bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Newly Formed and Aged Granules in the Neurohypophysis
Authors: Nordmann Jean J. , Cazalis Monique
Published: October 2006

Abstract Neurosecretory granules from the rat and bovine neurohypophysis were isolated and some of their biochemical and biophysical properties studied. Neurosecretory granules (NSG) from rat neurohypophysis were labeled, in vivo, with [35S]cysteine and isolated on isoosmotic gradients. Whereas 1 day after labeling most of the radioactivity was found in the lower part of the gradient, 35 days later the isotope was also located in the lighter NSG‐containing fraction. Different analytical procedures showed that the lighter fraction, both in bovine and rat NSG, contain more subpopulations of neurophysin‐like material than the heavier fraction. The first material to be released during stimulation of secretion, in vivo or in vitro, is mobilized from the heavy NSG. Isolation of rat NSG, at different times during and after dehydration of the animals, reveals that the newly synthesized material is found in the heavy NSG‐containing fraction. Furthermore, the results indicate that the newly synthesized NSG are more resistant to lysis than the lighter granules. The results are discussed in relation to the maturation and degradation processes of the granule content and to the functional state of the NSG.
Calcium ATPase Activities in Synaptic Plasma Membranes of Seizure‐Prone Mice
Authors: Palayoor Sanjeewani T. , Seyfried Thomas N. , Bernard David J.
Published: October 2006

Abstract: Audiogenic seizure (AGS)‐susceptible DBA/2 (D2) mice have a significant reduction in brain Ca2+‐ATPase activity compared to AGS‐resistant C57BL/6 (B6) mice. This reduction is inherited together with AGS susceptibility in B6 × D2 recombinant inbred strains. The Ca2+‐ATPase reduction occurs in microsomes and synaptosomes, but not in mitochondria. This enzyme activity is measured at a high Ca2+ concentration (2 mM) with no added Mg2+ or EGTA. We further studied this Ca2+‐ATPase activity and a Mg2+‐dependent (Ca2++ Mg2+‐ATPase activity in synaptic plasma membranes (SPM) from the B6 and D2 strains. Using EGTA or CDTA to adjust free Ca2+ concentrations, we measured Ca2+‐ATPase activities at Ca2+ concentrations from 0.8 μM to 436 μM. The Ca2+ATPase activity is consistently lower in the D2 than in the B6 SPM over all Ca2+ concentrations. The basal Mg2+ATPase activity measured at 2 mM MgCl2, is also lower in SPM of D2 than B6 mice. Calcium stimulates the basal Mg2+‐ATPase activity to the same extent in the SPM of the B6 and the D2 mice. Maximum stimulation in both strains occurs at 150 μM added CaCl2 (buffered with 100 μM EGTA). Higher Ca2+ concentrations inhibit this ATPase activity similarly in both strains. The EGTA‐EDTA washing of SPM significantly reduces by 50% the (Ca2+ Mg2+)‐ATPase activities of both strains, whereas calmodulin treatment restored these activities. Neither of these treatments, however, has any noticeable effects on the Ca2+‐ATPase activities of the strains. We conclude from this strain comparison that the Ca2+‐ATPase and the (Ca2++ Mg2+)ATPase activities are different enzymes and that the Ca2+‐ATPase activity in D2 mice is also deficient at low Ca2+ concentrations.
Asymmetry of Diacylglycerol Metabolism in Rat Cerebral Hemispheres
Authors: de Martínez Mercedes S. Ginóbili , de Turco Elena B. Rodríguez , Barrantes Francisco J.
Published: October 2006

Abstract: Diacylglycerols (DGs) were found to be asymmetrically distributed between the two cerebral hemispheres of rat brain. The left cerebral hemisphere (LCH) contained 100% more DG than the right cerebral hemisphere (RCH). The lateralization was enhanced in animals subjected to depolarization induced by a single electroconvulsive shock (ECS). During the acute phase of the convulsion, the DG pool increased in both hemispheres, with the LCH attaining a concentration 180% higher than the RCH. Stearate and arachidonate were the principal DG‐acyl groups accumulated in the RCH, whereas in the LCH stearate and palmitate were mainly involved. After the last of a series of five shocks (one per day) the laterialization of the “DG response’ was less accentuated during the acute phase of the ECS. Whereas DG release was drastically reduced in the LCH, in the RCH it was minimally affected. The DG sidedness after five shocks was nevertheless maintained at the level of arachidonate‐containing DGs, which showed a higher accumulation in the LCH than in the RCH. The kinetics of DG removal showed a rapid phase during the first minute following a single or five ECSs. Total DG levels returned to basal values in the RCH, whereas in the LCH they remained slightly increased with respect to the initial levels 1 min after the convulsive episode. Minimal changes occurred in the subsequent 4 min. Chronic ECS altered the endogenous DG content and composition. Thus, 24 h after the last of four ECSs, total levels of DGs diminished by 40% in the RCH, whereas they remained unchanged in the LCH. The asymmetry in the basal levels between hemispheres was therefore accentuated. with the LCH containing 160% more DG than the RCH. These results suggest the lateralization of biochemical events involved in DG metabolism in rat cerebral hemispheres, both under basal conditions and after electrical stimulation.
Major Central Nervous System Myelin Glycoprotein of the African Lungfish (Protopterus dolloi) Cross‐Reacts with Myelin Proteolipid Protein Antibodies, Indicating a Close Phylogenetic Relationship with Amphibians
Authors: Waehneldt T. V. , Matthieu J.‐M. , Jeserich †G.
Published: October 2006

Abstract: CNS myelin was isolated from the spinal cord of the African lungfish Protopterus dolloi. Its proteins consisted of (1) two basic proteins (16,000 and 18,500 apparent Mr) that reacted with anti‐human CNS myelin basic protein antibodies and (2) a major protein (29,000 apparent Mr) that stained with concanavalin A‐horseradish peroxidase and bound to anti‐rat CNS myelin proteolipid protein (PLP) antibodies. This dominant 29,000 Mr protein showed no reaction with antibodies against the major bovine PNS myelin glycoprotein P0. Following treatment with endoglycosidase F the 29,000 Mr protein was reduced in size to a 26,000 apparent Mr component that no longer bound concanavalin A but retained the anti‐PLP reactivity. These results agree with a concanavalin A‐binding oligosaccharide linked through asparagine to a protein backbone of PLP homology. The major 29,000 Mr lungfish CNS myelin protein was therefore termed g‐PLP (glycosylated proteolipid protein). This is the first report demonstrating the occurrence of a PLP‐cross‐reactive protein in CNS myelin of a fish. It attests to the close phylogenetic relationship of lungfishes to amphibians. Amphibians were previously recognized as the oldest class bearing PLP in its CNS myelin.
Release of Catecholamines from Superfused Bovine Adrenal Chromaffin Cells Cultured on Microcarrier Beads
Authors: Boarder M. R. , McArdle W.
Published: October 2006

Abstract: A procedure is described for the establishment of stable primary cultures of bovine chromaffin cells on microcarrier beads. The cells flatten and send out processes with varicosities over a few days and maintain their catecholamine content for 2 weeks. The beads may be incorporated into a superfusion apparatus with a chamber volume of about 150 μl, enabling the efficient perfusion of a high density of cells. The response to the introduction of nicotine and high potassium into the perfusing medium is shown to be more rapid and more transient than hitherto described, with each secretagogue producing a different degree of preferential stimulation of noradrenaline‐secreting cells.
Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase
Authors: Perez‐Reyes Edward , Cooper Dermot M. F.
Published: October 2006

Abstract: Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate‐solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide‐mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine‐5′‐(β‐γ‐imido)triphosphate [Gpp(NH)p] inhibited basal activity 40–60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine‐5′‐0‐(3‐thiotriphosphate) > Gpp(NH)p > GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70–90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin‐stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N‐ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibitin of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000‐ and 41,000‐dalton substrates comigrated with the functional Ni activity. These results demonstrate the feasibility of reconstituting Ni inhibition with resolved catalytic activity directly in solution.
Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain
Authors: Senders Mark , Weber Eckard
Published: October 2006

Abstract: Metorphamide is a [Met]‐enkephalin‐containing opioid octapeptide with a C‐terminal α‐amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for μ opioid (morphine) receptors, a somewhat lesser affinity for Kopioid receptors, and a relatively low affinity for δ opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared withthe concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]‐enkephaIyl‐Arg6‐Phe7 and [Met]‐enkephalyl‐Arg6‐Gly7‐Leu8 from proenkephalin; α‐ neoendorphin, β‐neoendorphin, dynorphin A(l‐8), dynorphin A(1–17), and dynorphin B from prodynorphin; and [Leu]‐enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalinderived opioid peptides. They were, however, similar to the concentrations of the prodynorphin‐derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin‐derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's μ opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous μ opioid receptor ligand.
Synergistic Action of Postsynaptic α‐Adrenergic Receptor Stimulation on Vasoactive Intestinal Polypeptide‐Induced Increases in Pineal N‐Acetyltransferase Activity
Authors: Yuwiler A.
Published: October 2006

Abstract: The α‐adrenergic agonists phenylephrine and methoxamine, at concentrations that have little effect on pineal N‐acetyltransferase activity, markedly enhance stimulation of this enzyme by vasoactive intestinal polypeptide (VIP). This augmentation can be blocked by the α1‐adrenergic antagonists phenoxybenzamine and prazosin and, at 10 but not 1 μM, by the α2‐antagonist yohimbine. The time course for VIP stimulation is not altered by concomitant α‐adrenergic stimulation. Augmented activity does not require concomitant α‐adrenergic stimulation, but α‐adrenergic agonists must be present for augmentation to be maintained. Phorbol 12, 13‐diacetate or ‐dibutyrate but not 4a‐phorbol can substitute for phenylephrine, a finding suggesting that protein kinase C is involved in the augmentation. These results are, in general, analogous to α‐adrenergic magnification of N‐acetyltransferase induction by β‐adrenergic agonists.
Affinity Shifts Induced by Cations Do Not Reliably Predict the Agonistic or Antagonistic Nature of Ligands at Brain Dopamine Receptors
Authors: Urwyler Stephan
Published: October 2006

Abstract: The effects of Ca2+ and Na+ ions on the affinities of rat striatal dopamine D1 and D2 receptors for a wide range of agonists and antagonists were investigated. These experiments were performed at 37°C, since it was found that at this physiological temperature D2 receptor affinities for dopamine and 2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahy‐dronaphthalene were clearly lower than at room tempera‐re. No correlation was found between the effects of the cations on the affinities of compounds for D1 and D2 receptors and their agonistic or antagonistic nature. On the other hand, the Hill coefficients of agonists but not of antagonists were consistently and significantly below unity, with either Ca2+ or Na+ ions present and at both receptors. This suggests the existence of yet another type of heterogeneity of D1 and D2 receptor forms, to which agonists but not antagonists are sensitive. It is thus concluded that changes in D1 and D2 receptor affinities induced by cations do not predict the agonistic or antagonistic nature of a compound. However, since dopamine itself was sensitive to Na+ or Ca2+ ions, this mechanism might play a role in the regulation of receptor affinities in synaptic transmission, in addition to that exerted by guanyl nucleotides.
Effect of Pressure on the Release of Radioactive Glycine and γ‐Aminobutyric Acid from Spinal Cord Synaptosomes
Authors: Gilman Sara C. , Colton Joel S. , Dutka Andrew J.
Published: October 2006

Abstract: Exposure to high hydrostatic pressure produces neurological changes referred to as the high‐pressure nervous syndrome (HPNS). Manifestations of HPNS include tremor, EEG changes, and convulsions. These symptoms suggest an alteration in synaptic transmission, particularly with inhibitory neural pathways. Because spinal cord transmission has been implicated in HPNS, this study investigated inhibitory neurotransmitter function in the cord at high pressure. Guinea pig spinal cord synaptosome preparations were used to study the effect of compression to 67.7 atmospheres absolute on [3H]glycine and [3H]γ‐aminobutyric acid ([3H]GABA) release. Pressure was found to exert a significant suppressive effect on the depolarization‐induced calcium‐dependent release of glycine and GABA by these spinal cord presynaptic nerve terminals. This study suggests that decreased tonic inhibitory regulation at the level of the spinal cord contributes to the hyperexciltability observed in animals with compression to high pressure.
Dolichol‐Linked Glycoprotein Synthesis in G1 Is Necessary for DNA Synthesis in Synchronized Primary Cultures of Cerebral Glia
Authors: Ishii Satoshi , Volpe Joseph J.
Published: October 2006

Abstract: Primary cultures of newborn rat cerebrum, which are composed of glial cells (principally astroglia), were used for examining the relationship between dolichol‐linked glycoprotein synthesis and DNA synthesis in developing cerebral glia. The cells were synchronized by reducing the content of fetal calf serum in the culture medium from 10 to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the quiescent state by return of the cultures to 10% serum causes a marked increase in DNA synthesis 12–24 h later. A sharp increase in glycoprotein synthesis (incorporation of [3H]mannose) occurred in the first 12 h after serum repletion, preceding the increase in DNA synthesis. Tunicamycin, an inhibitor of the dolichol‐linked pathway to glycoprotein synthesis at the first committed step in oligosaccharide formation, promptly and completely prevented the increase in glycoprotein synthesis and, in addition, the subsequent increase in DNA synthesis. The effects of tunicamycin on glycoprotein and DNA syntheses were reversible, and no comparable effect c n total protein synthesis was observed. When tunicamycin was added only during a temporally circumscribed peiod in G1, i.e., from 3 to 9 h after serum repletion, the increase in DNA synthesis between 12 and 24 h after repletion was still markedly inhibited, i.e., to ∼45% of the value in untreated cultures. The data thus show that there is a requirement for dolichol‐linked glycoprotein synthesis for the subsequent occurrence of DNA synthesis and that this requirement is expressed late in the G1 phase of the cell cycle.
Ubiquitin‐Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes
Authors: Meyer Edwin M. , M. West Christopher , Stevens Bruce R. , Chau Vincent , Nguyen Minh‐thanh , Judkins Jennifer H.
Published: October 2006

Affinity‐purified antibodies specific for ubiquitin were found to inhibit the sodium‐dependent uptake of [3H]choline, r‐[3H]aminobutyric acid ([3H]GABA), [3H]‐glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]scrotonin in rat cerebral cortical synaptosomes at a low concentration (10 μg/ml). These antibodies (termed anti‐Uh) had no effect on the sodium‐independent uptake of these substances or their calcium‐dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose‐free medium. Other nonneuronal sodium‐dependent transport processes were found to be unaffected by 10 μg/ml anti‐Ub, suggesting that anti‐Ub does not bind indiscriminantly to sodium‐binding sites on sodium‐dependent organic solute transporters. Finally, anti‐Ub inhibited sodium‐dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.
Further Characterization of the Avian Benzodiazepine Receptor Subunits Including Phylo‐and Ontogenetic Aspects
Authors: Hebebrand Johannes , Friedl Waltraut , Kessler Klaus , Hewing, Maria , Propping Peter
Published: October 2006

The two avian benzodiazepine binding proteins offer an opportunity for further studies concerning their regional variation and their phylo‐and ontogenetic development. Accordingly, regional variation of the benzodiazepine binding proteins is investigated further in two reptiles and chicken using photoaffinity labeling with [3H]flunitrazepam followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography. Whereas regional heterogeneity is pronounced in chicken, it is not readily apparent in the two reptiles. The ontogeny of the benzodiazepine binding proteins in chicken forebrain and cerebellum is remarkably similar to that previously reported in rodents. The results are discussed in light of the possible existence of the γ‐aminobutyric acid/benzodiazepine receptor as an isoreceptor complex.
Developmental Changes in Calmodulin‐Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition
Authors: Kelly Paul T. , Shields Steven , Conway Kathleen , Yip Rick , Burgin Karl
Published: October 2006

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin‐dependent protein kinase II (CaM‐kinase II). This kinase is comprised of a major 50‐kilodalton (kDa) and a minor 60‐kDa subunit. Experiments examined the developmental properties of CaM kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM‐kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that fore brain CaM‐kinase II undergoes a striking age‐dependent change in subunit composition. In early postnatal forebrain the 60‐kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM‐kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM‐kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.
Protein Carboxyl Methylation in Synaptic Membrane of Rat Brain: The Possible Presence of Adenosine‐Bound S‐Adenosyl‐L‐Homocysteine Hydrolase in the Membrane
Authors: Miyake Masaharu , Innami Toshihiko
Published: October 2006

The effects of some neurotransmitters, adenosine (Ad), and homocysteine (Hcys) on protein carboxyl methylation in synaptic plasma membranes from rat cerebral cortex were examined. Neither any of the neurotransmitters nor Ad had a detectable effect. Incubation of the membrane with DL‐Hcys alone (5 × 10‐5M), the combination of both Ad (5 × 10‐5) and DL‐Hcys (5 × 10‐5), or S‐adenosyl‐L‐homocysteine (SAH) (1 × 10‐6) strongly decreased the methyl ester formation. The inhibitory effect of the combination of both compounds may be interpreted in terms of the increased SAH concentration due to the presence of SAH hydrolase in the membrane. The inhibitory effect of Hcys alone was blocked by preincubation with Ad deaminase or Neplanocin A, a potent inhibitor of SAH hydrolase, suggesting the presence of Ad‐bound SAH hydrolase in the synaptic membrane. Ad‐bound SAH hydrolase activity estimated by the inhibition of methylation in the presence of Hcys was located in the membrane fractions including synaptosomes, myelin, and microsomes (about 70%), but the SAH hydrolase activity estimated on the basis of the inhibitory effect of the combination of both Ad and Hcys was localized exclusively in the soluble fraction (about 90%). The distribution of the latter activity is coincident with that of SAH hydrolase reported to date. Incubation of the synaptic membrane with Hcys alone and with the combination of both Ad and Hcys markedly increased the SAH concentration. The stimulatory effect of Hcys alone was blocked by Ad deaminase.
Phosphorylation Characteristics of Brain Clathrin‐Coated Vesicle Endogenous Proteins
Authors: Schook William J. , Parker Carlo , Silva Walter I. , Kohtz D. Stave , Puszkin Saul
Published: October 2006

Clathrin‐coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle‐associated substrates: a 50,000‐Mr poly‐peptide (pp50) and clathrin‐associated protein2 (CAP2; the faster‐migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin‐coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl‐dependent enzymes in the following order of potency: Zn2+ > Hg2+ > Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodini‐trobenzene, phenacyl bromide, and N‐ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from disassembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin‐coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.
Inactive Monomeric Acetyleholinesterase in the Low‐Salt‐Soluble Extract of the Electric Organ from Torpedo marmorata
Authors: Stieger S. , Brodbeck U. , Witzemann V.
Published: October 2006

Proteolytic fragmentation of (3H]diisopropylflu‐orophosphate‐labelled catalytic subunits of different molecular forms of acetylcholinesterase demonstrates that all forms extracted from the electric organ from Torpedo marmorata are true acetylcholinesterases. This is supported by immunochemical results showing that the radiolabelled polypeptides are readily recognized by specific anti‐acetyl‐cholinesterase antibodies. Although distinct structural differences exist, all forms contain a similar peptide carrying the serine hydroxyl of the esteratic subsite. Dimeric, detergent‐soluble acetylcholinesterase is present in the low‐salt‐soluble extract (Mr of the catalytic subunit 66,000) together with a monomeric form (apparent Mr 76,000). This monomeric polypeptide is hydrophilic, enzymatically inactive, and might represent a precursor of the asymmetric forms of acetylcholinesterase.
Effect of Postmortem Factors on Muscarinic Receptor Subtypes in Rat Brain
Authors: Burke Robert E. , Greenbaum Daniel
Published: October 2006

Although prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4°C and‐20°C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]‐Pir). We found that delay at room temperature up to 4 h, or storage at 4°C for 24 h or at‐20°C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]‐QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding.
Effect of the Tricyclic Antidepressant Desipramine on β‐Adrenergic Receptors in Cultured Rat Glioma C6 Cells
Authors: Fishman Peter H. , Finberg John P. M.
Published: October 2006

Abstract: Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of β‐adrenergic receptors in a time‐and dose‐dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic β‐adrenergic antagonist (±)‐[3H]4‐(3‐tert‐butylamino‐2‐hydroxypropoxyl)benzimidazole ‐2 ‐ on HCl ([3H]CGP‐12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine‐treated cells accumulated less cyclic AMP than control cells. The affinity of the β‐adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 μM. The presence in the culture medium of alprenolol or propranolol, potent β‐adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of β‐adrenergic receptors from cells maintained in serum‐free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of β‐adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.
D2 Dopamine Receptors on Bovine Chromaffin Cell Membranes: Identification and Characterization by [3H]N‐Methylspiperone Binding
Authors: Lyon Robert A. , Titeler Milt , Bigornia Luisa , Schneider Allan S.
Published: October 2006

Abstract: Although dopamine‐containing cells are known to be present in sympathetic ganglia, the site of action and the role of dopamine in ganglion function remain obscure. In the present work, we evaluated the interaction of dopamine receptor ligands with particulate membrane fractions from bovine chromaffin cells and adrenal medullary homogenates using the D2 dopamine receptor radioligand [3H]N‐methylspiperone ([3H]NMSP). Scatchard analysis of [3H]NMSP saturation experiments revealed a Bmax of 24.1 ± 1.6 fmol/mg of protein and a KD of 0.23 ± 0.03 nM in the particulate fraction from adrenal medulla homogenates and a Bmax of 26.5 ± 2.7 fmol/mg of membrane protein and a KD of 0.25 ± 0.02 nM in the particulate fraction prepared from isolated adrenal chromaffin cells. There were ∼ 1,000 receptors/cell. There were no detectable levels of specific [3H]NMSP binding in the particulates prepared from adrenal cortical or capsular homogenates. Competition studies with the nonradioactive D2 receptor antagonists spiperone, chlorpromazine, and (‐)‐sulpiride revealed K1 values of 0.28, 21, and 196 nM, respectively. The (+) isomer of butaclamol displayed a 604‐fold higher affinity than the (‐) isomer. Competition studies with the dopamine receptor agonists dopamine and apomorphine revealed affinities of 3,960 and 417 nM, respectively. A correlation coefficient of 0.96 was obtained in studies comparing the potencies of drugs in inhibiting specific [3H]NMSP binding in bovine adrenal medullary homogenates and in inhibiting specific [3H]NMSP binding to brain D2 dopamine receptors. In summary, radiolabeling studies using [3H]NMSP have revealed the presence of D2 dopamine receptors on bovine adrenal chromaffin cells. Dopamine may modulate adrenal medullary function, and possibly sympathetic ganglion function, through D2 dopamine receptors.
Characterization of the Binding of N‐Methyl‐4‐Phenylpyridine, the Toxic Metabolite of the Parkinsonian Neurotoxin N‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine, to Neuromelanin
Authors: D'Amato Robert J. , Benham David F. , Snyder Solomon H.
Published: October 2006

Abstract: N‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) selectively destroys neuronal cell bodies in the neuromelanin‐containing substantia nigra of humans and primates. We show that N‐methyl‐4‐phenylpyridine (MPP+), the active metabolite of MPTP, binds to neuromelanin with high affinity. This binding increases at higher pH and is displaced most potently by divalent cations and antimalarial drugs. MPP+ bound intracellularly to neuromelanin may be stored and released gradually, resulting in subsequent damage to neurons of the substantia nigra.
Posttranslational Protein Modification by Polyamines in Intact and Regenerating Nerves
Authors: Chakraborty G. , Leach T. , Zanakis M. F. , Sturman J. A. , Ingoglia N. A.
Published: October 2006

Abstract: A 150,000‐g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N′‐dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase‐mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N′‐dimethylcasein) showed labeling of 18‐, 46‐ and 200‐kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein‐bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.
Distribution of α‐(γ‐Aminobutyryl)‐Hypusine
Authors: Sano Akira , Kotani Kazuhiko , Ueno Shuichi , Kakimoto Yasuo
Published: October 2006

Abstract: The distribution of α‐(γ‐aminobutyryl)‐hypusine was examined in several organs of the rabbit and in the brain of the rat, rabbit, dog, ox, and monkey. The peptide occurred only in the brains, but appeared to be absent from dog brain. Concentrations were higher in the cerebral hemispheres than in other portions of the brain. No significant difference between white and gray matter was observed.
Enzyme Activities in Regions of the Hypothalamus
Authors: Martin R. J. , Bird M. I. , Saggerson E. D. , Munday L. A. , Clark J. B.
Published: October 2006

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3‐hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].
Monoclonal Antibodies Against the Voltage‐Sensitive Sodium Channel from Rat Skeletal Muscle: Mapping Antibody Binding Sites
Authors: Casadei Jan M. , Barchi Robert L.
Published: October 2006

Abstract: Twenty‐one monoclonal antibodies specific for the rat skeletal muscle voltage‐sensitive sodium channel have been characterized according to subunit reactivity, recognition of carbohydrates, and mutual binding interactions. All antibodies recognize the 260‐kDa α‐subunit of the sodium channel on immunoblots. N‐Acetylneuraminic acid inhibited the binding of five antibodies in a concentration‐dependent manner, but five other monosaccharides known to be components of the channel had no effect on antibody binding. Competition studies using biosynthetically labeled antibodies separated these 21 antibodies into groups recognizing at least nine distinct domains. Through common in teractions between domains, these could in turn be associated into two larger topologically related regions. One region encompasses seven interacting domains and 16 antibodies. This region is probably extracellular by virtue of the interaction of one subgroup with N‐acetylneuraminic acid, and may represent a particularly immunogenic region on this channel protein.
31P Nuclear Magnetic Resonance In Vivo Spectroscopy of the Metabolic Changes Induced in the Awake Rat Brain During KCN Intoxication and Its Reversal by Hydroxocobalamine
Authors: Benabid A. L. , Decorps M , Remy C. , Le Bas J. F. , Confort S. , Leviel J. L.
Published: October 2006

Abstract: Radiofrequency surface coils were chronically implanted in rats, which were subsequently subjected to 31P nuclear magnetic resonance (NMR) investigations at 4.7 T. The implanted coil allowed study of the animals without need for anesthesia, which is a prerequisite for studies of normal brain metabolism. The animals may be kept in the NMR probe for several hours. During subsequent experiments, they may be placed in the same position, therefore allowing follow‐up studies for periods as long as 2 months. This method has been used in the study of sublethal KCN intoxication. KCN, a cytochrome c oxidase inhibitor, induces a blockade of cell respiratory processes, which is reflected, in a dose‐dependent manner, by a decrease in phos‐phocreatine content and pH and an increase in inorganic phosphate content, whereas ATP levels remain constant until high doses of KCN (6 mg/kg i.p.) are reached. 31P NMR allows the time course of these metabolic changes to be followed. For high KCN doses, a new peak, termed X, is observed, which is interpreted as being due to a pool of inorganic phosphate at very low pH (5.65), corresponding to a subset of cells that did not survive KCN injury. Hydroxocobalamine, a specific antidote of KCN, suppresses the metabolic changes due to 6 mg/kg of KCN.
Lectin‐Induced Enhancement of Voltage‐Dependent Calcium Flux and Calcium Channel Antagonist Binding
Authors: Greenberg David A. , Carpenter Celia L. , Messing Robert O.
Published: October 2006

Abstract: Concanavalin A (Con A), a tetravalent lectin with preferential affinity for mannosyl and glucosyl residues of membrane glycoconjugates, increased K+ depolarization‐evoked uptake of 45Ca2+ in the PC12 neural cell line. Enhancement of uptake by Con A was concentration dependent, with maximal (24%) stimulation at 100 μg/ml of Con A, and was preferentially inhibited by mannoside and glu‐coside. Succinyl‐Con A, a divalent analog with reduced biological potency, increased uptake by only 7%. The effect of Con A on 45Ca2+ uptake was dependent on membrane depolarization, was abolished by ionic Ca2+ channel blockers and organic Ca2+ channel antagonists, and was accompanied by an equivalent increase in Ca2+ channel 3H‐labeled antagonist binding, observations suggesting that the voltage‐dependent Ca2+ channel was the site of Ca2+ entry. The mechanism for enhancement of 45Ca2+ uptake by Con A appeared to be separate from that used by the Ca2+ channel agonist BAY K 8644 and independent of that involved in Ca2+ channel regulation by phorbol esters. These findings suggest that voltage‐dependent Ca2+ channels may link cell surface carbohydrate interactions with intracellular effector processes.
Evaluation of the Biological Activity of Several Analogs of the Dopaminergic Neurotoxin 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine
Authors: Youngster Stephen K. , Somalia Patricia K. , Heikkila Richard E.
Published: October 2006

Abstract: Several analogs of l‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO‐A or MAO‐B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO‐A but many of the compounds were found to be good substrates for MAO‐B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1‐methyl‐4‐cyclohexyl‐1,2,3,6‐tetrahydropyridine, 1 ‐methyl‐4‐(2′‐methylphenyl)‐1,2,3,6‐tetrahydropyridine and 1‐methyl‐4‐(3′‐methoxyphenyl)‐1,2,3,6‐tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO‐B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO‐B. The capacity of the compounds studied to be oxidized by MAO‐B appears to be an important aspect of the neurotoxic process.
Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin‐Dependent Protein Kinase
Authors: Inoue Nobuhiro , Iwasa Takafumi , Fukunaga Kohji , Matsukado Yasuhiko , Miyamoto Eishichi
Published: October 2006

Abstract: Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin‐dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin‐dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.
Calcium/Calmodulin‐Dependent Protein Kinase II in Squid Synaptosomes
Authors: Bass Martha , Pant Harish C. , Gainer Harold , Soderling Thomas R.
Published: October 2006

Abstract: The Ca2+/calmodulin (CaM)‐dependent protein kinase II system in squid nervous tissue was investigated. The Ca2+/CaM‐dependent protein kinase II was found to be very active in the synaptosome preparation from optic lobe, where it was associated with the high‐speed particulate fraction. Incubation of the synaptosomal homogenate with calcium, calmodulin, magnesium, and ATP resulted in partial and reversible conversion of the Ca2+/CaM‐dependent protein kinase II from its calcium‐dependent form to a calcium‐independent species. The magnitude of this conversion reaction could be increased by inclusion of the protein phosphatase inhibitor NaF or by substitution of adenosine 5′‐O‐(3‐thiotriphosphate) for ATP. When [γ‐32P] ATP was used, proteins of 54 and 58 kilodaltons (kDa) as well as proteins >100 kDa were rapidly 32P‐labeled in a calcium‐dependent manner. Major 125I‐CaM binding proteins in the synaptosome membrane fraction were 38 and 54 kDa. The Ca2+/CaM‐dependent protein kinase II was purified from the squid synaptosome and was shown to consist of 54‐and 58–60‐kDa subunits. The purified kinase, like Ca2+/CaM‐dependent protein kinase II from rat brain, catalyzed auto‐phosphorylation associated with formation of the calcium‐independent form. These studies, characterizing the Ca2+/ CaM‐dependent protein kinase II in squid neural tissue, are supportive of the putative role of this kinase in regulating calcium‐dependent synaptic functions.
Calmodulin‐Binding Proteins in Chromaffin Cell Plasma Membranes
Authors: Fournier S. , Trifaró J.‐M.
Published: October 2006

Abstract: Calmodulin‐binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin‐binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine‐ to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X‐100 and applied to a calmodulin‐affinity column in the presence of calcium. Several major calmodulin‐binding proteins (240, 105, and 65 kilodaltons) were eluted by an EGTA‐containing buffer. 125I‐Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin‐binding proteins in common (65, 60, 53, and 50 kilodaltons), as well as unique calmodulin‐binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin‐binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two‐dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin‐binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin‐binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin‐binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.
Differentiation of Human Neuroblastoma Cells: Marked Potentiation of Prostaglandin E‐Stimulated Accumulation of Cyclic AMP by Retinoic Acid
Authors: Yu Victor C. , Hochhaus Güinther , Chang Fu‐Hsiung , Richards Mark L. , Bourne Henry R. , Sadée Wolfgang
Published: October 2006

Abstract: Neuroblastoma cells in culture contain low levels of cyclic AMP, a second messenger which plays a major role in neuronal maturation. In this study, human neuroblastoma cells, SK‐N‐SH‐SY5Y, were induced to differentiate by treatment with either nerve growth factor (50 ng/ml), retinoic acid (10 μM), dibutyryl cyclic AMP (1 mM), or 12‐O‐tetradecanoylphorbol‐13‐acetate (0.1 μM), and the ability of several neurotransmitters or hormones to stimulate adenylyl cyclase was tested. Although all four differentiation factors caused morphological changes towards a neuronal phenotype, only retinoic acid dramatically enhanced cyclic AMP accumulation, specifically upon stimulation with prostaglandin E1 (PGE,). PGE2 was also active, but less potent, than PGE1, whereas the other cyclic AMP‐stimulating agents tested were largely unaffected. Further, the rapid desensitization of the PGE1‐cyclic AMP response observed in control cells after 20 min of PGE1 exposure did not occur in retinoic acid‐treated cells, and the EQ50 values for PGE, were reduced from ∼240 to 14 nM after retinoic acid treatment. The increased sensitivity to PGE was associated with an increase of high‐affinity PGE1 binding sites, whereas the Gs coupling proteins and adenylyl cyclase were not measurably affected. A similar enhancement of the PGE1‐cyclic AMP response by retinoic acid was also observed in two additional human neuroblastoma cell lines tested, Kelly and IMR‐32, suggesting that up‐regulation of the prostaglandin response by retinoic acid is common among neuroblastoma cells. The ability of neuroblastoma cells to elevate cyclic AMP levels upon prostaglandin stimulation could modulate differentiation therapy with retinoic acid in vivo.