bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Ornithine Decarboxylase Antizyme in Mouse Brain
Authors: Hietala Oili A.
Published: October 2006

Ornithine decarboxylase, the rate‐limiting enzyme in polyamine synthesis, is known to be regulated by a macromolecular inhibitor, termed antizyme, in a number of cellular systems. The present results show that the antizyme is also a functional component of polyamine metabolism in the brain. It could be demonstrated both in normal randomly selected mice and in animals which had been subjected either to intracerebroventricular injection of saline, which is known to cause a transient activation of ornithine decarboxylase, or to l,3‐diamino‐2‐propanol, an antizyme‐inducing agent. When compared to tissues or cell systems studied so far, the cytosol fraction from mouse brain homogenate appeared to contain an exceptionally high amount of antizyme, that was bound to some material other than active ornithine decarboxylase. This feature was seen in all the animal groups studied, being most prominent after saline injection, when the amount of dissociable antizyme exceeded 14‐fold the corresponding released ornithine decarboxylase activity. In untreated animals the excess was about eightfold and after 1,3‐diamino‐2‐propanol about fivefold.
Ceramide Synthesis from Free Fatty Acids in Rat Brain: Function of NADPH and Substrate Specificity
Authors: Singh Inderjit
Published: October 2006

At the subcellular level, the synthesis of ceramide from free lignoceric acid and sphingosine in brain required reconstituted enzyme system (particulate fraction, heat‐stable and heat‐labile factors) and pyridine nucleotide (NADPH). The mitochondrial electron transfer inhibitors (KCN and antimycin A), energy uncouplers (oligomycin and 2,4‐dinitrophenol), and carboxyatractyloside, which prevents the transport of ATP and ADP through the mitochondrial wall, inhibit the synthesis of ceramide in the presence of NADPH but have very little effect in the presence of ATP. Similar to the synthesis of ceramide, the synthesis of ATP from NADPH and NADH by the particulate fraction also required cytoplasmic factors (heat‐stable and heat‐labile factors). Moreover, ATP, but not its analog (AMP‐CH2‐P‐O‐P), can replace NADPH, thus suggesting that the function of the pyridine nucleotide is to provide ATP for the synthesis of ceramide. The cytoplasmic factors were not required for the synthesis of ceramide in the presence of ATP. The maximum velocity for synthesis of ceramide from free fatty acids of different chain lengths (C16‐C26) was bimodal, with maxima around stearic acid (C18) and behenic acid (C22). The relative rate of synthesis of ceramide parallels the relative distribution of these fatty acids in brain cerebrosides and sulfatides.
Purification and Characterization of Two Distinct Isozymes of Protein Carboxymethylase from Bovine Brain
Authors: Aswad Dana W. , Deight Elizabeth A.
Published: October 2006

Protein carboxymethylase (EC 2.1.1.24) from cytosol of bovine brain was found to exist as two apparent isozymes that could be separated by chromatography on DEAE‐cellulose at pH 8.O. Rechromatography of the two forms, designated PCM I and PCM II, indicated that they are not interconvertible. Both enzymes have a molecular weight of 24,300 by sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis. PCM I consists mainly of one isoelectric form, pI 6.5, whereas PCM II resolves into two forms of pI 5.6 and 5.7. The relative amounts of PCM I and PCM II show a marked tissue dependence. Brain has approximately twice as much PCM I as II, whereas liver contains only the type II enzyme. The two enzymes were found to have similar substrate specificities when tested with five different methyl‐accepting proteins. Synapsin I, a basic protein associated with synaptic vesicles, was found to be an excellent methyl‐accepting protein with regard to its Km (1.2 μM), but it exhibited a low stoichiome‐try of methyl incorporation.
Inhibition by Quinacrine of Depolarization‐Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes
Authors: Baba Akemichi , Ohta Akira , Iwata Heitaroh
Published: October 2006

The effects of quinacrine on depolarization‐induced [3H]acetylcholine (ACh) release and 45Ca2+ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [3H]ACh by high K+ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187‐induced release of [3H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca2+ influx by veratridine and high K+ but not that by “Na+‐free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca2+ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca2+ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization‐induced Ca2+ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.
Elemental Analysis in Murine Central Nervous System: Elevation of Rubidium Subsequent to Newcastle Disease Virus Encephalopathy
Authors: Murray Ronald S. , Burks Jack S. , Smythe W. Rodman , Miller Nancy , Alfrey Allen C. , Gerdes John C.
Published: October 2006

Abstract: The Cg strain of Newcastle disease virus (NDV) produces neurologic signs and death in mice. This illness is unusual because of the lack of typical features of a viral encephalitis. Specifically, there is a paucity of infectious virus, detectable cellular inflammatory reaction, cytopathic effect, and viral antigen by immunofluorescence. We previously showed an elevation of α‐aminoisobutyric acid in the CNS of moribund NDV‐infected mice, indicating cellular membrane dysfunction. In an attempt to further our understanding of the pathogenesis of the illness, we evaluated CNS concentrations of sodium, potassium, iron, copper, zinc, magnesium, selenium, and rubidium. Elemental analysis revealed no difference between infected and control mice for all elements except for rubidium, which was significantly elevated in infected mice. Elevation in rubidium was detected in infected mice by X‐ray fluorescence and atomic absorption spectrophotometry, whereas rubidium concentrations for control mice were similar by both methods. Neurologic symptoms correlated directly with rising rubidium concentrations. Our data suggest that abnormal trace element levels during viral infection may be one mechanism responsible for the clinical symptoms.
Characterization and Partial Purification of a Ganglioside‐Associated Mitogen
Authors: Morgan James I. , Price Jack , Seifert Wilfried
Published: October 2006

Abstract: A nonganglioside factor(s) present in Sigma types II and III mixed bovine brain ganglioside preparations synergises with suboptimal amounts of serum to induce proliferation specifically in nondividing B103 neuroblastoma cultures. The active substance is nondialys‐able and soluble in water as well as in chloroform‐methanol mixtures of 1:1–4:1 (vol/vol). It is completely insoluble in ether and acetone at room temperature. Biological activity survives heating to 70°C in the presence of 0.1 M HC1 for 1 h as well as boiling at neutral pH. Loss of activity occurs on heating to 70°C for 1 h with 1 M HCl or 1 M NaOH. The activity is insensitive to digestion with neuraminidase, trypsin, pronase, and phospholipases A2 and C. The factor cochromatographs with gangliosides on Dowex AG 50W and Sephadex G100 and is partially recovered with GMI on DEAE‐Sepharose, but may be isolated in a ganglioside‐free fraction by sequential chromatography on Sephadex LH20 and silicic acid columns. The substance(s) has the properties of a water‐soluble proteolipid protein, the amino acid composition being reported. It is not immunologically cross‐reactive with antibodies to GMI ganglioside or the major proteolipid protein of myelin.
Further Characterization of In Vitro Conditions Appropriate for GABA Determination in Human CSF: Impact of Acid Deproteinization and Freeze/Thaw
Authors: Ferraro Thomas N. , Manyam Bala V. , Hare Theodore A.
Published: October 2006

Abstract: Recently established standardized protocols for collection, handling, and storage of CSF for measurement of γ‐aminobutyric acid (GABA) have proven valuable in the characterization of various CNS disorders. In response to two recent reports which may have an impact on certain widely used protocols, we have, using the confirmed ion‐exchange/fluorometric procedure, systematically evaluated the effects of deproteinization with various concentrations of sulfosalicylic acid (SSA) ranging from 0 to 10% (100 mg/ml), as well as the effects of freeze/ thaw (F/T) on CSF GABA levels. Results of F/T studies documented that levels are stable to freezing and thawing. Acid deproteinization studies revealed the presence of an equilibrium between strictly free GABA, demonstrable only in acid‐free CSF, and a very loosely bound form of GABA, fully demonstrable only in CSF deproteinized with concentrations of SSA above 1% (10 mg/ml). The relationship between GABA concentrations in undeproteinized and acid‐deproteinized CSF revealed a highly significant (p < .001) correlation, suggesting that alterations of central GABAergic activity would be reflected by either the level of strictly free GABA or free plus loosely bound GABA. This hypothesis was upheld in studies of patients with Parkinson's disease (PD) and Huntington's disease (HD), two neurologic disorders in which dysfunctions of the GABA system have been implicated. Results indicated that CSF GABA levels are significantly reduced in both PD and HD patients compared with neurologically normal controls, whether the measurement is of free GABA or free plus loosely bound GABA. Thus, we conclude that the level of strictly free GABA is stable to freezing and thawing and can only be accurately determined in nonacidified CSF; however, existing protocols employing deproteinization in 5% SSA yield data that provide an equally good reflection of central GABAergic transmission.
Sustained Elevation of Hippocampal Cyclic 3′‐5’ Adenosine Monophosphate Levels After Medial Septal Lesions
Authors: Bernard Estrada , Strauss Brian , Haring John , Davis James N.
Published: October 2006

Abstract: The cyclic 3′‐5’ adenosine monophosphate (cyclic AMP) content of the rat hippocampal formation doubles during the week following a medial septal lesion and remains elevated for at least 1 month, the longest time period studied. This elevation in cyclic AMP does not result from sympathetic in growth, as neither superior cervical ganglion stimulation nor ganglionectomy influences hippocampal cyclic AMP content after lesions. Interruption of the cholinergic septohippocampal pathway in the fornix did not elevate hippocampal cyclic AMP content. Further, treatment of septallesioned animals with oxotremorine or of normal animals with atropine did not influence hippocampal cyclic AMP content. Finally, neither locus ceruleus lesions nor treatment with propranolol affected hippocampal cyclic AMP content. We believe this to be the first report of a sustained elevation in hippocampal cyclic AMP content. Like other long‐term events, it is likely to have profound effects on hippocampal function and represents a remarkable brain adaptation to remote injury.
Cholesterol Esters in Rat Brain Infected with Acute Measles Encephalitis: Concentration and Fatty Acid Composition
Authors: Abe Toshiaki , Ogawa Kiyoko , Hiraiwa Mikio , Kobune Fumio , Nagashima Kazuo
Published: October 2006

Abstract: The present study deals with the concentration and fatty acid composition of cholesterol esters in rat brains infected experimentally with measles virus to induce acute encephalitis. The left side of the cerebrum, as well as other portions of the brain, when inoculated per‐cutaneously contained a large amount of cholesterol esters. The major fatty acids from the esters in the brain were C16:0, C16:1, C18:0, and C18:1; those from the serum were C18:1, C18:2, and C20:4 This result indicates that cholesterol esters may not come from serum but can be synthesized in situ, even in the brain with acute viral infection.
Evidence for Multiple Muscarinic Receptor Subtypes in Human Brain
Authors: Garvey Janice M. , Rossor Martin , Iversen Leslie L.
Published: October 2006

Abstract: Pirenzepine, a compound with selective anti‐muscarinic activity, was used to distinguish muscarinic acetylcholine receptor subtypes in normal human brain. Hill coefficients and IC50 values derived from the inhibition of specific [3H]l‐quinuclidinyl benzilate receptor binding suggest the presence of two muscarinic binding sites, differing both in affinity for pirenzepine and in tissue distribution.
Effect of Age on Vitamin E Concentrations in Various Regions of the Brain and a Few Selected Peripheral Tissues of the Rat, and on the Uptake of Radioactive Vitamin E by Various Regions of Rat Brain
Authors: Vatassery G. T. , Angerhofer C. K. , Knox C. A.
Published: October 2006

Abstract: The concentrations of tocopherols in selected areas of the brains and a few peripheral tissues of 3‐, 14‐, and 30‐month‐old male Fischer 344 rats were determined by a high‐performance liquid chromatographic method. Throughout the time period studied, α‐tocopherol was the only tocopherol detected in the brain. Concentrations of α‐tocopherol increased significantly with age in medulla and spinal cord whereas no such change was seen in other brain areas. Among the peripheral tisues, total tocopherol concentrations increased with age in the liver and adipose tissue while no significant changes were observed in the heart. The pattern of uptake of radioactive α‐tocopherol from the serum by the various areas of the brain was similar for the 3‐and 14‐month‐old animals even though the brains from the 14‐month‐old animals took up less of the radioactive compound. Measurable amounts of tocopherol esters were not present in the tissues of the 30‐month‐old animals.
Reaction of Opioid Peptides with Neutral Endopeptidase (‘Enkephalinase”)
Authors: Hersh Louis B.
Published: October 2006

Abstract: The kinetics of the reactions of nine opioid peptides with the neutral endopeptidase (“enkephalinase”) activities of human kidney, rat kidney, and rat brain have been determined. These opioid peptides can be divided into two classes, those that are good inhibitors of Leu5‐enkephalin hydrolysis (K1 < 75 μM) and good substrates for the enzyme, and those that are poor inhibitors (K1 > 500 μM) and are not substrates for the enzyme. The former group includes Leu5‐enkephalin, Met5‐enkephalin, Met5‐enkephalin‐Arg6‐Phe7, β‐lipotropin6169, and γ‐endorphin, while the nonreactive opioid peptides include α‐neo‐endorphin, β‐neo‐endorphin, dynorphin1–13, and β‐endorphin. These results suggest that those peptides containing the Met5‐enkephalin sequence are more reactive than those containing the Leu5‐enkephalin sequence. The lack of specificity of this neutral endopeptidase indicates that it may function in the degradation of a variety of biologically active peptides.
Regulation of Myelin Basic Protein (Arginine) Methyltransferase by Thyroid Hormone in Myelinogenic Cultures of Cells Dissociated from Embryonic Mouse Brain
Authors: Amur Shashi G. , Shanker Gouri , Pieringer Ronald A.
Published: October 2006

Abstract: The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 nMl‐3,5,3′‐triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin‐associated events in these cultures.
Vanillic Acid, a Metabolite of 4‐Hydroxy‐3‐Methoxyphenylglycol and 4‐Hydroxy‐3‐Methoxymandelic Acid in Man
Authors: Mådh Göran
Published: October 2006

Abstract: 4‐Hydroxy‐3‐methoxyphenylglycol (HMPG) labelled with 14C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4‐hydroxy‐3‐methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 ± 1.1% (mean ± SEM) was excreted as 14C‐labelled vantllic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium‐labelled HMPG ([2H3]HMPG) and HMMA ([2H6]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [2H3]VA from [2H3]HMPG was 8.3 ± 2.1% and the recovery of [2H6]VA from [2H6]HMMA was 9.0 ± 2.1%. The 2H‐labelled VAs were probably formed by a decar boxylation reaction, in the case of HMPG after previous oxidation to HMMA.
Bulk Separation and Long‐Term Culture of Oligodendrocytes from Adult Pig Brain.
Authors: Gebicke‐Härter P. J. , Althaus H.‐H. , Neuhoff V.
Published: October 2006

Abstract: Oligodendroglial proteins labeled with radioactive amino acids were subjected to one‐ and two‐dimensional polyacrylamide electrophoresis. Bands comigrating with myelin proteins, the basic protein (MBP), the proteolipid protein (PLP), and the Wolfgram protein (WP) doublet, were detected by Coomassie Blue staining and by autoradiography. The identity of the MBP and WP in the cellular material is evidenced by immunoblotting with specific antibodies. A comparative study of myelin samples from rat and pig CNS reveals that WP can be detected immunochemically in both species. Different protein patterns, however, are observed. Three protein bands are found with antibodies against the myelin‐associated glycoprotein (MAG). The high‐molecular‐weight component prevails in pig myelin, whereas the mediummolecular‐weight component is predominant in rat myelin. Moreover, two protein bands, of molecular weights 35,000 and 33,000 (011 and 01 2), are present in high amounts in oligodendroglial particulate material but are not detectable in myelin. These oligodendroglial characteristic proteins are not species‐specific, since they are found in preparations of cat oligodendrocytes as well. Activities of cerebroside sulfotransferase (EC 2.8.2.11) are low in freshly isolated cells and increase during the first week of culture. A reverse course of enzyme activities is observed with 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (EC 3.1.4.37). Values reach a minimum about day 5 in culture and recover their initial values. At day 10 they remain stable until the end of the third week of the culture period.
Evidence that a Cerebellum‐Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium‐Dependent Manner
Authors: Groswald Douglas E. , Kelly Paul T.
Published: October 2006

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP‐A. Previous results have shown that GP‐A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP‐A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP‐A is partially soluble in the non‐ionic detergent Triton X‐100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N‐lauryl sarcosinate. The solubilization of GP‐A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM‐1mM Ca2+ decreased the solubility of GP‐A in Triton by approximately threefold. GP‐A is a major concanavalin A (Con A)‐binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore‐brain synaptic fractions by two‐dimensional peptide mapping indicated that they were identical. The Con A‐binding property of GP‐A was exploited to purify it by affinity chromatography with agarose‐Con A. Peptide mapping comparisons between affinity‐purified GP‐A and GP‐A in SPM and SJ fractions indicated that GP‐A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP‐A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti‐fodrin antisera on Western blots; however, GP‐A failed to cross‐react. These observations, together with results from previous studies, indicate that GP‐A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP‐A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell‐surface glycoprotein at asymmetric synapses in cerebellum. GP‐A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP‐A in Triton that results from micromolar concentrations of calcium, suggest that GP‐A may play a role in stabilizing cerebellar synaptic junctions.
Brain and Kidney d‐Amino Acid Oxidases Are Coded by a Single Gene in the Mouse
Authors: Konno Ryuichi , Yasumura Yosihiro
Published: October 2006

Abstract: Mutant mice (ddYIDAO−) lacking d‐amino acid oxidase in the kidney also lacked this enzyme in the brain. Genetic cross experiments showed that the inheritance of the enzyme in the brain was the same as that in the kidney. The deficiency in the enzyme in the brain could not be separated from that in the kidney. The brain and kidney enzymes showed similar substrate specificities. These results suggest that brain and kidney d‐amino acid oxidases are coded by the same gene in the mouse.
Effect of Potassium on the Release of [3H]Noradrenaline from Rabbit and Human Pulmonary Artery
Authors: Vizi Elek Sylvester , Torok Tamas , Magyar Kalman
Published: October 2006

Abstract: The release of [3H]noradrenaline ([3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+ ‐free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic α2‐‐adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+ ‐free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of Na,K‐ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+‐free solution, the results may indicate involvement of the sodium pump in NA release.
On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol‐O‐Methyltransferase
Authors: Köster G. , Goede E. , Breuer H.
Published: October 2006

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t1) or 1 h (t2) after intraventricular injection of [3H]noradrenaline, they were decapitated. The total 3H activity and the recovery of [3H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total 3H activity was measured at t1 in all tropolone‐treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [3H]noradrenaline. At t1, 40–50% of the sum of [3H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [3H]noradrenaline was retarded after t1. Inhibition of catechol‐O‐methyltransferase by tropolone caused consistently elevated [3H]noradrenaline levels, but did not affect the metabolic rate after t1 when compared with similarly pretreated, but tropolone‐free rats. Thus, if catechol‐O‐methyltransferase was inhibited during the injection of [3H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol‐O‐methyltransferase is linked to the extraneuronal uptake2. By affecting the access of noradrenaline to non‐neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.
Effect of Ethanol on Rat Brain Polyphosphoinositides
Authors: Shah I. R. , Uma S. , Ramakrishnan C. V. , Hauser G.
Published: October 2006

Abstract: Female rats were allowed to consume ethanol during gestation and lactation, and brain polyphosphoinositides of the 21‐day‐old pups were quantified. Ethanol intake prevented the disappearance of the metabolically labile pools of phosphatidylinositol‐4‐phosphate and phosphatidylinositol‐4,5‐bis‐phosphate, which are rapidly degraded in the control group. In contrast, preweaning undernutrition left the size of these pools virtually unchanged, indicating a differential effect of the two nutritional regimens. Key Words: Polyphosphoinositides—Labile pools—Ethanol—Brain of offspring—Phosphati‐dylinositol ‐ 4 ‐ phosphate—Phosphatidylinositol ‐ 4,5 ‐bis‐phosphate—Ethanol feeding—Undernutrition (preweaning). Shah I. R. et al. Effect of ethanol on rat brain polyphosphoinositides.
Is Butyrylcholinesterase of the Rat CNS a Membrane‐Bound Enzyme?
Authors: Meisami Esmail
Published: October 2006

Abstract: The study of Arrhenius plots for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity from the rat brain and spinal cord revealed that in contrast to AChE, which exhibited biphasic Arrhenius plots with a distinct break (transition temperature) at about 16–18°C, BuChE showed no evidence of discontinuity and a higher activation energy in the physiological range of temperature. The results indicate lack of lipid‐protein interaction in the case of BuChE of the CNS tissue. It is inferred that BuChE, in contrast to AChE, is not bound in any significant way to cellular membranes of the CNS tissue.
Relation of Cholesterol to Astrocytic Differentiation in C‐6 Glial Cells
Authors: Bhat Narayan R. , Volpe Joseph J.
Published: October 2006

Abstract: The relation of cellular cholesterol content to a biochemical expression of astrocytic differentiation was investigated in cultured C‐6 glial cells. The astrocytic marker, glutamine synthetase, was studied. Cellular sterol content was perturbed with compactin, a specific inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and, thereby, cholesterol biosynthesis. Depletion of cellular sterol resulted in 72 h in a more than twofold increase in glutamine synthetase activity. Production of various degrees of sterol depletion with different concentrations of compactin demonstrated a striking inverse relationship between glutamine synthetase activity and the cellular sterol/phospholipid molar ratio. That the effect of compactin, in fact, is mediated by depletion of sterol was shown further by prevention of the compactin‐induced increase in synthetase activity by simultaneous addition of exogenous cholesterol. Moreover, addition of cholesterol alone to the culture medium led to both a decrease in glutamine synthetase activity and an increase in the sterol/phospholipid molar ratio. The possibility that the compactin‐induced increase in glutamine synthetase activity is caused by an increase in synthesis of the enzyme was suggested by prevention of the increase by cycloheximide. The data suggest that astrocytic differentiation is stimulated by a decrease in cellular sterol content. When considered with our previous observation that oli‐godendroglial differentiation is inhibited by such a decrease, the findings suggest that cellular sterol content is a critical determinant of the direction of glial differentiation, i.e., whether along astrocytic or oligodendroglial lines.
Immunoreactive Met‐Enkephalin Arg6 in Rat Brain, and Bovine Brain, Gut, and Adrenal
Authors: Giraud A. S. , Dockray G. J. , Williams R. G.
Published: October 2006

Abstract: Antibodies directed against the Met‐enkephalin‐related hexapeptide, Met‐enk Arg6, have been used in radioimmunoassays in the characterization of material in rat brain, and bovine striatum, colon, and adrenal medulla. Met‐enk Lys6 reacted 0.27 relative to Met‐enk Arg6, but Leu‐enk Arg6 and C‐terminal extensions or deletions of Met‐enk Arg6 showed less than 0.02 immunoreactivity. In rat brain, the concentration of Met‐enk Arg6‐like immunoreactivity was <20 pmol · g−1 in all regions, but after trypsinization of tissue extracts there were up to 80‐fold increases in immunoreactivity as a result of cleavage of C‐terminally extended forms. The tryptic product eluted as Met‐enk Arg6 on gel filtration. In control extracts of rat brain there were at least three immunoreactive forms of Met‐enk Arg6; one eluted in the position of the hexapeptide standard on gel filtration and HPLC while the others had properties of N‐terminally extended forms. In bovine striatum and colon the hexapeptide‐like material predominated; but in bovine adrenal extracts, there were relatively low concentrations of the hexapeptide and, instead, the dominant immunoreactive forms corresponded to two components that were probably N‐terminally extended variants. Trypsin again produced marked increases in immunoreactivity. HPLC studies indicated that Met‐enk Arg6Phe7‐and Met‐enk Arg6Gly7Leu8‐like immunoreactive peptides were important substrates in bovine brain for the production of hexapeptide immunoreactivity after trypsin. The differences in the patterns of immunoreactive forms in bovine adrenal, colon, and brain are consistent with tissue variations in the pathways of posttranslational processing of the precursor molecules.
High‐and Low‐Affinity States of Striatal D2 Receptors Are Not Affected by 6‐Hydroxydopamine or Chronic Haloperidol Treatment
Authors: MacKenzie Robert G. , Zigmond Michael J.
Published: October 2006

Abstract: Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5‐HT2 antagonist, but not in the presence of (+)‐butaclamol, a D2 and 5‐HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide‐insensitive whereas agonist binding was biphasic and guanine nucleotide‐sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6‐hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high‐and low‐affinity components associated with agonist binding to the D2 receptor.
Promotion of Microtubule Assembly by Neurofilament‐Associated Microtubule‐Associated Proteins
Authors: Leterrier Jean‐François , Wong Jackson , Liem Ronald K. H. , Shelanski Michael L.
Published: October 2006

Abstract: Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)‐purified tubulin. This assembly is temperature‐dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule‐associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC‐tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold‐sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC‐tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule‐assembly‐promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament‐associated MAPs.
Dural Mast Cells: Source of Contaminating Histamine in Analyses of Mouse Brain Histamine Levels
Authors: Orr Edward L.
Published: October 2006

Abstract: Histamine levels were determined in mouse brains from WBB6F1‐ +/+ (mast cell normal) and WBB6F1‐W/Wv (mast cell‐deficient) mice whose brains were dissected immediately after decapitation or after freezing the severed heads in liquid nitrogen for 10 s. In WBB6F1‐+/+ mice, brains obtained from frozen heads contained significantly higher levels of histamine than those obtained from unfrozen heads. The converse was found in brains obtained from the WBB6F1‐W/Wv mice. When CF‐1 mice (which also contain brain‐associated mast cells) were treated as described above, results very similar to those found with the WBB6F1‐ +/+ mice were obtained. Further, the high levels of histamine found in CF‐1 mice whose brains had been frozen in situ were accompanied by an extensive degranulation of mast cells in the dura mater of these mice. Because of this degranulation of mast cells, and the fact that increased levels of brain histamine were not found in mast cell‐deficient mice, it is concluded that dural mast cells are the likely source of the artifactually higher levels of histamine seen in brains frozen in situ.
Histamine Turnover in the Brain of Different Mammalian Species: Implications for Neuronal Histamine Half‐Life
Authors: Nishibori Masahiro , Oishi Ryozo , Saeki Kiyomi
Published: October 2006

The turnover of neuronal histamine (HA) in nine brain regions and the spinal cord of the guinea pig and the mouse was estimated and the values obtained were compared with data previously obtained in rats. The size of the neuronal HA pool was determined from the decrease in HA content, as induced by (S)‐α‐fluoromethylhistidine (α‐FMH), a suicide inhibitor of histidine decarboxylase. The ratios of neuronal HA to the total differed with the brain region. Pargyline hydrochloride increased the tele‐methylhistamine (t‐MH) levels linearly up to 2 h after administration in both the guinea pig and the mouse whole brain. Regional differences in the turnover rate of neuronal HA, calculated from the pargylineinduced accumulation of t‐MH, as well as in the size of the neuronal HA pool, were more marked in the mouse than in the guinea pig brain. The hypothalamus showed the highest rate in both species. There was a good correlation between the steady‐state t‐MH levels and the turnover rate in different brain regions. Neither the elevation of the t‐MH levels by pargyline nor the reduction of HA by α‐FMH was observed in the spinal cord, thereby suggesting that the HA present in this region is of mast cell origin. The half‐life of neuronal HA in different brain regions was in the range of 13‐38 min for the mouse and 24‐37 min for the guinea pig, except for HA from the guinea pig hypothalamus, which had an extraordinarily long value of 87 min. These results suggest that there are species differences in the function of the brain histaminergic system.
Subcellular Distribution of Opioid Peptides in Rat Hypothalamus and Pituitary
Authors: Molineaux C. J. , Rosenberger J. G. , Cox B. M.
Published: October 2006

Homogenates of rat anterior lobe (AL) and neurointermediate lobe (NIL) pituitary and rat hypothalamus were subjected to subcellular fractionation and density gradient centrifugation. The subcellular distribution of immunoreactive dynorophin A (ir‐Dyn A) in NIL was found to be similar to that of ir‐arginine vasopressin (ir‐AVP). ir‐Dyn A migrated as a discrete band on sucrose density gradients, which corresponded in sedimentation rate to that of ir‐AVP, suggesting that these two peptides are stored within organelles of similar size and density. Two other products of prodynorphin, ir‐α‐neoendorphin ir‐α‐nEND) and ir‐Dyn A‐(1–8) also comigrated with ir‐AVP. ir‐[Leu5]‐enkephalin (ir‐LE), which may be a product of prodynorphin or proenkephalin, was also found to migrate in this region of the gradient. When a homogenate of rat hypothalamus was prepared using a method that has been developed for synaptosome isolation, ir‐Dyn A was found to comigrate with Na+/K+‐activated adenosine triphosphatase (Na/K‐ATPase), a synaptosomal marker enzyme. Using a more concentrated homogenate ir‐Dyn A was found to migrate to a less dense region where peptide‐containing synaptic vesicles have previously been localized. When a synaptosomal preparation was lysed in hypotonic solution a shift was seen in the migration rate of (ir‐Dyn A to this region of the gradient (containing putative synaptic vesicles). Thus the bulk of hypothalamic dynorphin appears to be present within synaptosotne‐like structures which, upon lysis, release a less dense, smaller subcellular organelle corresponding in sedimentation characteristics to other types of peptide‐containing synaptic vesicles. Using either method ir‐AVP migrated to a position that was different from that of ir‐Dyn A and was unaffected by lysis conditions. These results indicate that the majority of the dynorphin in the hypothalamus is not stored with vasopressin at the subcellular level. Immunoreactivities associated with Dyn A‐(4–8), α‐nEND, LE, and [Met5]‐en‐kephalin‐Arg6‐Phe7 (ME‐RF) in hypothalamus were all found to migrate with ir‐Dyn A and all are shifted to approximately the same location by lysis. Thus all of the prodynorphin and proenkephalin products examined appear to be stored within synaptic vesicle‐like organelles present primarily within nerve terminals in the hypothalamus.
Simultaneous Measurement of 2′:3’ Cyclic‐Nucleotide 3’ Phosphodiesterase and RNase Activities in Sera and Spinal Fluids of Multiple Sclerosis Patients
Authors: Clapshaw Patric A. , Müller Hans Werner , Wiethölter Horst , Seifert Wilfried
Published: October 2006

Abstract: The enzymes 2′:3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and RNase were simultaneously measured in the sera and CSF of multiple sclerosis (MS) and non‐MS patients. No evidence of increased activity for these enzymes could be found regardless of pathology in either fluid source. Discrepancies between the present results and those from two previous studies that reported significant increases in CNPase activity in the CSF of patients with MS were carefully analyzed. It was concluded that the apparent increased CNPase activity correlated with MS in both previous studies was most probably the result of methodological and computational difficulties.
Mouse Monoclonal Antibodies Reacting with Human Brain Glial Fibrillary Acidic Protein
Authors: Albrechtsen M. , von Gerstenberg A. C. , Bock E.
Published: October 2006

Abstract: Three different epitopes on the glial fibrillary acidic protein (GFAP) have been identified by means of three monoclonal antibodies. The antibodies were named anti‐GFAP 1, anti‐GFAP 2, and anti‐GFAP 3. Antibody specificities were investigated by several techniques including indirect immunoprecipitation, immunoblotting, and immunohistochemistry. The anti‐GFAP 1 antibodies recognized an epitope found on GFAP from all three species tested: human, rat, and ox, but in addition a reaction was observed with cells not containing GFAP. The epitope recognized by anti‐GFAP 2 was present on GFAP from human and ox, but apparently not on rat GFAP; the anti‐GFAP 2 antibodies also reacted with antigen(s) other than GFAP. In contrast, the epitope defined by anti‐GFAP 3 has proved absolutely specific for GFAP in human, rat, and ox.
Solid‐Phase Immunoassay of PO Glycoprotein of Peripheral Nerve Myelin
Authors: Nunn D. J. , Mezei C.
Published: October 2006

Abstract: To explore the immunological properties of PO protein, antibodies were elicited in rabbits against the purified chick PO protein. Peripheral nervous system protein was fractionated on sodium dodecyl sulfate‐polyacrylamide slab gels and then transferred electrophoretically (“blotted”) onto nitrocellulose sheets. The PO protein was detected by its capacity to bind its specific antibody present in the rabbit serum. The PO‐specific antibody complex was then exposed to goat anti‐rabbit immunoglobulin G (IgG) coupled to peroxidase or labeled with 125I. The resulting PO antigen‐antibody “sandwich” was visualized and quantitated by densitometry of the colored peroxidase reaction product or by autoradiography and γ‐radiation counting of the 125I‐IgG complex. The methods permitted quantitation of the PO protein in various nerve extracts. The limit of detection of the PO antigen was about 1 ng of protein. The antibody was specific for the PO glycoprotein in the peripheral nerve extracts. The PO proteins from various species, including human, were also detected by the antibody to chick PO protein. Preliminary experiments indicate the solid‐phase immunoassay is a useful method for monitoring PO protein levels in small quantities of tissue extracts under various physiological and pathological conditions.
Temperature‐Dependent Kinetic Correlates of the Activation of the Glucocorticoid‐Receptor Complex
Authors: Luttge William G. , Densmore Charles L.
Published: October 2006

Abstract: The effects of temperature on the kinetics of activation were studied in [3H]triamcinoIone acetonide([3H]TA)‐labeled cytosol preparations from mouse whole brain. After removal of unbound [3H]TA and molybdate (which prevents activation) from the unactivated steroid‐receptor complex by gel exclusion chromatography, activation was initiated by incubation at 6–30°C for 0.75–24 min and then rapidly quenched at –5°C with Na2MoO4 (20 mM final concentration). The loss of the 9.2S (unactivated) form of the [3H]TA‐receptor complex and the concomitant formation of the 3.8S (activated) form increased dramatically with increases in the activation temperature. These hydrodynamic changes were correlated directly with rapid time‐ and temperature‐dependent increases in the binding of [3H]TA‐labeled cytosol to DNA‐cellulose (DNA‐C). Further analyses of these data revealed a >50‐fold increase in the apparent first‐order rate constant for the increased binding to DNA‐C as the activation temperature was increased from 6°C to 30°C. An Arrhenius plot of these temperature‐dependent kinetic constants revealed an energy of activation of 116 kJ. These data support a proposed model for activation of the glucocorticoid‐receptor complex that includes the splitting of a 297 kDa, unactivated species into a 92 kDa, activated species.
Multiple Defects in the Activity of Adenylate Cyclase from the Drosophila Memory Mutant Rutabaga
Authors: Dudai Yadin , Zvi Shoshana
Published: October 2006

Abstract: Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from ∼10−5M to ∼10−3M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole‐body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.
Isolation of Neuropeptide‐Containing Vesicles from the Guinea Pig Ileum
Authors: Ägoston Dénes V. , Ballmann M. , Conlon J. M. , Dowe G. H. C. , Whittaker V. P.
Published: October 2006

Abstract: Three distinct vesicle fractions enriched 40–60 times in the neuropeptides substance P, somatostatin, and vasoactive intestinal peptide (VIP) were prepared from the myenteric plexus of guinea pig ileum by density gradient centrifugation in a small zonal rotor. Mean densities (in g · ml−1) and diameters (in nm) of the three classes of vesicles were: substance P, 1.123, 65; somatostatin, 1.138, 37; VIP, 1.148, 110; standard deviations were about 5%. These peaks were distinct from the peak of acetylcholine‐containing vesicles of density 1.066 g · ml−1 and diameter 61 nm. When a relatively mild method of homogenization was used a second peak of acetylcholine appeared in the same region of the gradient as VIP and the VIP was larger. This may represent a class of vesicles containing both acetylcholine and VIP, though cosedimentation of two classes of vesicles of almost the same density and similar fragility, one containing VIP and the other acetylcholine, cannot be entirely excluded on present evidence.
Interaction of Hyaluronectin with Hyaluronic Acid Oligosaccharides
Authors: Bertrand Philippe , Delpech Bertrand
Published: October 2006

Abstract: Hyaluronic acid was digested by bovine testicular hyaluronidase, and oligomers were fractionated by gel permeation using AcA 202 Ultrogel, an acrylamide‐agarose matrix. Oligosaccharides composed of from two to six disaccharide repeating units were isolated. Two nonasaccharides were prepared by enzymatic or chemical modification of the decasaccharide. Oligosaccharides were compared by a competitive inhibition in the enzyme‐linked immunosorbent assay for their ability to inhibit the interaction of hyaluronectin (a hyaluronic acid‐binding brain glycoprotein) with hyaluronic acid. Among these oligosaccharides, decasaccharides were the smallest fragments that strongly inhibited the interaction. Octasaccharides inhibited with 700‐fold lower affinity than decasaccharides. Dodecasaccharides had the same effect as decasaccharides. Nonasaccharides obtained by β‐glucuronidase splitting of decasaccharides inhibited the interaction more than nonasaccharides prepared by an alkaline treatment.
Effects of 5,5′‐Dithiobis‐(2‐Nitrobenzoic Acid) on Opiate Binding to Both the Membrane‐Bound Receptor and the Partially Purified Opiate Receptor
Authors: Nozaki Masakatsu , Cho Tae Mook
Published: October 2006

Abstract: Pretreatment of partially purified opiate receptor from rat brains with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H‐agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane‐bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane‐bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.