bluuebunny/crossref_metadata_2025 huggingface dataset:
1 ‐Methyl‐4‐Phenyl‐ 1,2,3,6‐Tetrahydropyridine: Correspondence of Its Binding Sites to Monoamine Oxidase in Rat Brain, and Inhibition of Dopamine Oxidative Deamination In Vivo and In Vitro
Authors: Bocchetta A. , Piccardi M. P. , Del Zompo M. , Pintus S. , Corsini G. U.
Published: October 2006

Abstract: A saturable, specific, high‐affinity binding site for [3H] 1 ‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine was found in rat brain homogenates. The CNS regional distribution, the subcellular fractionation, and the displacement by pargyline, clorgyline, and deprenyl suggest that this binding site may correspond to monoamine oxidase. I ‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine inhibited the oxidative deamination of dopamine, both in vivo and in vitro. Striatal levels of 3,4‐dihydroxyphenylacetic acid were significantly reduced shortly after intravenous administration, and returned to normal values after a few hours. The in vitro formation of 3,4‐dihydroxyphenylacetic acid from dopamine was inhibited by concentrations of 1‐methyl‐4‐phenyl‐ 1,2,3,6‐tetrahydropyridine comparable to those of pargyline.
Wheat Germ Agglutinin Inhibits the Effects of Nerve Growth Factor on the Phosphorylation of Proteins in PC12h Cells
Authors: Hashimoto Seiichi , Ikeno Takeyuki , Kuzuya Hiroshi
Published: October 2006

Abstract: Treatment of PC12h cells in tissue culture with nerve growth factor (NGF) led to an increased incorporation of [32P]orthophosphoric acid into specific proteins. The increased phosphorylation of 60,000‐dalton and 20,000‐dalton proteins in the 0.2% Triton X‐100 detergent‐soluble fraction, of 35,000‐dalton protein in the 0.2% Triton X‐100 detergent‐insoluble fraction, and of slow migrating protein (SMP) in the nonhistone nuclear fraction was observed upon NGF treatment. On the other hand, wheat germ agglutinin (WGA) treatment of PC12h cells induced a slightly decreased phosphorylation of these NGF‐responsive proteins. Incubation of cell‐free extracts from PC12h cells with [γ‐32P]ATP led to the phosphorylation of a 100,000‐dalton protein. In extracts from cells treated with NGF, the labeling of the 100,000‐dalton protein was substantially and selectively reduced. In contrast, treatment of PC12h cells with WGA led to an increased phosphorylation of the 100,000‐dalton protein in cell‐free extracts. Thus, NGF and WGA showed opposite effects on the phosphorylation of specific proteins in both intact cells and cell‐free extracts. In addition, it was also observed in both systems that pre‐ and posttreatment of PC12h cells with WGA abolished the effects of NGF on the phosphorylation and produced a phosphorylation pattern similar to that from PC12h cells treated only with WGA. In parent PC12 cells, it has been reported that the treatment of cells with WGA inhibits NGF binding to its receptors and converts the rapidly dissociating receptors to slowly dissociating receptors. Thus, WGA in conjunction with NGF, results in the practical disappearance of rapidly dissociating receptors on cells. Therefore, the NGF‐induced alteration of the phosphorylation of proteins in PC12h cells, observed in our experiments, may be linked to the rapidly dissociating receptor.
Inhibition of Monoamine Oxidase by Phenyl Azides
Authors: Chen Shiuan , Shih Jean C. , Xu Qiu‐Ping
Published: October 2006

Abstract: We had previously shown that 4‐fluoro‐3‐nitrophenyl azide (FNPA) is a competitive inhibitor of both types of monoamine oxidase (MAO) in the dark, but it is a preferential photoaffinity label for only the type B MAO (MAO‐B). Recently we synthesized a number of arylazido compounds with structures related to FNPA and determined the effects of these compounds on the two types of MAO in rat brain cortex. We found that the fluoro group of FNPA was not required for the inhibition of MAO activities because neither the presence nor the position of the fluoro group affected its inhibition of MAO. On the other hand, both the nitro and the azido groups of FNPA were shown to be important for FNPA inactivation of two types of MAO. The inhibitory potency was significantly lower for compounds without either group. Furthermore, we found that all nitrophenyl azide isomers except 2‐nitrophenyl azide were photodependent inhibitors of MAO‐B. Under the same experimental conditions none of the compounds photoinactivated MAO‐A. On the basis of these findings, mechanisms for FNPA inhibition of the two types of MAO are discussed.
Regulation of Phosphate Incorporation into Four Brain Phosphoproteins that Are Affected by Experience
Authors: Lyn‐Cook Beverly D. , Ruder Franz J. , Wilson John Eric
Published: October 2006

Abstract: Various regulators of protein kinase activities were tested for their effects on the in vitro transfer of phosphate from [γ‐32P]ATP to four proteins of rat brain synaptic particulate preparations. One protein, of apparent molecular weight 44,000, accepted 32P in the presence of 8 mM EDTA and no added Mg2+. It was the major phosphoprotein of brain mitochondria. Its phosphorylation was inhibited by pyruvate and stimulated by K+, and it comigrated in electrophoretic gels with authentic α‐subunit of pyruvate: lipoamide oxidoreductase (decarboxylating) (EC 1.2.4.1) from bovine heart. The major kinase acting on three proteins of apparent molecular weights 24,000, 21,000, and 19,000 was stimulated by Ca2+, by preincubation with phospholipase C, and by 12‐tetradecanoyl 4‐β‐phorbol 13‐acetate. Phosphorylation of these lower‐molecular‐weight proteins was inhibited by ACTH1‐24, by cyclic 3′,5′‐adenosine monophosphate, and by 50μM trifluoperazine. The stimulatory effect of Ca2+ was antagonized by calmodulin. The kinase in question appears to be B‐50 protein kinase or protein kinase C.
Delta‐Sleep‐Inducing Peptide and Two of Its Analogs Reduce Nocturnal Increase of N‐Acetyltransferase Activity in Rat Pineal Gland
Authors: Graf Markus V. , Kastin Abba J. , Schoenenberger Guido A.
Published: October 2006

Abstract: Serotonin N‐acetyltransferase, an enzyme of the pineal gland, converts serotonin to N‐acetylserotonin. The activity of this enzyme is induced by norepinephrine in the evening to reach high levels during the dark phase. Delta‐sleep‐inducing peptide, a humoral sleep factor, also seems to affect circadian rhythms. Intravenous injection of this peptide or either of two of its analogs in the evening significantly reduced the increase of N‐acetyltransferase 4 h later. The dose‐response relationship of the peptides showed an inverted U‐shaped pattern with the active dose about 30 nmol/kg. The effect appears to be dependent on the time of day of administration, as injections in the morning did not change the enzymatic activity. These findings indicate that delta‐sleep‐inducing peptide (and two of its analogs) can affect enzymatic activities and that these influences probably vary throughout a time period of 24 h.
Correlation Between 2′,3′‐Cyclic Nucleotide 3′‐Phosphohydrolase Activity and Demyelination In Vitro Using a Syngeneic System
Authors: Roth German A. , Bornstein Murray B. , Röyttä Matias , Lyman William D. , Raine Cedric S.
Published: October 2006

Abstract: Cultures of myelinated SJL/J fetal mouse spinal cord were incubated with serum and lymphoid cells from syngeneic animals with experimental allergic encephalomyelitis (EAE) induced by syngeneic spinal cord homogenate (SSCH) in complete Freund's adjuvant or others injected with complete Freund's adjuvant alone. After 24 or 48 h of exposure, demyelination was determined by light microscopic examination and quantification of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase activity. Cultures exposed to spleen or lymph node cells from SSCH‐sensitized animals showed the greatest alterations in myelin and decreases in 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase activity whereas serum from these animals had less effect. Cells and serum from complete Freund's adjuvant‐injected control animals also induced structural changes in myelin that were significantly less than changes induced by cells and serum from animals with EAE. These experiments show that lymphoid cells and serum obtained from SJL/J mice with acute EAE affected myelin biochemistry and morphology in syngeneic CNS cultures.
Isolation of Diosgenin[(25R)‐Spirost‐5‐en‐3β‐o1] from a Lysate of a New Hypotensive Phospholipid Occurring in Bovine Brain
Authors: Tsukatani Hiroaki , Takauchi Kenkichi , Yoshida Junichi , Yamada Sadaji , Tokumura Akira , Hamaguchi Chitoshi
Published: October 2006

Abstract: Diosgenin[(25R)‐spirost‐5‐en‐3β‐o1] and cholesterol were detected as trimethylsilyl derivatives by gas chromatography‐mass spectrometry in an alkaline lysate of a new hypotensive phospholipid from the lipid fraction of bovine brain. These steroids seemed to be present in the hypotensive phospholipid fraction in some bound forms.
Newly Synthesized and Preformed Acetylcholine Are Released from Torpedo Synaptosomes by Different Pathways
Authors: Luz Shira , Pinchasi Irit , Michaelson Daniel M.
Published: October 2006

Abstract: In this study, we investigated the mechanisms underlying the release of preformed and of newly synthesized acetylcholine (ACh) from isolated Torpedo nerve terminals (synaptosomes). This was pursued by examining and comparing the effects of anticytoskeletal and anticalmodulin drugs and of activating the presynaptic muscarinic ACh receptors on the release of preformed endogenous ACh and of newly synthesized radiolabeled ACh. The anticytoskeletal drugs vinblastine, cytochalasin B, and colchicine inhibit the Ca2+‐dependent K+mediated release of newly synthesized radiolabeled ACh, but have no effect on the release of preformed ACh. By contrast, the muscarinic agonist oxotremorine markedly inhibits the release of preformed ACh, but has little effect on the release of newly formed ACh. Treatment of the synaptosomes with the calmodulin antagonist trifluoperazine inhibits the release of both ACh pools concomitantly. These findings show that preformed and newly synthesized ACh are released by different routes and suggest that their secretion is mediated by converging pathways. The significance of these results in view of the previously demonstrated preferential release of newly synthesized ACh is discussed.
Comparative Studies of the Binding of Dimeric and Monomeric Enkephalins to Neuroblastoma‐Glioma NG108–15 Cells
Authors: Krumins Solveig A. , Rodbard David
Published: October 2006

Abstract: Binding activity of the enkephalin dimer [d‐Ala2, Leu5‐NH‐CH2‐‐]2 (DPE2) to NG108–15 hybrid cells was compared to that of the monomer [d‐Ala2, Leu5]enkephalin amide (DALEA). At 25°C, the values of the apparent affinity constant for DPE2, measured to intact and lysed cells and membranes, was 5.0 (±0.09) × 109M−1 for n = 28 experiments, as compared to 0.9 (±0.08) × 109M−1 (n = 16) for DALEA. At 4°C, the binding affinity of DPE2 decreased by 43% and that of DALEA by 33%. An important difference between the binding of DPE2 and DALEA was that, after necessary corrections for difference in maximal “bindability” of the respective tritiated enkephalins, the molar binding capacity for DALEA was twofold higher than for DPE2, although mutual cross‐displacement studies indicated that binding occurred to one class of noninteracting homogeneous receptors. The binding capacity for intact and lysed cells and membranes was 20 (±2) × 10−‐11M for DPE2 and 43 (±2) × 10−‐11M for DALEA. The enkephalin monomers [d‐Ala2, d‐Leu5]enkephalin (DADLE) and [d‐Ala2, Met5]enkephalin amide (DAMEA) showed binding characteristics similar to those of DALEA.
Sequential Limited Proteolysis of Myelin Basic Protein by Neutral Protease Activities of Bovine Brain
Authors: Berlet Hans H. , Ilzenhöfer Heike
Published: October 2006

Abstract: Acid extracts of delipidated white matter of bovine brain were prepared, and their proteolytic activities toward myelin basic protein (MBP) were evaluated at pH 3 and pH 7. This was done by measuring changes in total protein using a selective dye‐binding assay, and by evaluating peptide patterns by sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis and densitometry. At pH 7 >50% of total protein and about 75% of MBP were degraded after 48 h, whereas at pH 3 it was <20% altogether. Neutral proteolysis of MBP entailed up to 12 different proteolytic peptide fragments in the molecular weight range of 17.5 to 6 kd. Its enzymatic nature was verified using protease inhibitors, including N‐ethylmale‐imide, phenylmethylsulfonyl fluoride, o‐phenanthroline, and EDTA, as well as pepstatin A and α2macroglobulin. Both transient changes in percentages of some intermediate peptides and differential effects of individual inhibitors on electrophoretic peptide patterns strongly suggest a sequential type of limited proteolysis. The results also indicate that acid extracts contained several endopepti‐dases of which a cysteine protease appears to initiate the breakdown of MBP.
Characterization of FMRF Amide‐Like Immunoreactivity in Rat Spinal Cord by Region‐Specific Antibodies in Radioimmunoassay and HPLC
Authors: Dockray G. J.
Published: October 2006

Abstract: Material in rat spinal cord extracts that reacts with antibodies to the molluscan tetrapeptide FMRF amide (Phe‐Met‐Arg‐Phe‐NH2) has been characterized by HPLC and radioimmunoassay using region specific antibodies. An antibody to the N‐terminally extended analogue, Tyr‐Gly‐Gly‐Phe‐Met‐Arg‐Phe‐NH2 (YGGFMRF amide), did not react with the rat material. Two antibodies to FMRF amide were characterized that differed markedly in their affinities for analogues with substitutions in the second and third positions from the C‐ter‐minus; both required the C‐terminal amide, and neither showed appreciable sensitivity to substitutions in the fourth position from the C‐terminus. With both antibodies the relative potency of the avian brain peptide, LPLRF amide, was about 0.1. Both antibodies revealed similar concentrations of immunoreactive material in rat spinal cord extracts. On reversed‐phase HPLC using Techsil C18 and Spherisorb‐phenyl columns, two peaks were separated that could be distinguished in retention times from FMRF amide, Leu‐Pro‐Leu‐Arg‐Phe‐NH2 (LPLRF amide), and YGGFMRF amide. The results suggest that the rat spinal cord peptides are structurally related to the C‐terminal tripeptide of FMRF amide and are probably extended at the N‐terminus by sequences immunochem‐ically distinct from other known peptides.
Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington's Disease
Authors: Foster Alan C. , Schwarcz Robert
Published: October 2006

Abstract: Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50= 6.1 μM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89–3.75 μM and Vmax values of 33.4–72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2‐fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at ‐20°C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.
γ‐Aminobutyric Acid: Temperature Dependence in Benzodiazepine Receptor Binding
Authors: Chweh Andrew Y. , Swinyard Ewart A. , Wolf Harold H.
Published: October 2006

Abstract: The effects of muscimol and/or incubation temperature on the inhibition of [3H]flunitrazepam receptor binding by benzodiazepine receptor ligands were investigated. At 0°C muscimol decreased the Ki values for some ligands as displacers of [3H]flunitrazepam binding to brain‐specific sites while increasing or having no effect on the Ki values for other ligands. The Ki values for some ligands are higher at 37°C than at 0°C but are reduced by muscimol at both 0° and 37°C. In contrast, the ligands whose Kt values are increased by muscimol either decreased or did not alter the Ki values at 37°C as compared to those at 0°C. Incubation of membranes at 37°C for 30 min accelerated γ‐aminobutyric acid (GABA) release by 221% over that at 0°C. These results indicate that changes in incubation temperature alter benzodiazepine receptor affinity for ligands via GABA.
Changes in Presynaptic Release of Acetylcholine During Development of Tolerance to the Anticholinesterase, DFP
Authors: Russell Roger W. , Booth Ruth A. , Jenden Donald J. , Roch Margareth , Rice Kathleen M.
Published: October 2006

Abstract: The rat myenteric plexus was used as a peripheral model for studying muscarinic modulation of acetylcholine (ACh) release from presynaptic muscarinic neurons during development of tolerance to the anticholinesterase agent, diisopropylfluorophosphate (DFP). DFP in arachis oil was administered subcutaneously to intact animals according to both acute and chronic regimens, with arachis oil injections serving as controls. Postmortem analyses showed that the mean AChE activity level in whole brain was reduced under all DFP conditions to 18.0 ± 1.4% when compared with the control level. After 10 days of DFP treatment, the AChE level was 22.3 ± 2.1% of control in the myenteric plexus. There were no significant differences among the treatment groups in resting ACh release. Release evoked by electrical stimulation (difference between stimulated and resting release) in the absence of atropine, i.e., “basal rate,” for strips taken at various times after a single injection of DFP did not differ from that for strips from animals receiving arachis oil only. However, basal release for strips from chronically treated subjects was significantly greater than that of controls (p < 10−3), although not different from each other. Analysis of variance (ANOVA) for repeated measures showed that there existed a highly significant atropine dependency in strips from all treatments when they were stimulated in concentrations of atropine from 10−9 to 10−5M (p < 10−10). Further analyses established that the increases in rates of evoked ACh release as concentrations of atropine increased were similar for strips from chronically treated DFP and arachis oil animals. The overall results show that a significant effect of chronic depression in AChE activity is disinhibition of ACh‐evoked release from presynaptic neurons. This is consistent with the model in which muscarinic autoreceptors control evoked ACh release. The results are also consistent with time parameters for maximum decreases in [3H]quinuclidinyl benzilate binding in brain during chronic DFP treatments such as the regimens used in the present experiments.
Kinetic Analysis of Cerebrovascular Isoleucine Transport from Saline and Plasma
Authors: Takasato Yoshio , Momma Seiji , Smith Quentin R.
Published: October 2006

Abstract: The concentration dependence of regional isoleucine transport across the blood–brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [Am. J. Physiol.247, H484–493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis‐Menten saturation kinetics where Vmax= 9–11 × 10−4μmol · s−1· g−1 and Km= 0.054–0.068 μmol · ml−1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to · 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent Km for isoleucine transport from plasma by ≥24‐fold to 1.5–1.7 μmol · ml−1. These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood–brain barrier. In addition, they indicate that the cerebrovascular transfer‐site affinity (1/Km) for isoleucine is ∼fivefold greater than previously reported with the brain uptake index technique.
Circannual Rhythm of Free Fatty Acids and Diacylglycerols in 5‐Day‐Old Rat Cerebrum During Pentylenetetrazol‐Induced Convulsions
Authors: de Liberti Susana A. Morelli , de Schaub Elisa B. de los Santos , de Turco Elena B. Rodriguez
Published: October 2006

Abstract: Free fatty acids (FFA) and diacylglycerol (DG) content and composition in the cerebrum of 5‐day‐old rats were studied after pentylenetetrazol (PTZ)‐induced convulsions. A threefold increase in brain FFA was observed 30 min after PTZ injection in experiments carried out in spring. In contrast. a 50% decrease in FFA content was observed during summer. These changes were accounted for by saturated and monoenoic fatty acids, whereas arachidonic and docosahexaenoic acids were not affected during the convulsive episode in either season. The effect of PTZ on brain DG was much smaller than it was on FFA, and less sensitive to seasonal influence. However, DG released in the summer was significantly less enriched in arachidonic acid than in the spring. Levels of FFA and DG in untreated animals were found to be subject to a circannual rhythm. Both the levels of FFA and their degree of unsaturation (unsaturated fatty acids/total FFA) were highest in summer and lowest in winter. whereas the opposite was true for DG. Circannual variations in these metabolites may be the manifestation of a programmed biological calendar regulating enzymes of brain lipid metabolism in homeotherms that under natural conditions must adapt to changing environmental temperatures.
Ontogeny of Glucose Metabolism in Sympathetic Ganglia of Chickens. Changes in the Carbon Fluxes to CO2, Lactate, and Tissue Constituents from 8 to 19 Days of Embryonic Age
Authors: Larrabee Martin G.
Published: October 2006

Abstract: Chains of sympathetic ganglia were excised from the lumbar region of white Leghorn chicken embryos, 8–19 days of age. The chains were incubated for 5 h at 36°C in a bicarbonate‐buffered physiological salt solution containing 5.55 mM unlabeled glucose and tracer amounts of glucose labeled either uniformly or at carbon‐1 or carbon‐6. Glucose uptake and labeled lactate output were both highest in ganglia from the youngest embryos studied and declined progressively with increasing age. The output of labeled CO2 rose to a peak rate at an incubation age of 10–12 days in the presence of either [U‐14C]glucose or [1‐14C]glucose, but changed relatively little with age in the presence of [6‐14C]glucose. The incorporation of 14C into tissue constituents was fastest at 10–12 days with all three labeled glucoses. It is concluded that the hexosemonophosphate shunt is most active at an incubation age of 10–12 days, after glycolysis has greatly slowed. The literature on morphological and biochemical changes in the sympathetic ganglia during development is briefly reviewed and discussed in relation to the observed metabolic changes. The early high glycolytic rate may be related to the normal developmental delay in vascularization of the sympathetic chains.
Retrograde Axonal Transport of Gangliosides and Glycoproteins in the Motoneurons of Rat Sciatic Nerve
Authors: Aquino Dennis A. , Bisby Mark A. , Ledeen Robert W.
Published: October 2006

Abstract: Axonal transport of glycoconjugates was studied in the motoneurons of rat sciatic nerve following injection of [3H]glucosamine into the lumbosacral spinal cord. After varying time intervals, the sciatic nerve was exposed, and two ligatures were tied for collection of materials undergoing anterograde and retrograde transport. Gangliosides and glycoproteins were found to undergo fast anterograde transport, estimated at 284–446 mm/day. Both classes underwent retrograde transport as well, with labeled glycoproteins returning slightly ahead of labeled gangliosides. Only minor quantities of labeled proteoglycans were detected. Purified gangliosides extracted from nerve segments were fractionated according to sialic acid number on diethylaminoethyl‐Sephadex; the distributional pattern tended to resemble that of brain gangliosides. The similarity between anterograde and retrograde patterns suggested absence of metabolic changes in gangliosides entering and leaving the axon‐nerve terminal structures.
Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein
Authors: Baudier Jacques , Labourdette Gérard , Gerard Dominique
Published: October 2006

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea‐sodium dodecyl sulfate‐polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal‐free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCI, rat brain S100b protein is characterized by two high‐affinity Ca2+ binding sites with a KD of 2 + 10−5M and four lower affinity sites with KD about 10−4M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low‐affinity calcium binding sites whereas similar Ca2+ ‐induced conformational changes were observed for both proteins. In the presence of 120 mM KCI rat brain S100b protein bound two Zn2+‐ ions/mol of protein with a KD of 10 −7M and four other with lower affinity (KD+ 10−6M). The occupancy of the two high‐affinity Zn2+ binding sites was responsible for most of the Zn2+‐induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed. Such results significantly differ from that obtained with bovine S100b protein, which on binding of eight Zn2+ equivalents/mol undergoes maximal conformational changes associated with a sixfold increase of the tyrosine fluorescence quantum yield. Finally, we report in this work that zinc binding on rat S100b protein regulates calcium binding by increasing the calcium affinity of the protein and reducing the antagonistic effect of K+ on calcium binding.
Amino Acid Sequence of Porcine Myelin Basic Protein
Authors: Kira Jun‐ichi , Deibler Gladys E. , Krutzsch Henry C. , Martenson Russell E.
Published: October 2006

The myelin basic protein (BP) of pig brain was cleaved into its constituent tryptic peptides and the amino acid composition of each was determined. Those tryptic peptides that had not been sequenced previously were cleaved with dipeptidyl peptidases and the resulting dipeptides were trimethylsilated, separated by gas chromatography, and identified by mass spectrometry. Carboxypeptidases B and Y were used to establish the COOH‐terminal sequences of some of the tryptic peptides; one tryptic peptide (sequence 76–92) was cleaved with thermolysin and the thermolytic peptides were analyzed. From the results of the present study together with those reported previously, it has been possible to determine the complete amino acid sequence of the protein. The protein consists of 172 residues and has a theoretical molecular weight of 18,604. Its amino acid sequence is identical with that reported for the homologous bovine protein with the following exceptions: Ser replaces (bovine) Ala2; His‐Gly is inserted between Arg9 and Ser10; Ala replaces Ser45; His and Gly replace Gly76 and His77, respectively; Pro replaces Ser131 and Ser135; Ala is inserted between Gly142 and His143; and Gin replaces His143.
Identification of the Adenosine Uptake Sites in Guinea Pig Brain
Authors: Jarvis Simon M. , Ng Andrew S.
Published: October 2006

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high‐affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 ± 0.012 nM; Bmax values of 0.24 ± 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high‐affinity binding. Low concentrations of cyclohexoadenosine (10–300 nM) had no effect on NBMPR binding. These properties of the high‐affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free‐radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000–45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzyl‐thioguanosine.
The Subcellular Distribution of Peptide Histidine Isoleucine Amide‐27‐Like Peptides in Rat Brain and Their Release from Rat Cerebral Cortical Slices In Vitro
Authors: Korchak Diana M. , Gysling Katia , Beinfeld Margery C.
Published: October 2006

The subcellular distribution of peptide histidine isoleucine amide (PHI)‐27‐like peptides (PLP) was investigated in rat cerebral cortex and whole rat brain in comparison with the distribution of vasoactive intestinal peptide (VIP). The highest content of PLP was found in the crude mitochondrial fraction (P2) and was also detected in the microsomal pellet. PLP was recovered in synaptosomes when further fractionation of P2 was performed. This distribution of PLP closely follows that of VIP and is suggestive of possible storage in vesicles at the nerve terminal. Basal release of PLP from rat cerebral cortical slices was below the detection limit of the PHI radioimmunoassay. However, depolarization by 55 mM potassium induced measurable PLP release. This release was calcium‐dependent. These findings support the hypothesis that PLP could play a role in neurotransmission.
GABA‐Modulin: A Synaptosomal Basic Protein that Differs from Small Myelin Basic Protein of Rat Brain
Authors: Vaccarino Flora , Tronconi Bianca M. Conti , Panula Pertti , Guidotti Alessandro , Costa Erminio
Published: October 2006

GABA‐modulin, a basic protein that allosterically inhibits the high‐affinity binding of GABA to its recognition sites, has been extracted and purified from the synaptosomal fraction of rat brain where it represents approximately 0.5% of the total synaptosomal proteins. GABA‐modulin has characteristics in common to the class of highly basic proteins isolated from myelin, in particular to the rat small myelin basic protein (SMBP). However, GABA‐modulin is located selectively in synaptosomes, whereas the SMBP is located in myelin. Moreover, synaptosomal GABA‐modulin is different from SMBP in amino acid composition (it contains more Glx and Lys and fewer Arg residues) and in apparent molecular weight (17,000 and 15,000 for GABA‐modulin and SMBP, respectively). Synaptosomal GABA‐modulin fails to bind [3H]muscimol per se but noncompetitively inhibits (IC50˜ 0.5 μM) the binding of [3H]muscimol to purified synaptic membranes. Cyanogen bromide treatment generated a 13,000 MW major fragment from both SMBP and GABA‐modulin. These two fragments were compared and showed differences in amino acid composition and sequence. Moreover, the peptide maps generated from GABA‐modulin and SMBP by trypsin and staphylococcal V8 protease digestion are different. The high concentration of GABA‐modulin in synaptosomal membranes, its high potency in the inhibition of GABA binding, and its neuronal specificity suggest that GABA‐modulin plays an important role in neuronal membrane function linked to the modulation of GABA and perhaps other neurotransmitter receptors.
A Study on Erythrocyte Membrane Plasmalogen in Myotonic Dystrophy
Authors: Antoku Yasunobu , Sakai Tetsuo , Tsukamoto Kohsuke , Goto Ikuo , Iwashita Hiroshi , Kuroiwa Yoshigoro
Published: October 2006

Phospholipid classes that included plasmalogens of erythrocyte membranes in seven myotonic dystrophy (MyD) patients and seven normal controls were analyzed by HPLC. No significant difference in phospholipid classes was found between patients with MyD and normal controls, but there was a visible difference in peak profiles of compounds of the phosphatidylethanolamine class. In the study of plasmalogens, we used two preparation methods: exposure to HCl and deacylation with mild alkaline. The area ratio of the plasmalogen form to the diacyl form in the phosphatidylethanolamine class of MyD erythrocyte membranes was significantly lower than that of normal controls. Fatty acid analyses showed that fatty acids of both phosphatidylethanolamine subclasses have less unsaturation in MyD.
Translational Activity of mRNA Coding for Cytoskeletal Brain Proteins in Newborn and Adult Mice: A Comparative Study
Authors: Darmon Michèle C. , Paulin Denise J.
Published: October 2006

Translational activity of mRNA coding for cytoskeletal brain proteins was used to determine the relative abundance of the mRNA in the brains of newborn and adult mice. mRNA was translated in a cell‐free system containing rabbit reticulocyte factors. The products of translation were analyzed by two‐dimensional gel electrophoresis and characterized by peptide map analysis. Comparison of the products of translation from newborn and from adult brain mRNA shows a 50% decrease in actin and tubulin from newborn to the adult stage. In contrast, the 70 kd neurofilament protein and glial fibrillary acidic protein show a twofold increase in the adult stage. The heat‐shock protein HSP70 increases slightly (30%) whereas the brain isozyme of creatine kinase and the heat‐shock protein HSP90 are three times as high in adult subject as in newborns.
Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin‐Containing Peptides
Authors: Fischer‐Colbrie R. , Frischenschlager I.
Published: October 2006

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by twodimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine β‐hydroxylase reacted with one protein of Mr 75,000. Met‐enkephalin antisera labeled eight proteins of Mr 23,000–14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine β‐hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.
Cerebellum Lipids in Rats After Chronic Ethanol Treatment
Authors: Vrbaški SneŽana R. , Ristić Milosav
Published: October 2006

Eighteen male Wistar rats weighing approximately 200 g were divided into three groups of six animals each. The experimental animals were maintained on nutritionally complete diets in which ethanol comprised 45% of the available energy. Control animals were pair‐fed an equivalent diet in which sucrose was substituted isocalorically for ethanol. An additional control group received unlimited access to standard pelleted laboratory food and water. The investigations were carried out over 24 weeks. The effects on phospholipid, monogalactosyl glycolipid, and ganglioside composition after 24 weeks of feeding 43% alcohol were studied. There is abundant evidence that the changes in the cerebellum membrane phospholipids (phosphatidylethanolamine and phosphatidylcholine), gangliosides (GTlb), and myelin lipids (phosphatidylserine, sphingophospholipid, phosphatidylinositol, cerebrosides with hydroxy fatty acids, sulfoglycolipids, and monosialoganglioside GM1) occur as a result of chronic ethanol treatment.
Studies on the Alkylation of Choline Acetyltransferase by Choline Mustard Aziridinium Ion
Authors: Rylett R. Jane , Colhoun E. Howard
Published: October 2006

Although a potent irreversible inhibitor of high‐affinity choline transport in rat brain synaptosomes, choline mustard aziridinium ion (ChM Az) appeared to be a relatively weak inhibitor of choline acetyltransferase (ChAT) in rat brain homogenates, and evidence for irreversible binding of this compound to the enzyme had not been established. Accordingly, the irreversible inactivation of partially purified rat brain ChAT by ChM Az was studied. This compound is a rather weak inhibitor of the enzyme, with 50% inhibition of ChAT activity achieved following 30 min incubation at 37°C with 0.6 mM ChM Az. This result indicates that although ChM Az has affinity for many nucleophiles there was little diluting effect of the inhibitor in the crude brain homogenate which could be attributed to such reactions (50% inhibition caused by 1.8 mM ChM Az following 10 min incubation). Although the initial binding of ChM Az to ChAT may be of a competitive nature, irreversible bond formation resulted. The time‐dependent alkylation reaction conformed to pseudo‐first‐order kinetics with an observed forward rate constant (kobs) of 0.173 min−1; the half‐time (t1/2) for irreversible binding was about 4 min. The irreversible inactivation of ChAT by ChM Az would appear to be slower than the alkylation of high‐affinity choline carriers in synaptosomes by this compound, and the relatively weak inhibitory action of ChM Az against either partially purified ChAT or ChAT activity in crude rat brain homogenates is in striking contrast to previous evidence that ChAT in intact synaptosomes was inhibited irreversibly by lower concentrations of the inhibitor.
Relationship Between the Regulation of Enkephalin‐Containing Peptide and Dopamine β‐Hydroxylase Levels in Cultured Adrenal Chromaffin Cells
Authors: Wilson Steven P. , Viveros O. Humberto , Kirshner Norman
Published: October 2006

Abstract: Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin‐containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine β‐hydroxylase, were examined. Catecholamine‐depleting drugs, such as tetrabenazine, and cyclic nucleotide‐elevating drugs, including forskolin, 8bromo‐cyclic AMP, and theophylline, increase chromaffin cell enkephalin‐containing peptide levels but fail to increase dopamine β‐hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin‐containing peptides and dopamine β‐hydroxylase. The increased amounts of enkephalin‐containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density‐gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin‐containing peptides, but that after treatment with catecholamine‐depleting or cyclic nucleotide‐related agents enkephalin‐containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.
Thyroid Hormone Binding and Regulation of Adrenergic Enzymes in Two Neuroblastoma Cell Lines
Authors: Safaei Roohangiz , Timiras Paola S.
Published: October 2006

Abstract: Two neuroblastoma cell lines were cultured in control (euthyroid) and hypothyroid media and examined for protein, RNA and DNA content, activity of the catecholaminergic enzymes tyrosine hydroxylase (TH, EC 1.14. 16.2) and monoamine oxidase‐A (MAO‐A, EC 1.4.3.4), and for L‐triiodothyronine (T3) nuclear receptors. In the hypothyroid condition, the rate of cell division and the levels of RNA and protein as well as the activities of TH and MAO were lower than in the euthyroid condition, the reduction being more marked in the Ē than in the A2(1) cell line. T3 nuclear receptors, unaltered in affinity, were increased in number in the hypothyroid medium, possibly as a regulatory response to hormonal deficiency. Examination of a possible relationship between T3 occupancy and TH activity in the Ē cells, most sensitive to thyroid hormone deficiency, revealed that induction of TH activity by T3 is dose‐dependent and correlates with the number of nuclear sites occupied by the hormone. When neuroblastoma cells were induced to differentiate by the addition of sodium butyrate to the medium, parameters of cell growth (protein, RNA) and enzyme activity (TH and MAO‐A) increased in both cell lines irrespective of the presence of thyroid hormones. These data indicate that thyroid hormones, through their nuclear receptors, directly affect the activity of catecholaminergic enzymes in cultured, immature (undifferentiated) neurons.
Calmodulin‐Dependent Protein Phosphorylation in Synaptic Junctions
Authors: Kelly Paul T. , Yip Rick K. , Shields Steven M. , Hay Michael
Published: October 2006

Abstract: Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)‐dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25‐fold greater amounts of Ca2+/CAM‐dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10‐fold more than PSDs. The phosphorylation of SJ proteins of 60‐ and 50‐kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23‐ and 17‐fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6–15‐fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54‐kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170‐kilodalton component is a cell‐surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM‐dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+‐dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 μg CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ‐associated CaM involved in regulating Ca2+‐dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM‐dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM‐dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.
Transient States of Adenylate Cyclase in Brain Membranes
Authors: Tamir Amichai , Tolkovsky Aviva M.
Published: October 2006

Abstract: Basal activity of adenylate cyclase from the amygdala of sheep brain and the neostriatum of turkey brain decays in two phases at 37°C. The first phase is rapid (t1/2= 2.3 ± 0.3 min) and results in the loss of 60–70% of basal activity. The second phase is slow (t1/2± 100 min) during which time the catalytic units denature irreversibly. The GTP analogue guanosine‐5’(β‐γ‐imino) triphosphate (p[NH]ppG) prevents the rapid decay by stabilizing the enzyme at its initial level of activity and also reactivates the enzyme to initial levels during or immediately following the early phase, indicating that denaturation of neither the guanylnucleotide units nor the catalytic units causes the rapid decline in basal activity. Activation by p[NH]ppG is rapid at 37°C, but the binding of p[NH]ppG to the guanylnucleotide subunit also occurs at nonactivatory temperatures. This is determined by the protection of catalytic units from thermal or N‐ethylmaleimide inactivation after extensive washing. Thus, at 25°C all of the catalytic units can be stabilized by saturating p[NH]ppG concentrations. At 0°C, 35% of the catalytic units can be stabilized by saturating p[NH]ppG concentrations within 30 s. The half‐saturation constant for the binding of p[NH]ppG at 0°C is identical to that derived in an assay at 37°C, or after an incubation of the membranes for 10 min at 45°C, when the process of thermal denaturation is 80% complete (K1/2∼ 3 ± 2 μM). Taken together these results suggest that the catalytic units of adenylate cyclase in brain membranes are coupled to a freely accessible, preactive state of guanylnucleotide units when the membranes are prepared, leading to an initial state of high activity.
Metabolism of Catecholamines in the Developing Spinal Cord of the Rat
Authors: Commission John W.
Published: October 2006

Abstract: The metabolism of 3,4‐dihydroxyphenylethylamine (DA, dopamine) and norepinephrine (NE) both normally, and after the administration of levo‐3,4‐dihydroxyphenylalanine (l‐DOPA), has been studied in several regions of the developing spinal cord of the rat from fetal day (FD) 16 to the young adult stage. During late fetal (from FD 16) and most of neonatal life [to neonatal day (ND) 20], dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were either just detectable or present in very low concentration in all regions in the untreated developing rat. However, the developing spinal cord possesses an enormous capacity to metabolize the large amounts of DA synthesized from injected l‐DOPA. At the end of 1 h after 100 mg/kg i.p. of l‐DOPA, DOPAC and HVA are 54 ± 14 (n = 5) and 16 ± 5 (n = 5) nmol/g, respectively, in the thoracic zona intermedia in the 12h‐old (ND 0.5) rat. This metabolic capability is already highly developed as early as FD 16, peaks during the first half of neonatal life (ND 4 for DOPAC, and ND 15 for HVA), and is considerably reduced toward the end of neonatal life (approximately ND 28) and in the young adult. Control experiments suggest that a substantial part of this synthesis (from l‐DOPA) and metabolism of DA occurs in elements other than the descending monoaminergic nerve fibers. By comparison, the synthesis and metabolism of NE develop more slowly, peak in the latter half of neonatal life, and then decline to the level found in the young adult. In the ventral horn of the lumbar region, however, the capacity to metabolize NE develops more rapidly than in the dorsal horn and zona intermedia. The results suggest that the activities of several enzymes involved in the synthesis (aromatic amino acid decarboxylase) and inactivation of catecholamines (monoamine oxidase and catechol‐O‐methyltransferase) are already highly developed by FD 16, and achieve their highest activities in the spinal cord during early neonatal life.