bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of S100 Protein on Free and Membrane‐Bound Polysomes of the Rabbit Brain
Authors: Cosgrove James W. , Heikkila John J. , Marks Alexander , Brown Ian R.
Published: October 2006

Abstract: Free and membrane‐bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA‐dependent cell‐free system derived from rabbit reticulocytes. Analysis of the [35S]methionine‐labeled translation products on two‐dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane‐ bound polysomes was determined by direct immuno‐precipitation of the cell‐free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane‐bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly‐ (A+)mRNA coding for SlOO protein was also twofold greater in membrane‐bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane‐bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.
Specific Binding of a 125I‐Substance P Derivative to Rat Brain Synaptosomes
Authors: Viger A. , Beaujouan J. C. , Torrens Y. , Glowinski J.
Published: October 2006

Binding characteristics of 125I‐labeled Bolton‐Hunter substance P (125I‐BHSP), a radioactive analogue of substance P, were studied with a crude syn‐aptosomal fraction (P2) of the rat brain. A specific binding of 125I‐BHSP that was saturable, reversible, and temperature dependent was demonstrated. Under the conditions of the binding assay (20°C, 5 min), nonspecific binding represented no more than 25% of total binding, and in contrast to [3H]dopamine 125I‐BHSP was not taken up within synaptosomes by a ouabain‐sensitive process. Scatchard analysis indicated the existence of a single population of noninteracting sites with a high affinity (KD, 470 pM) and a low‐density (Bmax, 13 fmol/mg protein). Substance P and different substance P analogues were tested for their competitive potencies in inhibiting 125I‐BHSP binding to synaptosomes. (Tyr8) substance P, substance P, and BHSP strongly inhibited 125I‐BHSP specific binding. Among various tachykinins, physalaemin was the most potent (one fourth the potency of substance P). When substance P C‐terminal fragments were tested for their ability to compete with 125I‐BHSP binding, a good relationship was found between competitive activity and peptide length. N‐terminal fragments of substance P were ineffective. However, (nor‐Leu11)substance P and the methyl ester of substance P1‐11 slightly reduced 125I‐BHSP binding. (D‐Pro2, D‐Phe7, D‐Trp9) substance P and (D‐Pro2, D‐Trp7,9)substance P, two substance P antagonists, were also tested. (D‐Pro2, D‐Trp7,9) substance P was the only one to inhibit 125I‐BHSP binding even slightly. Results were compared with those previously obtained concerning 125I‐BHSP binding to mesencephalic embryonic cells in primary cultures using similar conditions for the binding assay.
Purification and Characterization of Myosin from Calf Brain
Authors: Malik Mazhar N. , Fenko Michael D. , Scotto Lois , Merz Pat , Rothman Jeffrey , Tuzio Helen , Wisniewski Henryk M.
Published: October 2006

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (>200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was >95% pure as judged by electron microscopy and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate‐polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KC1 and MgCl2. At low ionic strength, the Mg2+‐ATPase activity of myosin was stimulated 3‐ to 3.5‐fold in the presence of skeletal muscle f‐actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP > ATP ∼ CTP < GTP ∼ UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KC1 was complex and consisted of plateau regions. The Arrhenius plot of the Ca‐ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20°C.
Uptake of Adenosine by Cultured Cerebral Vascular Smooth Muscle Cells
Authors: Beck David W. , Vinters Harry V. , Moore Steven A. , Hart Michael N. , Cancilla Pasquale A.
Published: October 2006

Abstract: Adenosine uptake by cerebral smooth muscle cells is a carrier‐mediated process. The Km value for adenosine uptake is 10.0 μM and the Vmax is 0.95 nmol/ min‐mg cell protein. This uptake system is inhibited by the adenosine analog 2‐chloroadenosine at low adenosine concentrations. These results prove the existence of a nucleoside transport system associated with cerebral smooth muscle.
Impairment of Blood‐Cerebrospinal Fluid Barrier in Multiple Sclerosis
Authors: Takeoka Tsuneyuki , Shinohara Yukito , Furumi Koichi , Mori Kosuke
Published: October 2006

Abstract: The blood‐CSF barrier (BCB) function in active multiple sclerosis (MS) was studied by means of CSF proteins analysis using disc electrophoresis and immu‐nofixation. Forty‐five CSF samples were obtained by repeat lumbar punctures at various intervals, from four autopsy‐proven cases and three male and nine female patients with clinically definite MS. When total protein content was increased, the percentages of prealbumin and t fraction were decreased significantly in association with the presence of haptoglobin (Hp) polymers in nearly all the samples, as a result of increased permeability of the BCB. Even when the total protein content was normal, Hp polymers were detected in 56% of the samples, and the t fraction tended to be decreased. Monoclonal immunoglobulin and Hp polymers were both recognized in some cases. The results suggested a more frequent occurrence of BCB impairment in MS than had formerly been revealed by CSF albumin analysis, and accorded with the recent reports of contrast‐enhancing lesions of MS brain in computerized tomography.
The Synthesis and Release of [3H‐Tyrosine1]Methionine5‐Enkephalin from Guinea Pig Brain Slices
Authors: Jones C. A. , Marchbanks R. M.
Published: October 2006

Abstract: Stores of methionine‐enkephalin were labelled on the N‐terminal by incubation of whole brain slices with [3H]tyrosine (10 °Ci/ml). The 3H radioactivity corresponding to the position of authentic Met‐enkephalin after extraction on Amberlite XAD2 and separation by thin‐layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met‐enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μM cycloheximide. Isolated nerve terminals failed to incorporate any [3H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N‐terminus and removal of this tyrosine resulted in loss of opiate‐like activity. The incorporation of [14C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N‐terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met‐enkephalin. KCl (50 mM) elicited a Ca2+‐dependent release of the synthesised [3H]Met‐enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met‐enkephalin radioimmunoactivity paralleled that of [3H]met‐enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.
Determination of Brain 5‐Hydroxytryptamine Turnover in Freely Moving Rats Using Repeated Sampling of Cerebrospinal Fluid
Authors: Sarna G. S. , Hutson P. H. , Tricklebank M. D. , Curzon G.
Published: October 2006

Abstract: A simple technique is described for repeated sampling of cerebrospinal fluid (CSF) from the freely moving rat and its use in the determination of 5‐hydroxytryptamine (5‐HT) turnover validated. A catheter, constructed from polyethylene tubing (PP10) was implanted via a cranial approach into the cisterna magna and x‐ray studies confirmed that the catheter avoided the cerebellum. 5‐HT turnover was determined from the rate of rise of 5‐hydroxyindoleacetic acid (5‐HIAA) in both CSF and brain following an injection of probenecid (200 mg/kg i.p.), Concentrations of 5‐HIAA, 5‐HT and trypto‐phan were determined by high pressure liquid chromatog‐raphy. Turnover values for individual rats were obtained using CSF samples. After p‐chlorophenylalanine treatment (when brain 5‐HT was depleted by 43%) 5‐HT turnover values obtained were comparably reduced whether determined from CSF (67%) or brain (74%). Thus differences of rat brain 5‐HT turnover are proportionately reflected by CSF measurements. The method for sampling of CSF should be applicable in a wide range of pharmacological and physiological situations.
[3H]Dopamine Depletion from Osmotically Defined Storage Sites: Effects of Reserpine, 53 mM KC1, and d‐Amphetamine
Authors: Takimoto Glenn S. , Stittsworth James D. , Stephens Janet K.
Published: October 2006

Abstract: A crude synaptosome‐containing fraction (P2′) prepared from rat striatal slices incubated with [3H)dop‐amine was exposed to hypoosmotic conditions and rapidly subjected to Millipore filtration. P2′‐associated [3H]dopamine trapped on the filters was defined as hypoosmotic resistant, whereas P2′‐associated [3H]dop‐amine that washed through the filters was defined as hypoosmotic sensitive. Electron microscopic examination of sections prepared from a P2’pellet that had been exposed to hypoosmotic conditions revealed extensive synaptosomal lysis. [3H]Dopamine accumulation and retention by the hypoosmotic‐resistant fraction were reduced by reserpine. The proportional distribution of [3H]dopamine between hypoosmotic‐resistant and ‐sensitive fractions was measured following in vitro exposure of the preloaded P2’fraction to reserpine, 53 mM KC1, and d‐amphetamine. Each of these treatments resulted in a time‐dependent loss of [3H]dopamine from the loaded P2’fraction without eliciting an alteration in the proportional distribution of [3H]dopamine between hypoosmotic‐resistant and ‐sensitive fractions. Release induced by reserpine and d‐amphetamine was independent of extrasyn‐aptosomal Ca2+, whereas 53 mM KCl‐induced release was dependent on extrasynaptosomal Ca2+. These results suggest that dopamine may be rapidly equilibrated between osmotically defined storage compartments, and thus specific compartmental depletion of loaded [3H)dop‐amine cannot be identified on the basis of osmotic lability.
Regional Distribution of Methionine‐Enkephalin‐Arg6‐Phe7 in the Rat Brain: Comparative Study with the Distribution of Other Opioid Peptides
Authors: Giraud P. , Castanas E. , Patey G. , Oliver C. , Rossier J.
Published: October 2006

Abstract: The distribution of the opioid peptide methionine‐enkephalin‐arginine6‐phenylalanine7 (M‐Enk‐Arg6‐Phe7) has been investigated in various structures of the rat brain by using a highly specific radioimmunoassay (RIA). Immunoreactive M‐Enk‐Arg6‐Phe7 has been further characterized by high performance liquid chromatography. The levels of M‐Enk‐Arg6‐‐Phe7 in various structures of the rat brain were compared with the levels of several other opioid peptides, including methionineenkephalin (M‐Enk), leucine‐enkephalin (L‐Enk), dynorphin 1–13, and α‐neoendorphin, which were also measured by RIA. There was a close relationship between the distribution of M‐Enk‐Arg6‐‐Phe7 immunoreactive material (ir), M‐Enk ir, and L‐Enk ir. The distribution of dynorphin 1–13 ir and α‐neoendorphin ir appeared to be distinct from that of the enkephalin group. These results are in agreement with recent reports on the cloning and sequencing of the c‐DNA coding for the prohormones, in which it has been hypothesized that M‐Enk‐Arg6‐Phe7 and M‐Enk are synthesized by the same precursor, called proenkephalin, and that dynorphin‐related peptides and α‐neoendorphin arise from a separate precursor, prodynorphin.
Calmodulin Stimulation of Ca2+‐Dependent ATP Hydrolysis and ATP‐Dependent Ca2+ Transport in Synaptic Membranes
Authors: Ross D. H. , Cardenas H. L.
Published: October 2006

Abstract: We report here characterization of cal‐modulin‐stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+‐stimulated, Mg2+‐dependent ATP hydrolysis coupled with ATP‐dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synap‐tosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM‐1 and SPM‐2. Both Ca2+‐ATPase and ATP‐dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 μM for Ca2+‐ATPase and 0.4 μM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high‐affinity Ca2++ Mg2+ ATPase as a regulator for cytosolic Ca2+.
Modulation of GABA Receptor Binding by Ca+2
Authors: Corda M. G. , Guidotti A.
Published: October 2006

Abstract: In frozen‐thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1–5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]γ‐aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]mus‐cimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated 3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 μg/ml), trifluoperazine (10‐‐5M), verapamil (10‐‐6M), quinacrine (10‐‐4M), cordycepin (0.1 mM), leupeptin (20 μM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA‐modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.
β‐Adrenergic Binding Sites in Fetal Rat Brain
Authors: Bruinink A. , Lichtensteiger W.
Published: October 2006

Abstract: The ontogeny of β‐adrenergic binding sites was studied in forebrain homogenates from male and female rats. Specific [3H]dihydroalprenolol binding was defined by the difference between binding in the presence and in the absence of 330 nM (+)oxprenolol. Significant binding was detected at prenatal stages. The dissociation constants (KD of [3H]dihydroalprenolol binding were similar at gestational day (GD) 193/4 and postnatal day (PN) 31. Binding was first detected in forebrain at GD 153/4. The amount of binding sites increased until PN 31, when adult values were reached. No sex differences could be detected at any of the stages tested (GD 193/4‐PN 31).
Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1
Authors: Facci Laura , Leon Alberta , Toffano Gino , Sonnino Sandro , Ghidoni Riccardo , Tettamanti Guido
Published: October 2006

Abstract: Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro‐2a clone, in monolayer culture. GM1 bound to neuro‐2a cells in three distinct forms, one removable by treatment with serum‐containing solutions, one serum‐resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell‐associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10−6M); the trypsin‐labile and ‐stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro‐2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite‐bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10−4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite‐bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin‐labile and ‐stable forms of cell‐associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, <1:20,000–1:50,000. Therefore the neuritogenic effect can be attributed to ganglioside itself. The results obtained suggest that under the experimental conditions used the stimulation of neuro‐2a cell differentiation by GM1 is related to changes of the plasma membrane properties following association of exogenous GM1 molecules. This would facilitate the spontaneous process of differentiation, or enhance cell responsiveness to differentiating factors present in the serum.
Characteristics of Tryptophan Accumulation by Isolated Rat Forebrain Synaptosomes
Authors: Knowles Richard G. , Pogson Christopher I.
Published: October 2006

Abstract: Uptake of 10 μMl‐tryptophan into isolated rat brain synaptosomes was studied to assess its effect on the rate of serotonin synthesis from tryptophan. The initial rate of uptake was rapid, being two orders of magnitude above the rate of tryptophan hydroxylation. Uptake was highly concentrative, the concentration ratio across the plasma membrane at equilibrium being approximately 9. This concentration ratio was decreased to about 1 in the presence of high concentrations of amino acids transported by the L‐type neutral amino acid uptake system. A mixture of the large neutral amino acids at physiological concentrations decreased the internal tryptophan concentration to 58% of that in their absence. Large tryptophan concentration ratios were observed in experiments in which Na+ in the medium was replaced with choline+. The concentrative uptake of tryptophan was energy‐dependent, being decreased by inclusion of cyanide and omission of glucose. The concentration gradient was abolished by veratridine or rotenone. Time courses of the changes in ATP content and tryptophan concentration ratio on addition of these and other agents established that tryptophan uptake is probably not driven by ATP hydrolysis or efflux of other amino acids, but by the plasma membrane potential.
Distribution of Glycine, γ‐Aminobutyric Acid, Glutamate Decarboxylase, and γ‐Aminobutyric Acid Transaminase in Rabbit and Mudpuppy Retinas
Authors: Dick Evan , Lowry Oliver H.
Published: October 2006

Abstract: The distributions of glycine, γ‐aminobutyric acid (GABA), glutamate decarboxylase (EC 4.1.1.15), and GABA transaminase (EC 2.6.1.19) were determined in rabbit and mudpuppy retinas. In both species, peak levels of the amino acids and the enzymes occurred in the inner plexiform layer. Glutamate decarboxylase was almost entirely confined to the inner plexiform layer. Determinations were also made of the GABA content of 107 individual putative amacrine cell somas from mudpuppy retina. About 30% of those somas were found to have high endogenous GABA levels.
Characterization and Regulation of Insulin Receptors in Rat Brain
Authors: Zahniser Nancy R. , Goens M. Beth , Hanaway Patrick J. , Vinych John V.
Published: October 2006

Abstract: An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD= 3–11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four‐fold greater (Bmax= 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 μM insulin for 3 h at 37°C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.
Reduction of Noradrenaline in Cerebellum of Patients with Olivopontocerebellar Atrophy
Authors: Kish Stephen J. , Shannak Kathleen S. , Hornykiewicz Oleh
Published: October 2006

Abstract: Noradrenaline (NA) was measured in postmortem cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The mean cerebellar cortical NA level was significantly reduced (by 40%) in OPCA as compared with control values. The NA deficit most likely reflects a degeneration of the locus caeruleus noradrenergic system that is known to occur in some patients with OPCA. The relationship between the altered cerebellar NA levels and the clinical symptomatology of OPCA remains to be determined.
Effects of Various Forms of Stimulation on the Content of Enolase Isozymes and S‐100 Protein in Superior Cervical Sympathetic Ganglia Excised from Rats
Authors: Nagata Yutaka , Ando Masato , Miwa Masato , Kato Kanefusa
Published: October 2006

Abstract: Contents of the three forms (αα, αγ, and γγ) of enolase isozymes and S‐100 protein in superior cervical sympathetic ganglia (SCG) excised from rats were determined by the sensitive method of enzyme immunoassay, after application of various forms of stimulation, during incubation for 3 h at 37°C in vitro. The amounts of the three forms of enolase isozymes and of S‐100 protein in the SCG were not altered by preganglionic or postganglionic stimulation (10 Hz) or by the addition of acetylcholine (1 mM) or a high concentration of K+ (70 mM) to the incubation medium. Norepinephrine (NE; 50 μM), as well as isoproterenol (200 μM) or 3,4‐dihydroxy phenylethylamine (dopamine; 200 μM), increased the ganglionic αα and αγ enolase content to 1.5 to 2.0 times the control level, whereas NE tended to slightly decrease the γγ enolase content. The increase in the α isozymes did not appear until after 2 to 3 h of incubation with this agent as a result of an increase in protein synthesis. Propranolol, an adrenergic antagonist, partly inhibited the NE‐induced increase in both αα and αγ enolases. NE and its agonists also considerably increased the S‐100 protein level in the SCG; however, the effect developed within half an hour of incubation as a result of the conversion of the bound S‐100 protein to the water‐soluble form, and did not greatly increase thereafter. Cyclic AMP (1 mM) produced the same kind of increase in the ganglionic S‐100 protein content as NE did. Propranolol, as well as cycloheximide, completely suppressed the NE‐evoked increase in S‐100 protein during 3 h incubation by inducing leakage of the protein into the medium. Axotomy caused greater NE‐induced increases in both act and αγ enolases and a greater decrease in γγ enolase in the SCG than denervation did, especially if examined 1 or 4 days after the operation. Axotomy alone also produced a delayed rise in NE‐induced ganglionic S‐100 protein concentration 7 days after the operation. These results suggest that there are no interconversions between αγ enolase and the αα or γγ forms in response to cholinergic stimulation or cell depolarization in the SCG. Instead, they suggest that NE acts on nonneuronal, glial rather than on neuronal components by increasing the biosynthesis of the α isozymes of enolase proteins, and that NE increases S‐100 protein content in nonneuronal, satellite cells through a receptor‐mediated, cyclic AMP‐dependent solubilization of the bound protein.
Effect of Monocular Deprivation on Uptake and Binding of [3H]Glutamate in the Visual System of Rat Brain
Authors: Schliebs Reinhard , Kunert Erich , Bigl Volker
Published: October 2006

Abstract: [3H]Glutamate uptake and binding studies were performed in the visual cortices, lateral geniculate nuclei (LGN), and superior colliculi of 3‐month‐old rats with one eyelid surgically closed from postnatal day 10 (monocular deprivation). Uptake and binding were highest in the lateral geniculate nucleus followed by the visual cortex (69% and 15%, respectively compared to LGN values) and the superior colliculus (32% and 59% of LGN values). Monocular deprivation did not affect [3H]glutamate uptake in any of the visual regions examined. However, a 46% decrease in [3H]glutamate binding in the lateral geniculate nucleus ipsilateral to the sutured eye was detected. Binding levels in other regions were not affected.
Mass Spectrometric Analyses of Brain Synaptic Peptides Containing Taurine
Authors: Marnela Kirsi‐Marja , Timonen Maritta , Lähdesmäki Pekka
Published: October 2006

The structure of a number of low‐molecular‐weight acidic peptides containing taurine prepared from calf brain synaptosomes and their subcellular vesicles was studied using electron impact mass spectrometry. At least seven sequences could be identified: N‐acetylas‐partyl‐glutamyl‐taurine, N‐acetylaspartyl‐taurine, N‐ace‐tylglutamyl‐taurine, glutamyl‐taurine, aspartyl‐taurine, seryl‐glutamyl‐seryl‐taurine, and seryl‐taurine.
Concentrations of Homovanillic Acid and 5‐Hydroxyindoleacetic Acid in Cerebrospinal Fluid from Human Infants in the Perinatal Period
Authors: Silverstein Faye S. , Donn Steven , Buchanan Karen , Johnston Michael V.
Published: October 2006

To assess maturation of central serotonin and catecholamine pathways at birth, we measured lumbar CSF homovanillic acid (HVA) and 5‐hydroxyindoleacetic acid (5‐HIAA), stable acid metabolites of dopamine and serotonin, using HPLC with electrochemical detection. CSFs from 57 neonates (38 premature and 19 at term) and 13 infants 1–6 months old were studied. HVA levels increased with maturity (p < 0.05; ANOVA), whereas, 5‐HIAA levels were similar in all these subjects. HVA/5‐HIAA ratios increased markedly from 1 ± 0.12 in the most premature neonates to 1.98 ± 0.17 in the older infants (p < 0.01; t test). There were no sex differences for these values.
5‐Hydroxytryptophol in Human Cerebrospinal Fluid: Conjugation, Concentration Gradient, Relationship to 5‐Hydroxyindoleacetic Acid, and Influence of Hereditary Factors
Authors: Beck Olof , Borg Stefan , Edman Gunnar , Fyrö Bengt , Oxenstierna Gabriella , Sedvall Göran
Published: October 2006

Abstract: The serotonin metabolite 5‐hydroxyiryptophol was studied in human cerebrospinal fluid. A minor fraction (∼13%) was found in conjugated form from which it was liberated by treatment with sulphatase containing 3‐ glucuronidase activity. A concentration gradient of 5‐hydroxytryptophol concentration was shown on lumbar tapping and the concentration in ventricular CSF was about 2.5 times higher than that in lumbar CSF. 5‐Hydroxytryptophol and 5‐hydroxyindoleacetic acid concentrations were significantly correlated in healthy, psychotic, and depressed subjects, but not in alcoholics. 5‐Hydroxytryptophol concentrations in CSF of psychotic and depressed subjects were not different from those of healthy controls (4.22 pmol/ml ± 0.15, SEM). In healthy subjects, hereditary factors seemed to have little influence on the CSF level of 5‐hydroxytryptophol.
Dopamine Receptor Parameters Detected by [3H]Spiperone Depend on Tissue Concentration: Analysis and Examples
Authors: Seeman Philip , Ulpian Carla , Wreggett Keith A. , Wells James W.
Published: October 2006

The binding of lipophilic radioligands to homogenized tissue was investigated with the help of a simple, two‐component model: a specific component reflects binding to a single and uniform population of sites; a nonspecific component reflects partitioning into the membrane and the entrapment of some drug present in the aqueous phase prior to separation of the pariculate fraction. The results indicate that the capacity and the affinity of the receptor may be underestimated when the data are analyzed in terms of total rather than free radioligand. Errors in capacity arise when for a significant fraction of the radioligand access to the receptor is blocked by an unlabelled drug and this appears as nonspecific binding. This is most likely to occur when the partition coefficient is such that the free radioligand is located pre‐dominantly in the particulate phase. Errors in affinity reflect the tendency of the membrane to reduce the free concentration of a lipophilic drug in the aqueous phase. A further complication arises when a significant fraction of the total radioligand binds to the receptor. [3H]Spiperone binds to dopamine D2 receptors with a dissociation constant of about 50 pM and partitions into the particulate phase of brain homogenates with a membrane/buffer partition coefficient of 410. As expected, both capacity and affinity can appear to depend on the concentration of tissue used in the assay. If the partition coefficient is known, corrected estimates of both parameters can be obtained knowing only the total concentration of radioligand; if the partition coefficient is not known, the free concentration of radioligand in the aqueous phase must be measured independently. The former procedure requires that the aqueous and particulate components of the system be separated by centrifugation; with filtration, the removal of an indeterminate amount of radioligand from the membrane during washing precludes any correction based on the partition coefficient. For the specific example of [3H]spiperone in human brain, the artifacts become negligible at concentrations of protein below 0.1 mg/ml of incubate. The capacity per unit of original tissue is best determined using unwashed preparations, since about 30% of the total protein and a comparable percentage of the receptors are lost on washing.
Effect of Lesions of the Olfactory Bulb on the Levels of Amino Acids and Related Enzymes in the Olfactory Cortex of the Guinea Pig
Authors: Sandberg M. , Bradford H. F. , Richards C. D.
Published: October 2006

Abstract: Olfactory bulb removal and consequential degeneration of the lateral olfactory tract led to a decreasein the levels of glutaminase and malate dehydrogenase inthe ipsilateral olfactory cortex. These changes in enzyme activity may account for the well established decrease inthe levels of aspartate and glutamate in the olfactory cortex following ipsilateral bulbectomy. The level of glutamine synthetase, a glial marker enzyme, was slightly‐increased while the activities of glutamate decarboxylase, glutamate dehydrogenase, and glutamate oxaloacetic transaminase were unchanged.
Assessment of Noradrenaline and 5‐Hydroxytryptamine Uptake in Intact Rat Brain
Authors: Wyllie M. G. , Wood M. D. , Moser P. C.
Published: October 2006

Abstract: A method for the measurement of 3H‐monoamine [noradrenaline (NA) and 5‐hydroxytryptamine (5‐HT)] uptake into intact brain following intracerebroventricular injection is described. Most of the accumulated monoamine was associated with nerve terminals (synaptosomes). Radioactivity in the synaptosomal fraction was retained by 0.45‐μm filters and was osmotically sensitive, features indicative of accumulation within particles rather than binding to membranes. [3H]5‐HT associated with synaptosomes was reduced in animals pretreated with the neurotoxin 5,7‐dihydroxytryptamine, in parallel with a reduction in endogenous 5‐HT levels. Oral administration of tricyclic antidepressants including clomipramine, desipramine, and imipramine inhibited the synaptosomal accumulation of 3H‐monoamines. Overall, the features of the accumulation of 3H‐monoamines, after intracerebroventricular administration, displayed many of the characteristics expected of a physiological monoamine reuptake system.
Dopamine Sulfoconjugation in the Rat Brain: Regulation by Monoamine Oxidase
Authors: Buu Nguyen T.
Published: October 2006

Abstract: An increase of free 3,4‐dihydroxyphenylethylamine (DA, dopamine) in the rat brain such as is found following 3,4‐dihydroxyphenylalanine (L‐DOPA) administration or an intraventricular injection of free dopamine did not result in DA sulfate formation, despite the presence of phenolsulfotransferase activity in various regions of the brain and the high affinity of DA for this enzyme. However, when rats were pretreated with pargyline, a monoamine oxidase inhibitor, the same treatment with L‐DOPA or free DA led to active synthesis of DA sulfate. The increase in DA sulfate was significantly correlated with the degree of monoamine oxidase inhibition and directly proportional to free DA concentrations in the hypothalamus (r = 0.86), striatum (r = 0.54), and brain‐stem (r = 0.89). The highest ratio of DA sulfate to free DA was found in the hypothalamus, suggesting that sulfoconjugation is most active in this region. Prior treatment of rats with 6‐hydroxydopamine did not decrease DA sulfate concentrations, indicating that sulfoconjugation occurs most likely in extraneuronal tissues not destroyed by the neurotoxin. The results are compatible with the notion that phenolsulfotransferase may be highly compartmentalized and that inhibition of monoamine oxidase allows the newly generated free DA to become accessible to the sulfoconjugating enzyme, resulting in increase in DA sulfation.
Inhibition of Calmodulin‐Stimulated Phosphodiesterase Activity by Vasoactive Intestinal Peptide
Authors: Barnette Mary S. , Weiss Benjamin
Published: October 2006

Abstract: The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin‐dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 μM and 2 μM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 μM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 μM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.
A D‐Glucose‐Sensitive Cytochalasin B Binding Component of Cerebral Microvessels
Authors: Baldwin S. A. , Cairns M. T. , Gardiner R. M. , Ruggier R.
Published: October 2006

Abstract: The technique of photoaffinity labelling with [4‐3H]cytochalasin B was applied to osmotically lysed cerebral microvessels isolated from sheep brain. Cytochalasin B was photo‐incorporated into a membrane protein of average apparent Mr 53,000. Incorporation of cytochalasin B was inhibited by D‐glucose, but not by L‐glucose, which strongly suggests that the labelled protein is, or is a component of, the glucose transporter of the blood–brain barrier. Investigation of noncovalent [4‐3H]cytochalasin B binding to cerebral microvessels by equilibrium dialysis indicated the presence of a single set of high‐affinity binding sites with an association constant of 9.8 ± 1.7 (SE) μM−1. This noncovalent binding was inhibited by D‐glucose, with a Ki of 23 mM. These results provide preliminary identification of the glucose transporter of the ovine blood–brain barrier, and reveal both structural and functional similarities to the glucose transport protein of the human erythrocyte.
Existence of Sites for Anions and Divalent Cations in the Solubilized γ‐Aminobutyric Acid/Benzodiazepine Receptor Complex
Authors: Gavish M. , Awad M. , Fares F.
Published: October 2006

Abstract: This study evaluated the ability of γ‐aminobutyric acid (GABA), baclofen, monovalent anions, divalent cations, and various combinations thereof to protect solubilized benzodiazepine (BZ) receptors of types 1 and 2, when contained together on the complex, against heat inactivation. Neither anions, cations, nor GABA alone provided significant protection of solubilized BZ receptors against heat, but inclusion of monovalent anions or divalent cations together with 500 μM GABA did afford protection. Monovalent anions combined with GABA (500 μM) provided 50% to full protection. Divalent cations, such as CaCl2 (2.5 mM) or MgCl2 (2.5 mM) in the presence of GABA (500 μM) yielded 45% and 24% protection, respectively. Other divalent cations tested (Zn2+, Hg2+, Co2+, and Ni2+) were poor protectors, even when combined with GABA. Monovalent anions (200 mM NaCl) and divalent cations (5 mM CaCl2) when tested together provided no protection. Similarly, baclofen (the GABA‐B agonist) provided no protection, either alone or together with anions or divalent cations. These results indicate that the independent but interacting recognition sites of GABA, BZ, anions, and divalent cations, previously detected in the membrane‐bound state, are retained in the solubilized state.
CCK26–33 Degrading Activity in Brain and Nonneural Tissue: A Metalloendopeptidase
Authors: Steardo L. , Knight M. , Tamminga C. A. , Barone P. , Kask A. M. , Chase T. N.
Published: October 2006

Abstract: Cholecystokinin octapeptide (CCK26–33) is metabolized by neural membranes with an initial cleavage to CCK29–33 and subsequent breakdown to CCK31–33 and CCK32–33; this pattern of proteolysis occurs on incubation with either P2 or purified lysed synaptosomal membranes. To determine whether the pattern of CCK26–33 proteolysis is unique to the brain and whether regional brain differences in its pathway or rate exist, we analyzed the proteolysis of CCK by synaptic membranes of various brain areas and cellular membranes of peripheral tissue. The pattern of degradation in brain did not differ among the regions studied. The overall proteolysis rate, as measured by the formation of tryptophan, was higher in the striatum than in the cortex, although CCK29–33 was formed at the same rate in both areas. In nonneural tissue, the rate of degradation was highest in liver membranes and lowest in pancreatic acinar cell preparations. Thus, it appears that degradative peptidases are not necessarily colocalized with CCK receptors. The pattern of product formation is the same in peripheral compared with CNS membranes; thus, the degradative pathway does not appear to be unique to brain tissue. The enzyme present in synaptic membranes that is responsible for CCK29–33 formation requires a metal ion and sulfydryl groups for the catalysis and thus is a metalloendopeptidase. Furthermore, its activity is inhibited by Ac‐Gly‐Phe‐Nle‐al, a peptide aldehyde whose sequence bears some homology to the amino acid sequence in the region of CCK26–33 that is cleaved by this enzyme.
Quinolinic Acid Phosphoribosyltransferase in Rat Brain
Authors: Foster Alan C. , Zinkand William C. , Schwarcz Robert
Published: October 2006

Abstract: Because of the possible participation of quinolinic acid in brain function and/or dysfunction, the characteristics of its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRTase; EC 2.4.2.19), were examined in rat brain tissue. For this purpose, a sensitive radiochemical assay method, based on the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN), was developed. For brain QPRTase, the Mg2+ dependency, substrate specificity, and optimal assay conditions were virtually identical to those of the liver enzyme. Kinetic analyses of brain QPRTase revealed a Km of 3.17 ± 0.30 μM for quinolinic acid and Km= 65.13 ± 13.74 μM for the cosubstrate phosphoribosylpyrophosphate. The respective Vmax values were: 0.91 ± 0.08 pmol NAMN/h/mg tissue for quinolinic acid and 11.65 ± 1.55 fmol NAMN/h/mg tissue for phosphoribosylpyrophosphate. All kinetic parameters measured for the brain enzyme were significantly different from those determined for liver QPRTase, indicating structural differences or distinct regulatory processes for the brain and liver enzymes. Phthalic acid was a potent competitive inhibitor of brain QPRTase. Examination of the regional distribution of QPRTase in the rat CNS and retina indicated a >20‐fold difference between the area displaying the highest activity (olfactory bulb) and those of only moderate activity (frontal cortex, striatum, retina, hippocampus). Enzyme activity was present at the earliest age tested, 2 days, and tended to increase in older animals. Brain QPRTase activity was preferentially located in the nerve‐ending (synaptosomal) fraction. Enzyme activity was stable over extensive periods of storage at –80°C. These data suggest that further investigations of brain QPRTase (including studies with human brain material) may reveal clues as to the role of quinolinic acid in normal and pathologic brain.
Enzyme‐Linked Immunosorbent Assay for the Human Glial Fibrillary Acidic Protein Using a Mouse Monoclonal Antibody
Authors: Albrechtsen Merete , Massaro Angelo , Bock Elisabeth
Published: October 2006

Abstract: Two enzyme‐linked immunosorbent assays (ELISAs) have been developed for the quantification of soluble human glial fibrillary acidic protein (GFAP). The specificity of the assays for GFAP is ensured by the use of a monoclonal antibody directed against a GFAP‐specific antigenic determinant. One ELISA is a four‐layer system working in the concentration range 5–600 ng GFAP/ml. The other ELISA is a five‐layer system and includes a biotin/avidin binding reaction. The latter assay has a working range of 0.5‐60 ng GFAP/ml. The assays may be used for quantification of GFAP in CSFs, amniotic fluids, and extracts or homogenates of normal and pathological brain material. GFAP in serum could not be quantified because of unidentified interference. CSFs from 18 nonneurological subjects were found to contain 2–14 ng GFAP/ml (mean 4.1 ng/ml), whereas amniotic fluids from 50 normal pregnant women contained up to 24 ng GFAP/ml (mean 12.4 ng/ml). GFAP concentrations in CSFs from 32 multiple sclerosis patients were found not to be elevated compared to the control group.
Immunoassay of the Neuronal and Neuroendocrine Marker PGP 9.5 in Human Tissues
Authors: Bradbury Jane M. , Thompson R. J.
Published: October 2006

Abstract: An inhibitory, coated‐well immunoassay for the neurone‐specific protein PGP 9.5 has been devised and used to measure the concentrations of the protein in human tissues. Concentrations of PGP 9.5 between 40 ng/ml and 10 μg/ml could be measured using this assay. In brain PGP 9.5 was present at 100.58 ± 16.18 μg/mg protein. Of the other organs examined only kidney and testis showed significant concentrations of PGP 9.5 (3.97 ± 0.87 μg/mg protein and 3.25 ± 0.36 μg/mg protein, respectively). All other organs contained <2% of the brain level. The tissue levels determined by coated‐well immunoassay confirmed the tissue specificity of PGP 9.5 originally determined by high‐resolution two‐dimensional gel electrophoresis.
Regulation of Cerebroside and Sulfatide Metabolism in Glia Cells
Authors: Jungalwala Firoze B. , Koul Omanand , Stoolmiller Allan , Sapirstein Victor S.
Published: October 2006

Abstract: Mouse oligodendroglioma cells, G‐26 clone 20 and 24, contain galactosylceramide (cerebroside) and sul‐fogalactosylceramide (sulfatide) as determined by an HPLC technique. The synthesis of both these lipids was stimulated by 10−‐6M hydrocortisone (cortisol) and also by the removal of serum from the culture medium. Forty‐eight hours after the addition of cortisol the incorporation of H235SO4 into sulfatide, the level of sulfatide and the specific activity of the enzyme 3′‐phosphoadenosine 5′‐phosphosulfate:galactosylceramide sulfotransferase in the cells increased three‐ to fourfold. The level of cerebroside and the specific activity of UDP‐galactose: hydroxyacyl sphingosine galactosyltransferase also increased threefold in the cells on treatment with cortisol. The effect of the hormone on the synthesis of cerebroside preceded the increase in sulfatide synthesis. Experiments with cycloheximide and actinomycin D showed that the effect of the hormone on glycolipid synthesis in these cells was mediated through de novo messenger RNA and protein synthesis. Removal of serum from the culture medium resulted in an approximately twofold enhancement of H235SO4 incorporation into sulfatide within 24 h. The levels of sulfatide and cerebroside and the specific activity of the galactosyltransferase and sulfotransferase also increased significantly after serum removal. However, in contrast to the effect of the steroid, the sulfotransferase activity and the level of sulfatide increased prior to elevations in galactosyltransferase and cerebroside. The effect of serum removal was also found to be mediated by de novo RNA and protein synthesis. The effects of cortisol and serum removal on the synthesis of cerebroside and sulfatide were strictly additive. This observation plus the different temporal patterns of induction suggest that two separate regulatory mechanisms at the nuclear level exist in oligodendroglioma cells for the biosynthesis of these glycolipids.