bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the Major Proteins of Shark Myelin with the Proteins of Higher Vertebrates
Authors: Tai F. L. , Smith Ross
Published: October 2006

Abstract: On gel electrophoresis in dodecyl sulphate solutions shark CNS myelin showed four bands close in mobility to the proteolipid protein of bovine CNS myelin. They had apparent molecular weights of 21,000, 26,000, 27,000, and 31,500. Unlike bovine proteolipid protein, all of these shark proteins were shown to be glycosylated by staining gels with the periodate‐Schiff reagent. Amino acid analyses of the polypeptides eluted from polyacrylamide gels indicated a high content of apolar amino acids and a composition approximating that of the Po protein of bovine peripheral nervous system (PNS) myelin, rather than that of the CNS proteolipid protein. The shark poly‐peptide of apparent molecular weight 31,500 was obtained by elution from dodecyl sulphate gels and antibodies raised against it in rabbits. By probing of electroblots with this antiserum the four shark CNS bands were shown to share common determinants with each other, with a major shark PNS protein and with sheep and chicken major PNS glycoproteins (Po). The binding of antibody was unaffected by deglycosylation of the shark CNS polypeptides with anhydrous hydrogen fluoride. Together, these results appeared to establish that shark CNS myelin contains four proteins that are closely related to a major shark PNS protein and to the Po protein of higher species.
Cleavage of Rabbit Myelin Basic Protein by Thrombin
Authors: Law Mona J. , Martenson Russell E. , Deibler Gladys E.
Published: October 2006

Abstract: Rabbit myelin basic protein (BP) contains several Arg‐X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin‐to‐substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95) ‐ Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme‐to‐substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)‐Phe(44) and Arg(128)‐Ala(129) bonds, but with some cleavage at the Arg(31)‐His(32) and Arg(63)‐Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96–168), which we examined in detail, nearly complete cleavage of the Arg(128)‐Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)‐Gly(106), Arg(111)‐Phe(112), Arg(150)‐Leu(151), and Arg(160)‐Ser(161) bonds. The susceptibilities to cleavage of the Arg‐X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)‐Ala(129) and Arg(160)‐Ser(161), suggests the occurrence of a chain reversal or β‐turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe‐Phe(87–88) sequence is left intact.
Identification and Comparison of Protein I in Chick and Rat Forebrain
Authors: Sorensen Roger G. , Babitch Joseph A.
Published: October 2006

Abstract: Protein I has been identified and compared in membranes prepared from chick and rat forebrain. Based upon five criteria known to characterize protein I, namely, (1) its ability to serve as a substrate for both the cyclic AMP‐dependent protein kinase and (2) the Ca2+‐dependent, calmodulin‐requiring protein kinase, (3) its ability to be extracted from membranes at low pH, (4) its characteristic pattern of digestion by collagenase, and (5) its existence as a basic protein, we have determined that although protein I of rat brain consists of the usual doublet polypeptides la and Ib, only a single chick forebrain polypeptide is detectable which possesses protein Mike properties.
Free Fatty Acids and Energy Metabolites in Ischemic Cerebral Cortex with Noradrenaline Depletion
Authors: Yoshida Shinichi , Harik Sami I. , Busto Raul , Santiso Mercedes , Martinez Elena , Ginsberg Myron D.
Published: October 2006

Abstract: We tested whether cerebral noradrenaline (NA) may play a central role in mediating the increased production of free fatty acids (FFAs) during cerebral ischemia. Levels of FFAs, cyclic AMP, and NA, as well as ATP, ADP, and AMP, were measured in cerebral cortex during decapitation ischemia in rats 2 weeks after unilateral locus ceruleus lesion. Comparisons were made between the results obtained from the contralateral cortex with normal NA content and the NA‐depleted ipsilateral cortex. Although NA depletion was associated with a diminished transient rise of cyclic AMP in response to ischemia, it failed to influence the magnitude of FFA increase or the decline of energy state within the 15‐min period of ischemia. A more than twofold increase of total FFAs (sum of palmitic, stearic, oleic, arachidonic, and docosahexaenoic acids) was observed in both hemispheres at 1 min after decapitation, when energy failure became manifest. The increased production of FFAs continued throughout the 15 min of ischemia, with a preferential rise in the levels of stearic and arachidonic acids. There was an inverse correlation between FFA levels and total adenylate pool. The results do not support a major role for NA and cyclic AMP in increasing cortical FFAs during complete ischemia. Instead, they are consistent with the view that impaired oxidative phosphorylation activates deacylating enzymes. Disturbance of reacylation due to energy depletion is probably another factor contributing to the continuous increase of FFAs during prolonged ischemia.
A Study of Somatostatin Receptors in Amygdaloid‐Kindled Rat Brain
Authors: Higuchi T. , Kokubu T. , Sikand G. S. , Wada J. A. , Friesen H. G.
Published: October 2006

Abstract: Kindling is an animal model of epilepsy. Previously somatostatin (SRIF) was implicated in seizure activity in the brain. Recently we reported a significant increase in brain SRIF content in the temporal cortices and cortices of kindled rats. Since the interaction between the neurotransmitter and the receptor eventually is responsible for the biological response, the present study was undertaken to examine evidence for the participation of SRIF receptor in the kindled state. In this study we present a procedure for detection of SRIF receptors using radiolabeled (D‐Tyr8)‐SRIF as a tracer. The present study indicates that in kindled rats there are no differences in the total number or affinity of the binding sites in the temporal cortex and a slight increase in the total number of binding sites in the cortex when compared with controls. These results, in view of our other observations, suggest that in kindled rat brain there may be an increased release of SRIF but no down‐regulation of SRIF receptors in temporal cortex and cortex. There appears to be a significant decrease in the number of SRIF receptors in kindled hippocampus. The mechanism by which this occurs remains unclear.
Unique Features of the Insulin Receptor in Rat Brain
Authors: Hendricks S. Anne , Agardh Carl‐David , Taylor Simeon I. , Roth Jesse
Published: October 2006

Abstract: We examined the structure of the affinity‐labeled insulin receptors in rat brain, rat liver, and human IM‐9 lymphocytes using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In gels run under reducing conditions, the α‐subunit of the insulin receptor in brain had an apparent Mr of 127,000, distinctly lower than that seen in both rat liver and human lymphocytes (apparent Mr= 136,000). Exposure to neuraminidase increased the electrophoretic mobility of the liver receptor, but had no effect on the insulin receptor in brain. The carbohydrate moieties of the insulin receptors in rat brain and liver were further examined by chromatography on wheat‐germ agglutinin agarose. The receptors in both tissues adsorbed to the wheat‐germ agglutinin; elution with 0.3 MN‐acetyl glucosamine resulted in slightly better recovery of the brain than of the liver receptor. Exposure to neuraminidase virtually abolished the interaction of the liver receptor with the lectin, whereas adsorption of the brain receptor was unaffected by neuraminidase. These results indicate that the insulin receptor in brain is distinguished from those in peripheral tissues by structural alterations, including changes in the carbohydrate moiety of the receptor. Such alterations contrast sharply with the previously observed similarities in insulin binding properties between insulin receptors in brain and other tissues. The implications of such structural alterations for the program of insulin action expressed by the receptors in brain remain to be explored.
Alteration of Striatal Glutamate Release After Glutamine Synthetase Inhibition
Authors: Rothstein Jeffrey D. , Tabakoff Boris
Published: October 2006

Abstract: The effect of the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), on glutamate levels in, and glutamate release from, rat striatal tissue was examined. Tissue levels of glutamate were unchanged 24 h after an intraventricular injection of MSO, but tissue glutamine levels were decreased 50%. Calcium‐dependent, potassium‐stimulated glutamate release was diminished in tissue prisms from animals pretreated with MSO compared to controls. The decreased release of glutamate correlated over time with the inhibition of GS following an intraventricular injection of MSO. The maximum diminution of calcium‐dependent, potassium‐stimulated glutamate release (50%) and the maximum inhibition of GS activity (51%) were observed 24 h after MSO. The addition of 0.5 mM glutamine to the perfusion medium completely reversed the effects of MSO pretreatment on calcium‐dependent, potassium‐stimulated glutamate release. Since GS is localized in glial cells and the measured glutamate release is presumed to occur from neurons, the data support the contention that astroglial glutamine synthesis is an important contributor to normal neuronal neurotransmitter release.
Photoaffinity Identification of Colchicine‐Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase
Authors: Rasenick Mark M. , Wheeler George L. , Bitensky Mark W. , Kosack Carolyn M. , Malina Rachel L. , Stein Peter J.
Published: October 2006

Abstract: Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [3H] or [32P](P3‐azido‐anilido)‐P1‐5’GTP (AAGTP). When AAGTP‐treated membranes were incubated with colchicine or vinblastine and subsequently washed, a single AAGTP‐labelled protein of 42 kD was released into the supernatant. About 30% of the total labelled 42‐kD protein was released into supernatants from membranes pretreated with colchicine or vinblastine compared with 15% released from control membranes. The amount of adenylate cyclase regulatory subunit (G unit) remaining in these membranes was assessed with reconstitution studies after inactivating the adenylate cyclase catalytic moiety with N‐ethylmaleimide (NEM). Forty to fifty percent of functional G units were lost from membranes treated with colchicine prior to washing. This 40–50% loss of functional G unit after colchicine treatment corresponds to the previously observed 42% loss of NaF and guanylyl‐5′‐imidodiphosphate [Gpp(NH)p]‐activated adenylate cyclase. Release of the AAGTP‐labelled 42‐kD protein from colchicine‐treated synaptic membranes is double that from lumicolchicine‐treated membranes. This colchicine‐mediated release of 42‐kD protein correlates with a doubling of functional G unit released from synaptic membranes after colchicine treatment. These findings suggest multiple populations of the G unit within the synaptic plasma membrane, some of which may interact with cytoskeletal components.
Effects of Chronic (Dietary) Choline Availability on the Transport of Choline Across the Blood‐Brain Barrier
Authors: Wecker L. , Trommer B. A.
Published: October 2006

The effects of dietary choline availability on the transport of choline across the blood‐brain barrier (BBB) were investigated using the intracarotid injection technique. Maintenance of rats on choline‐deficient, basal choline, or choline‐supplemented diets for 28–32 days led to respective increases in blood levels of choline and correlative increases in the velocity of transport of choline measured using a buffer injectate. When serum from these rats was included in the injectate and transport determined in control animals, there was a marked inhibition of choline transport that was related to the concentration of choline in the diets. Results suggest that the activity of the choline carrier at the BBB is antagonized by an inhibitory substance in serum whose concentration or activity may be modified by chronic alterations in circulating levels of choline and whose presence may normally regulate the velocity of choline transport.
Distribution and Properties of Aldehyde Dehydrogenase in Regions of Rat Brain
Authors: Weiner Henry , Ardelt Barbara
Published: October 2006

Abstract: NAD‐dependent aldehyde dehydrogenases (EC 1.2.1.3) were isolated from various subcellular organelles as well as from different regions of rat brain. The mitochondrial, microsomal, and cytosolic fractions were found to contain 40%, 28%, and 12%, respectively, of the total aldehyde dehydrogenase (5.28 ± 0.44 nmol NADH/min/g tissue) found in rat brain homogenate when assayed with 70 μ.M propionaldehyde at pH 7.5. The total activity increased to 17.3 ± 2.7 nmol NADH/min/g tissue when assayed with 5 mM propionaldehyde. Under these conditions the three organelles contained 49%, 23%, and 9%, respectively, of the activity. The enzyme isolated from cytosol possessed the lowest Km. The molecular weight of the enzyme isolated from all three subcellular organelles was ∼100,000. Four activity bands were found by electrophoresis of crude homogenates, isolated mitochondria, or microsomes on cellulose acetate strips. Cytosol possessed just two of the forms. The total activity was essentially the same in homogenates obtained from cortex, subcortex, pons‐medulla, or cerebellum. Further, the enzyme had the same molecular distribution and total activity in each of these four brain regions. Disulfiram was found to be an in vivo and in vitro inhibitor of the enzymes obtained from these brain regions. Mercaptoethanol, required for the stability of the enzyme, reversed the inhibition produced by disulfiram. The effect was greater for enzyme isolated from cytosol than from mitochondria. Calculations led to the prediction that aldehydes such as acetaldehyde are oxidized in cytosol.
Modifications of Synaptosomal Na+‐K+‐ATPase Activity During Vasogenic Brain Edema in the Rabbit
Authors: Averet N. , Rigoulet M. , Cohadon F.
Published: October 2006

Abstract: This study investigates the functioning of synaptosomal ouabain‐sensitive Na+‐K+‐ATPase in cold‐induced edema. During vasogenic brain edema development, the enzyme affinities for Na+ and K+ are progressively decreased paralleling the increase in the tissue water content, whereas maximal velocity of the reaction is not changed. On the basis of these data, it is likely that Na+‐K+‐ATPase impairment accounts for the intracellular uptake of water in this model of edema.
Decreased High‐Affinity Binding of [3H]Muscimol to Cerebral Synaptic Membranes of Scrapie‐Infected Mice
Authors: Quinn Michael R. , Somerville Robert A.
Published: October 2006

Abstract: Scrapie is a transmissible disease that results in progressive degeneration of the central nervous system and death. Although scrapie has been studied histopathologically, relatively little is known concerning neurotransmitter alterations. Specific [3H]muscimol binding to whole brain crude synaptic membranes (CSM) from mice clinically affected with scrapie was significantly (p < 0.01) reduced, to approximately 73% of that of the controls. Of the brain regions examined, binding to only cerebral CSM was significantly (p < 0.0001) decreased. Scatchard analyses of saturation curves revealed that the high‐affinity (KD= 8 ± 3 nM) site for muscimol was abolished in cerebral CSM from scrapie‐infected mice, while the low‐affinity site was unaffected. Binding of [3H]flunitrazepam to cerebral CSM was unaffected by scrapie and was stimulated by GABA to the same extent in both scrapie and control mice. These results suggest that scrapie agent 139A in C57BL/6J mice manifests a portion of its CNS pathology via a high‐affinity GABA binding site that is unassociated with the benzodiazepine receptor.
Changes of Enzymes Involved in Prostaglandin Metabolism and Prostaglandin Binding Proteins in Rat Brain During Development and Aging
Authors: Ueno Ryuji , Osama Hiroyoshi , Urade Yoshihiro , Hayaishi Osamu
Published: October 2006

Abstract: In the developing rat brain, the enzymatic formation of prostaglandin D2 from prostaglandin H2 increased 60‐fold from day 12 of gestation to birth. The activity still rose gradually to the highest level (90 nmol/min/g wet tissue) at day 7 after birth. The activities of prostaglandin E2 and F2α synthetases in rat brain were highest at gestational age 19 days (30 nmol/min/g wet tissue) and at gestational age 14 days (15 nmol/min/g wet tissue), respectively. The specific activity of NADP‐dependent 15‐hydroxy‐prostaglandin D2 dehydrogenase in rat brain was highest at the earliest gestational age we examined (day 12 of gestation), The specific bindings of prostaglandin D2 and E2 to the crude mitochondrial fraction of rat brain were observed from day 16 of gestation and increased to day 7 after birth. Although the activities of the enzymes responsible for prostaglandin metabolism were unchanged postmaturationally, the maximal concentrations of the binding sites on the synaptic membrane for both prostaglandins D2 and E2 decreased with constant affinity to less than one‐sixth with age from 1 week to 24 months after birth. These results indicate that prostaglandins may play important roles during maturation and aging in rat brain.
Reduction of Muscarinic Acetylcholine Receptor Number and Affinity by an Endogenous Substance
Authors: Creazzo Tony L. , Hartzell H. Criss
Published: October 2006

Abstract: We examined the soluble fraction from homogenates of 12‐day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ([3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six‐ to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from < 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was < 10,000 daltons and probably <700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex‐100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.
t‐[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ‐Aminobutyric Acid–Regulated Chloride Ionophore
Authors: Lawrence Lowell J. , Palmer Christopher J. , Gee Kelvin W. , Wang Xiaoyu , Yamamura Henry I. , Casida John E.
Published: October 2006

Abstract: t[3H]Butylbicycloorthobenzoate ([3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4‐chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium‐potassium phosphate buffer (pH 7.4) at 25°C, with 80–90% specific relative to total binding, a KD of 61 ± 15 nM, and a Bmax of 1.6 ± 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0°C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37°C with similar KD values. [3H]TBOB with its long radioactive half‐life and temperature‐independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the γ‐aminobutyric acid receptor‐ionophore complex.
Phosphorylation of Microtubule‐Associated Protein 2 at Distinct Sites by Calmodulin‐Dependent and Cyclic‐AMP‐Dependent Kinases
Authors: Goldenring James R. , Vallano Mary Lou , DeLorenzo Robert J.
Published: October 2006

Abstract: Microtubule‐associated protein 2 (MAP2) is an excellent substrate for both cyclic‐AMP (cAMP)‐dependent and Ca2+/calmodulin‐dependent kinases. A recently purified cytosolic Ca2+/calmodulin‐dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule‐associated cAMP‐dependent and calmodulin‐dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two‐dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule‐associated cAMP‐dependent kinase and five major peptide species phosphorylated by calmodulin‐dependent kinase. All 11 of the cAMP‐dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin‐dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP‐dependent and calmodulin‐dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.
Glucocorticoids Potentiate the Prostaglandin E1‐Mediated Cyclic AMP Formation by a Cultured Murine Neuroblastoma Clone
Authors: Forray Carlos , Richelson Elliott
Published: October 2006

Abstract: The effect of steroid hormones on the prostaglandin E1 (PGE1)‐mediated cyclic AMP formation by murine neuroblastoma clone N1E‐115 was studied. Dexamethasone at submicromolar concentrations and corticosterone at micromolar concentrations (steroids with glucocorticoid activity) were able to modify the PGE1‐mediated response whereas testosterone, progesterone, and estradiol each at 10 μM had no effect. Glucocorticoids added to the culture medium of N1E‐115 cells produced an increase in the maximal response to PGE1 only after long‐term (4 h) incubation with the hormone. Inhibitors of protein and RNA synthesis blocked this effect of glucocorticoids. Basal activity of adenylate cyclase in treated cells was twofold higher than that in control cells, and this enzyme seemed to be the primary target for the hormone action, since the activity of 3′:5′‐cyclic AMP phosphodiesterase and the binding of [3H]PGE1 to its receptors were not altered by glucocorticoid treatment. Our results indicate that glucocorticoids modulate receptor‐mediated responses in cells of neural origin through a mechanism that involves induction of protein synthesis.
Developmental Expression of the Myelin‐Associated Glycoprotein in the Peripheral Nervous System Is Different from That in the Central Nervous System
Authors: Frail Donald E. , Webster Henry de F. , Braun Peter E.
Published: October 2006

Abstract: We recently characterized two developmentally regulated myelin‐associated glycoprotein (MAG) polypeptides synthesized by mouse brain mRNA in vitro. We now extended these studies to include the peripheral nervous system (PNS). Total cytoplasmic RNA was isolated from the sciatic nerves of 7‐, 12‐, and 17‐day‐old and adult rats and translated in vitro in a rabbit reticulocyte lysate system. In contrast to results in the CNS, it appears that only one MAG polypeptide, p67MAG, is synthesized by PNS mRNA at all ages. The implications of these findings are discussed with respect to recent observations concerning both the localization of MAG and the synthesis of MAG in the PNS of dysmyelinating mutant mice.
Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion
Authors: White Jeffrey D. , Krause James E. , Karten Harvey J. , McKelvy Jeffrey F.
Published: October 2006

Abstract: The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5‐and for Leu5‐enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5‐ and Leu5‐enkephalin and with substance P. The ontogeny of Met5‐enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5‐enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.
GABA‐Mimetic Activity and Effects on Diazepam Binding of Aminosulphonic Acids Structurally Related to Piperidine‐4‐Sulphonic Acid
Authors: Falch Erik , Jacobsen Poul , Krogsgaard‐Larsen Povl , Curtis David R.
Published: October 2006

The relationship between structure, in vivo activity, and in vitro activity of some analogues of the γ‐aminobutyric acid (GABA) agonist piperidine‐4‐sulphonic acid (P4S) was studied. The syntheses of 1,2,3,6‐tetrahydropyridine‐4‐sulphonic acid (DH‐P4S) and (RS)‐pyrrolidin‐3‐yl‐methanesulphonamide (PMSA‐amide) are described. Like P4S, its unsaturated analogue DH‐P4S and the five‐ring isomer (RS)‐pyrrolidin‐3‐yl‐methanesulphonic acid (PMSA) were bicuculline methochloride (BMC)‐sensitive inhibitors of the firing of neurones in the cat spinal cord. Whereas isonipecotic acid was less potent than its unsaturated analogue isoguvacine as a GABA‐mimetic and as an inhibitor of GABA binding, the opposite relative potencies of P4S and DH‐P4S were observed, P4S being proportionally more potent than DH‐P4S. In contrast with P4S and DH‐P4S, PMSA, which is an analogue of the potent GABA uptake inhibitor and BMC‐sensitive GABA‐mimetic homo‐β‐proline, was a relatively weak inhibitor of GABA uptake in vitro. PMSA‐amide was more than two orders of magnitude weaker than PMSA as an inhibitor of GABA binding and did not significantly affect GABA uptake in vitro. The effects of 3‐aminopropanesulphonic acid (3‐APS), PMSA, P4S, and DH‐P4S on the binding of [3H]diazepam in vitro at 30°C, in the presence or absence of chloride ions, were studied and compared with those of the structurally related amino acids GABA, homo‐β‐proline, isonipecotic acid, and isoguvacine. Under these conditions the aminosulphonic acids were weaker than the respective amino acids in enhancing [3H]diazepam binding, the difference being more pronounced in the absence of chloride.
Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP‐Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme
Authors: Ghalayini Abdallah , Eichberg Joseph
Published: October 2006

A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP‐1,2‐diacylsn‐glycerol:myo‐inositol 3‐phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200–250‐fold from the homogenate by solubilization with Triton X‐100 from microsomal membranes and affinity chromatography on CDP‐diacylglycerol‐Sepharose. Elution of enzyme activity required the presence of Triton X‐100, CDP‐diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5–10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The final preparation contained levels of CDP‐diacylglycerol hydrolase and CDP‐diacylglycerol: sn‐glycero‐3‐phosphate 3‐phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5–9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo‐inositol.
Arachidonic Acid Release and Catecholamine Secretion from Digitonin‐Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents
Authors: Frye Roy A. , Holz Ronald W.
Published: October 2006

The relationship between catecholamine secretion and arachidonic acid release from digitonin‐treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin‐treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin‐treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N‐ethylmaleimide (NEM) or p‐bromophenacyl bromide (BPB) resulted in dose‐dependent inhibition of 10 μM Ca2+‐stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 μM. The IC50 of BPB for both events was 25 μM. High concentrations (5–20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin‐treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.
Effects of 1‐Methyl‐4‐Phenyl‐1,2,5,6‐Tetrahydropyridine and Related Compounds on the Uptake of [3H]3,4‐Dihydroxyphenylethylamine and [3H]5‐Hydroxytryptamine in Neostriatal Synaptosomal Preparations
Authors: Heikkila Richard E. , Youngster Stephen K. , Manzino Lawrence , Cabbat Felicitas S. , Duvoisin Roger C.
Published: October 2006

1‐Methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine (MPTP) is known to cause a destruction of the dopaminergic nigrostriatal pathway in certain animal species including mice. MPTP and some structurally related analogs were tested in vitro for their capacity to inhibit the uptake of [3H]3,4‐dihydroxyphenylethylamine‐([3H]DA), [3H]5‐hydroxytryptamine ([3H]5‐HT), and [3H]γ‐aminobutyric acid ([3H]GABA) in mouse neostriatal synaptosomal preparations. MPTP was a very potent inhibitor of [3H]5‐HT uptake (IC50 value 0.14 μM), a moderate inhibitor of [3H]DA uptake (IC50 value 2.6 μM), and a very weak inhibitor of [3H]GABA uptake (no significant inhibition observed at 10 μM MPTP). In other experiments, MPTP caused some release of previously accumulated [3H]DA and [3H]5‐HT, but in each case MPTP was considerably better as an uptake inhibitor than as a releasing agent. The 4‐electron oxidation product of MPTP, i.e., 1‐methyl‐4‐phenyl‐pyridinium iodide (MPP+), was a very potent inhibitor of [3H]DA uptake (IC50 value 0.45 μM) and of [3H]5‐HT uptake (IC50 value 0.78 μM) but MPP+ was a very weak inhibitor of [3H]GABA uptake. These data may have relevance to the neurotoxic actions of MPTP.
Myelin‐Associated Galactolipids in Primary Cultures from Dissociated Fetal Rat Brain: Biosynthesis, Accumulation, and Cell Surface Expression
Authors: Singh H. , Pfeiffer S. E.
Published: October 2006

Abstract: Galactolipid metabolism was investigated as a function of development in primary cultures initiated from 19–21‐day‐old dissociated fetal rat brain. Significant amounts of galactocerebrosides, sulfatides, and monogalactosylglycerides were synthesized and accumulated by 8 days in culture. Thereafter the synthetic rates and levels of these galactolipids increased rapidly, reaching maximal values ∼22–29 days in culture. Galactolipids containing nonhydroxy or 2‐hydroxy fatty acid were both synthesized at approximately equal rates. The initial rates of synthesis, investigated at 15, 29, and 50 days in culture, were three‐ to fivefold higher for galactocerebrosides than for sulfatides and two‐ to threefold higher than for monogalactosylglycerides. The total number of cells staining with antisera against galactocerebroside of sulfatide also increased very rapidly between 8 and 22 days in culture, reaching levels of 4–5 million cells per seeded fetal brain. The amount of galactocerebroside or sulfatide per cell stained with the corresponding antiserum increased severalfold from 10 to 27 days in culture and remained high until at least 36 days in culture (the latest time point examined). Thus, the temporal expression of galactolipid accumulation in the cell cultures was comparable to that occurring in rat brain, but some important quantitative reductions in the levels of accumulation per cell in culture were noted. In addition, in contrast to normal brain in which galactolipid synthetic rates are reduced after the period of most active myelination, in culture both synthesis and turnover of these galactolipids remained high, suggestive of a partial arrest in myelin maturation.
Extraction of Glycolytic Enzymes: myo‐Inositol as a Marker of Membrane Porosity
Authors: Knull Harvey R.
Published: October 2006

Abstract: Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X‐100 and Brij 58, led to similar results for extraction of myo‐inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X‐100 in isotonic phosphate‐buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with nonextractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X‐100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X‐100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X‐100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent‐mediated alterations in membrane porosity than glycolytic enzyme leakage. In addition, it may be suggested that Brij 58 caused plasma membrane perforations prior to destruction of the cytomatrix, whereas Triton X‐100 appeared to affect both the membrane integrity and the cytomatrix as indicated by dramatic losses of both inositol and glycolytic enzymes. This distinction between detergents should be considered and used to advantage in the design of histochemical, immunocytochemical, or further biochemical studies.
Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan
Authors: Falkensammer G. , Fischer‐Colbrie R. , Winkler H.
Published: October 2006

Abstract: The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse‐labeled with [35S]sulfate. The radioactively labeled products were characterized by one‐ and two‐dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein. much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000 100,000; pI 4.3–5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue‐stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.
Relationship Between Catechol‐O‐Methyltransferase and Phenolsulfotransferase in the Metabolism of Dopamine in the Rat Brain
Authors: Buu Nguyen T.
Published: October 2006

Abstract: The relationship between phenolsulfotransferase (PST) and catechol‐O‐methyltransferase (COMT) in the metabolism of free 3,4‐dihydroxyphenylethylamine (DA, dopamine) in the rat brain was studied. In rats not pretreated with a monoamine oxidase (MAO) inhibitor a huge increase of free DA in the brain, following an intraperitoneal injection of L‐3,4‐dihydroxyphenylalanine (L‐DOPA) or an intraventricular injection of free DA, did not lead to any noticeable change in DA sulfate or 3‐methoxytyramine (3‐MT), which remained undetectable by the present HPLC method. However, in rats previously treated with the MAO inhibitors pargyline or tranylcypromine, the same L‐DOPA or free DA treatment resulted in significant increases in both 3‐MT and DA sulfate in the hypothalamus, brainstem, and striatum. This response of COMT and PST was not affected by prior treatment of the rats with 6‐hydroxydopamine, which suggests that O‐methylation and sulfoconjugation occur outside adrenergic neurons not destroyed by the neurotoxin. Inhibition of COMT activity did not lead to any increase in DA sulfate, which showed that despite their common mode of action (both enzymes react preferentially at the same hydroxyl group in the DA molecule), the two enzymes are not competitive. After MAO inhibition there were strong correlations between an increase in DA sulfate and 3‐MT on the one hand, and between free DA and 3‐MT on the other. Because 3‐MT is a marker of central DA release, these data suggest that inhibition of MAO activity not only affects DA metabolism by this enzyme but also influences DA release in the rat brain.
Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells
Authors: Poduslo Joseph F.
Published: October 2006

Abstract: The posttranslational processing of the asparagine‐linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A‐Sepharose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high‐mannose‐type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9–91.9% of the [3H]mannose radioactivity as the complex‐type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high‐mannose‐type glycopeptides of the transected nerve isolated from the concanavalin A‐Sepharose column were hydrolyzed by endo‐β‐N‐acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean α‐d‐mannosidase gave the expected yields of free Man and ManGlcNAc from these high‐mannose‐type oligosaccharides. The data support the notion that at least two α‐1,2‐mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I‐B) may be an important rate‐limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this highmannose‐type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high‐mannose‐type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex‐type oligosaccharide chain and to contain fucose attached to the core asparagine‐linked GlcNAc residue. The extent of oligosaccharide processing of this down‐regulated glycoprotein remains to be determined.
Alteration of Amino Acid Metabolism in Epileptogenic Mice by Elevation of Brain Pyridoxal Phosphate
Authors: Norris D. K. , Murphy R. A. , Chung S. H.
Published: October 2006

Abstract: A single intraperitoneal injection of pyridoxal‐5′‐phosphate (PLP) in a species of mouse, DBA/2J, that is normally susceptible to sound‐induced convulsion exacerbated its epileptic condition. The effect of injection was most pronounced about 30 min after the administration and subsided gradually within the following 4 h. Correlated with this increased seizure susceptibility were enhanced levels of synaptosomal aspartate and glutamate, and a diminished γ‐aminobutyric acid (GABA) level. The concentrations of nonneuroactive amino acids remained unchanged. When stimulated with veratrine, synaptosomes prepared from PLP‐injected mice showed an increased release of aspartate and glutamate and a decreased release of GABA compared to those prepared from control mice. The activity of glutamate decarboxylase in the brains of PLP‐treated mice was lowered, whereas the activity of GABA‐transaminase was enhanced. Finally, the epileptic condition of DBA mice could be ameliorated by maintenance on a diet composed of vitamin B6‐deficient feed and cellulose.
Pre‐ Versus Postsynaptic Localization of Benzodiazepine and β‐Carboline Binding Sites
Authors: Tietz Elizabeth I. , Chiu Ted H. , Rosenberg Howard C.
Published: October 2006

Abstract: [3H]Flunitrazepam (FNP) and [3H]methyl β‐carboline‐3‐carboxylate (MCC) binding was examined in soluble and particulate fractions from membranes solubilized with Triton X‐100 or in subfractions of synaptosomal membranes obtained by a physical separation technique. Results using both methods demonstrate that benzodiazepine and β‐carboline sites reside on both pre‐ and postsynaptic membranes. Further, subfractionation experiments indicate that the binding sites for both ligands are unequally distributed within the synapse and among brain regions. For example, in cerebral cortical presynaptic membranes there are twice as many FNP as MCC sites whereas in postsynaptic membranes this ratio is reversed. The number of FNP and MCC sites are equal in the presynaptic fraction from cerebellum. The postsynaptic membranes derived from cerebellum have three times the number of FNP compared to MCC sites. In hippocampus this ratio varies between 1.5 and 2.8 in each subfraction. These results support the idea that benzodiazepine and β‐carboline binding sites represent different recognition sites.
Interaction Between Tetanus Toxin and Rabbit Kidney: A Comparison with Rat Brain Preparations
Authors: Habermann Ernst , Albus Udo
Published: October 2006

Abstract: 125I‐Tetanus toxin is bound by basolateral membranes from rabbit kidneys. Fixation is specific, as it is minimally inhibited by the nonbinding (fragment B) moiety of tetanus toxin, whereas the binding moiety (fragment C) is equivalent to the native toxin in inhibiting fixation. Competition is also pronounced with mildly toxoided toxin. Association and dissociation of 125I‐toxin are delayed in kidney when compared to brain membranes. The binding sites in kidney membranes are partially sensitive to neuraminidase and resist heating to 56°C, in contrast to those in brain membranes which are very sensitive to both treatments. The binding sites of the two preparations can be discriminated further by variation of the ionic environment. Sodium dodecyl sulfate‐disc gel electrophoresis followed by transfer to nitrocellulose, and TLC with consecutive overlay indicate that tetanus toxin exclusively binds to long‐chain gangliosides from rat brain. Binding sites in kidney membranes from rabbits and rats can be made visible by the overlay technique. They are apparently heterogeneous and more hydrophobic. We conclude that rabbit kidney contains binding sites for tetanus toxin which resemble gangliosides but differ from the major gangliosides in brain both chemically and with respect to their interaction with tetanus toxin.