergent extracts are incubated in the presence of exogenous PME, [CH3‐3H]PDE represents 86% of the enzymatically labeled products. In the presence of exogenous PDE, more than 97% of the label is incorporated into PC. Optimal conditions for the membrane‐bound and detergent‐solubilized PME‐ and PDE‐N‐methyltransferase activity have been established. These conditions have been used as a basis for testing the hypothesis that the conversion of PME to PC is catalyzed by a single enzyme in calf brain. In these studies, PME‐ and PDE‐N‐methyltransferase activities have been found to be similar, if not identical, with respect to: (1) extractability with Triton X‐100; (2) pH optimum; (3) response to divalent cations; (4) apparent Km, for S‐adenosyl‐l‐methionine and KI for S‐adenosyl‐l‐homocysteine, (5) sensitivity to N‐ethylmaleimide; and (6) thermal inactivation at 55°. Overall, these results are consistent with the conclusion that in calf brain, PME and PDE are methylated by the same enzyme or by two phospholipid N‐methyltransferases having very similar properties.
Identification of Actin in Highly Purified Synaptic Vesicles from the Electric Organ of Torpedo marmorata
Authors: Zechel Kasper , Stadler Herbert
Published: October 2006

Abstract: Evidence has been obtained that actin is a major constituent of highly purified synaptic vesicles isolated from the electric organ of Torpedo marmorata. The mobility of a prominent spot in the polypeptide pattern of vesicles in high‐resolution two‐dimensional polyacrylamide gel electrophoresis is very similar to the mobility of the main component in the actin preparation purified from the whole electric organ by affinity chromatography on immobilized pancreatic deoxyribonuclease I. The comparison of tryptic peptide maps obtained from the putative vesicle actin and authentic actin from the electric organ, both purified by two‐dimensional gel electrophoresis and labeled in situ with 125I, showed about 88% homology, thereby supporting the conclusion that the vesicle actin is indeed an actin isoform.
In Vitro Synthesis of the Four Mouse Myelin Basic Proteins: Evidence for the Lack of a Metabolic Relationship
Authors: Yu Yie‐Teh , Campagnoni Anthony T.
Published: October 2006

Abstract: Studies on the synthesis of the four immunologically related mouse myelin basic proteins (MBPs) were carried out to determine if these proteins were metabolically related. Two in vitro systems were used: (a) a homologous brain system consisting of free polysomes, pH 5 enzymes, and initiation factors; and (b) a reticulocyte lysate system directed with mRNA and supplemented with brain factors. Incorporation of [35S]methionine into the four MBPs (14K, 17K, 18.5K, and 21.5K) was detected by immunoprecipitation of the in vitro products of synthesis followed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. The four MBPs were identified by cross‐reactivity with purified anti‐MBP antibodies and their apparent molecular weights in SDS gels. Synthesis of all four proteins was detected in both systems soon after the incubations were begun. The kinetics of the labeling of the proteins showed no evidence of a precursor‐product relationship (i.e., 21.5K→ 18.5K; 17K → 14K) in either system. Inhibition studies with puromycin and “chase” experiments with unlabeled methionine demonstrated that neither system contained posttranslational “processing” activity. Thus, the 21.5K and 17K proteins were not being processed into the 18.5K and 14K MBPs by either . in vitro system. Detection of the synthesis of all four proteins in the reticulocyte system programmed with brain mRNA indicates that the four proteins are probably coded for by separate mRNAs. This conclusion was supported by studies using polyribosomes separated into different size classes, which suggest that the mRNAs for the four proteins may be translated on proteins of differing size class. It is proposed, therefore, that the four MBPs are the primary translation products of independent brain mRNAs and are not metabolically related.
Method for Lyophilizing Brain Proteolipid Preparation That Increases Subsequent Solubilization by Detergent
Authors: Aguilar J. S. , Cózar M. de , Criado M. , Monreal J.
Published: October 2006

Abstract: A frozen mixture of solubilized brain proteolipid proteins in chloroform‐methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform‐methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2 mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X‐100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X‐100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed.
Cholinergic Neuronotrophic Factors. Concurrent Activities on Certain Nerve Growth Factor‐Responsive Neurons
Authors: Manthorpe Marston , Skaper Stephen D. , Barbin Gilles , Varon Silvio
Published: October 2006

Abstract: We had previously reported that in vitro survival of chick embryo ciliary ganglionic neurons can be assured by the addition to the culture medium of appropriate amounts of soluble macromolecular agents termed ciliary neuronotrophic factors. Particularly rich sources of one such factor are aqueous extracts from chick embryo intraocular tissues that include the smooth and striated musculature innervated by ciliary ganglionic neurons. We report here that this eye extract also contains agents that we term ganglionic neuronotrophic factors that support the survival of 11‐day chick embryo sympathetic and neonatal mouse dorsal root ganglionic neurons, two traditional targets of nerve growth factor (NGF). Using a recently developed microassay procedure we found that these ganglionic activities are not inactivated by rabbit, rat, or guinea pig antisera raised against the 2.5S (beta) subunit of male mouse submaxillary NGF, rabbit antisera against 7S NGF, or quail antisera against cobra venom NGF. Both the ciliary and ganglionic activities can be quantitated simultaneously by using 24‐h in vitro microassays, thus permitting a direct comparison of their respective properties. Both activities were found to (a) adsorb to DE52 cellulose and coelute at a similar salt concentration, (b) focus and be recovered from isoelectric polyacrylamide gels at exactly the same pH region, (c) be heat‐and partially acid‐labile, but base‐stable, and (d) be inactivated by exposure to trypsin. These results suggest that the ciliary and ganglionic neuronotrophic activities are associated with the same protein.
Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain
Authors: Sims N. R. , Marek K. L. , Bowen D. M. , Davison A. N.
Published: October 2006

Abstract: Production of [14C]acetylcholine and 14CO2 was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [14C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 mm‐ and 31 mm‐K+, contrasting with the lack of significant change in 14CO2 production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13‐month and senescent animals. Measurements of the release of [14C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.
Phospholipid Degradation and Cellular Edema Induced by Free Radicals in Brain Cortical Slices
Authors: Chan Pak Hoo , Yurko Mary , Fishman Robert A.
Published: October 2006

Abstract: Cellular edema and increased lactate production were induced in rat brain cortical slices by xanthine oxidase and xanthine, in the presence of ferric ions. Lipid peroxidation, as measured by thiobarbituric acid‐reactive malon‐dialdehyde, was increased 174%. Among the various subcellular fractions of brain cortex, xanthine oxidase‐stimulated lipid peroxidation was highest in myelin, mitochondria, and synaptosomes, followed by microsomes and nuclei. Antioxidants, catalase, chlorpromazine, and butylated hydroxytoluene inhibited lipid peroxidation in both homogenates and synaptosomes, indicating H2O2 and radicals were involved. Further, several free fatty acids, especially oleic acid (18:1), arachidonic acid (20:4), and docosahexaenoic acid (22:6) were released from the phospholipid pool concomitant with the degradation of membrane phospholipids in xanthine oxidase‐treated synaptosomes. These data suggest that Upases are activated by free radicals and lipid peroxides in the pathogenesis of cellular swelling.