bluuebunny/crossref_metadata_2025 huggingface dataset:
Large‐Scale, One‐Step Purification of Oxidized and Reduced Forms of Bovine Brain S100b Protein by HPLC
Authors: Mely Y. , Gérard D.
Published: October 2006

A rapid and simple method, using a reverse‐phase column in a HPLC system, has been developed to purify high yields of both oxidized and reduced S100b proteins from a bovine brain S100 protein mixture. The final proteins were characterized by amino acid analysis, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and ab‐sorbance and fluorescence spectroscopy. Both S100b subtypes appeared highly purified and differed only by their oxidation state: all four cysteinyl sulfhydryl groups were free in reduced S100b protein whereas two of them gave disulfide bridges in oxidized S100b protein. The stability of the oxidation state of the two isolated subtypes suggests that the two forms were not in rapid equilibrium and probably coexisted in vivo.
Protein Turnover and Growth of the Rat Brain from the Foetus to Old Age
Authors: Goldspink David F.
Published: October 2006

Abstract: Growth of the rat brain was studied between 16 days of foetal life and old age (105 weeks). Developmental changes in cerebral RNA, DNA, and protein contents are described. The age‐related decline in brain growth rates correlates with progressive decreases in the fractional rates of protein synthesis (from 58 to 6.8% per day) and break down (from 36.4 to 4.1% per day).
Utilization of the Synthetic Phosphagen Cyclocreatine Phosphate by a Simple Brain Model During Stimulation by Neuroexcitatory Amino Acids
Authors: Woznicki Dennis T. , Walker James B.
Published: October 2006

Abstract: The ability of 1‐carboxymethyl‐2‐imino‐3‐phosphonoimidazolidine (cyclocreatine‐P), accumulated by a simple brain model, to function as a supplemental synthetic phosphagen and respond to the decreases in cytosolic ATP/free ADP ratios that occur during prolonged stimulation by various excitatory amino acids was investigated. Suspensions of chopped whole brain from 11‐ to 14‐day‐old chick embryos were incubated with 30 mM cyclocreatine for 90 min, resulting in accumulation of 100 μmol/g dry weight of cyclocreatine‐P, and then incubated for up to 1 h with a series of excitatory amino acids of widely differing potencies. Under these conditions net utilization of cyclocreatine‐P was detected in response to stimulation by the following neuroexcitatory compounds at the indicated threshold concentrations: kainate (20 μM), N‐methyl‐dl‐aspartate (20 μM), l‐homocysteate (20 μM), l‐glutamate (200 μM), d‐glutamate (200 μM), l‐aspartate (2 mM), dl‐2‐amino‐3‐phosphonopropionate (2 mM), and dl‐2‐amino‐4‐phosphonobutyrate (2 mM). Significant increases in water content of chick embryo brain minces accompanied stimulation by excitatory amino acids. It is suggested that changes in water content or cyclocreatine‐P levels in this sensitive brain model might be utilized in automatable screening procedures for detecting novel antagonists and/or new agonists of excitatory amino acids.
Pharmacological and Biochemical Characterization of Rat Hippocampal 5‐Hydroxytryptamine1A Receptors Solubilized by 3–[3–(Cholamidopropyl)dimethylammonio]‐1‐Propane Sulfonate (CHAPS)
Authors: El Mestikawy S. , Cognard C. , Gozlan H. , Hamon M.
Published: October 2006

Abstract: Rat hippocampal 5‐hydroxytryptamine1A (5‐HT1A) binding sites were solubilized with a yield of 34% using 3–[3–(cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8‐hydroxy‐2–(di‐n‐propylamino)tetralin ([3H]8‐OH‐DPAT) binding indicated that the 5‐HT1A sites exhibit the same properties in the soluble form as in the membrane‐bound form. Furthermore, a positive correlation (r= 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8‐OH‐DPAT binding to either soluble or membrane‐bound 5‐HT1A sites. Gel filtration through Sephacryl S‐400 as well as chromatography on wheat germ agglutinin (WGA)‐agarose did not affect the modulation by guanine nucleotides (5′‐guanylylimidodi‐phosphate) of [3H]8‐OH‐DPAT binding which suggests that the 5‐HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8‐OH‐DPAT binding material eluted from Sephacryl S‐400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (∼60 kilodaltons) and one G protein (∼80 kilodaltons). Marked enrichment in 5‐HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S‐400 (by sixfold) and WGA‐agarose (by 26‐fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5–HT1A receptors.
Neuropathy Target Esterase: Rates of Turnover In Vivo Following Covalent Inhibition with Phenyl Di‐n‐Pentylphosphinate
Authors: Meredith Clive , Johnson Martin K.
Published: October 2006

Abstract: Phenyl di‐n‐pentylphosphinate is a reasonably stable easily synthesized inhibitor of neuropathy target esterase (NTE) with low anticholinesterase activity. Like phenyl‐methylsulphonyl fluoride it protects hens against neuropathic effects of compounds such as diisopropylphosphorofluoridate. At intervals up to 15 days after dosing hens (10 mg/kg s.c. to inhibit 90% NTE) assays were made of catalytically active and of phosphinylated NTE in autopsy tissue. The sum of these components was always within the range of catalytic activity in undosed controls. However, the half‐life of reappearance of active NTE was 2.07 days ±0.13 (SD, n = 6) for brain and 3.62 days ± 0.23 (SD, n = 6) for spinal cord‐shorter than after dosing with phenylmethylsulphonyl fluoride. It is proposed that: (1) The physiological turnover mechanism cannot distinguish between catalytically active and di‐n‐pentylphosphinylated NTE although initiation of organophosphate‐induced delayed polyneuropathy might involve recognition of aged di‐alkyl‐phosphorylated NTE as “foreign”. (2) The short half‐lives indicate a slow spontaneous dephosphinylation of inhibited NTE occurs in vivo as well as de novo synthesis. The difference in half‐lives for brain and spinal cord NTE may be due to different rates of synthesis de novo or (more likely) to different rates of spontaneous reactivation of the inhibited NTE in the two tissues.
Differential Response of Adenylate Cyclase and ATPase Activities After Chronic Ethanol Exposure of PC12 Cells
Authors: Rabin Richard A.
Published: October 2006

Abstract: The direct effects of chronic ethanol administration on adenylate cyclase, Na,K‐ATPase, and Mg‐ATPase activities in a cell containing neuronal characteristics were investigated using PC 12 pheochromocytoma cells. Exposure of PC12 cells to0, 75, and 150 mM ethanol for 4 days caused a dose‐dependent increase in the stimulation of adenylate cyclase by in vitro ethanol without altering activation of the enzyme by GTP, NaF, MnCl2, or 2‐chloroadenosine. Conversely, a 4‐day treatment with 150 mM ethanol increased Na,K‐ATPase and Mg‐ATPase activities without altering the inhibitory effects of in vitro ethanol. The increase in Na,K‐ATPase activity was associated with an increase in Vmax without any change in the Km for KC1. Chronic ethanol exposure also increased the amount of [3H]ouabain specifically bound to PC 12 cell membranes. Except for the increase in Mg‐ATPase activity, the above results were also observed when chronic ethanol treatment was carried out in the presence of pyrazole. Although ethanol slowed PC 12 cell growth, observed changes were not due to an ethanol‐induced reduction in cellular density. A 4‐day exposure of a nonneuronal cell line (Madin Darby canine kidney cell) to 150 mM ethanol did not alter adenylate cyclase or ATPase activities. The present study indicates that the direct effects of chronic ethanol exposure of a neuronal‐like cell involve an increase in the density of sodium pumps per cell and an enhanced sensitivity of adenylate cyclase to activation by ethanol.
Effects of Protonophores on the Synthesis of Catecholamines and the Intracellular pH in Cultured Bovine Adrenal Medullary Cells
Authors: Yokota Kenji , Yanagihara Nobuyuki , Izumi Futoshi , Wada Akihiko
Published: October 2006

The protonophores carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m‐chlorophenylhydrazone (CCCP) stimulated the synthesis of 14C‐catecholamines from [14C]tyrosine in cultured bovine adrenal medullary cells. The stimulatory effect of CCCP but not of FCCP was partially dependent on extracellular Ca2+. CCCP but not FCCP increased the influx of 45Ca2+ to the cells. When cells were incubated with either CCCP or FCCP (0.01–0.2 μg/ml), the intracellular pH fell from 7.2 to 6.3–6.5 and catecholamine synthesis increased. Tyrosine hydroxylase activity in a soluble fraction prepared from cultured adrenal medullary cells was measured after incubation of the cells with FCCP or CCCP. Although FCCP did not affect the activity of the enzyme, CCCP caused a stable activation of it which was dependent on extracellular Ca2+. Since the optimal pH of soluble tyrosine hydroxylase is around 6.0 in adrenal medullary cells, FCCP may increase the synthesis of catecholamines by shifting the intracellular pH toward it. In addition to this mechanism, CCCP may enhance the synthesis of catecholamines by a Ca2+‐dependent mechanism.
Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms
Authors: Stieger S. , Bütikofer P. , Wiesmann U. N. , Brodbeck U.
Published: October 2006

Abstract The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell‐associated enzyme could be subdivided into soluble AChE (25%) and detergent‐soluble AChE (75%). Both extracts contained predominantly monomelic AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretary and the intracellular soluble tetramers were hydrophilic, whereas the detergent‐soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent‐soluble monomelic forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomelic and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate‐labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.
Optic Nerve Regeneration in Adult Fish and Apolipoprotein A‐I
Authors: Harel A. , Fainaru M. , Shafer Z. , Hernandez M. , Cohen A. , Schwartz M.
Published: October 2006

Abstract Fish optic nerves, unlike mammalian optic nerves, are endowed with a high capacity to regenerate. Injury to fish optic nerves causes pronounced changes in the composition of pulse‐labeled substances derived from the surrounding non‐neuronal cells. The most prominent of these injury‐induced changes is in a 28‐kilodalton (kDa) polypeptide whose level increases after injury, as revealed by one‐dimensional gel electrophoresis and autoradiography. The present study identified as apolipoprotein A‐I (apo‐A‐I) a polypeptide of 28 kDa in media conditioned by regenerating fish optic nerves. The level of this polypeptide increased after injury by approximately 35%. Apo‐A‐I was isolated by gel‐permeation chromatography from delipidated high‐density lipoproteins (HDL) that had been obtained from carp plasma by sequential ultracentrifugation. Further identification of the purified protein as apo‐A‐I was based on its molecular mass (28 kDa) as determined by gel electrophoresis, amino acid composition, and microheterogeneity studies. The isolated protein was further analyzed by immunoblots of two‐dimensional gels and was found to contain six isoforms. Western blot analysis using antibodies directed against the isolated plasma protein showed that the 28‐kDa polypeptide in the preparation of soluble substances derived from the fish optic nerves (conditioned media, CM) cross‐reacted immunologically with the isolated fish plasma apo‐A‐I. Immunoblots of two‐dimensional gels revealed the presence of three apo‐A‐I isoforms in the CM of regenerating fish optic nerves (pIs: 6.49, 6.64, and 6.73). At least some of the apo‐A‐I found in the CM is derived from the nerve, as was shown by pulse labeling with [35S]methionine, followed by immunoprecipitation. The apo‐A‐I immunoactive polypeptides in the CM of the fish optic nerve were found in high molecular‐weight, putative HDL‐like particles. Immunocytochemical staining revealed that apo‐A‐I immunoreactive sites were present in the fish optic nerves. Higher labeling was found in injured nerves (between the site of injury and the brain) than in non‐injured nerves. The accumulation of apo‐A‐I in nerves that are capable of regenerating may be similar to that of apo‐E in sciatic nerves of mammals (a regenerative system); in contrast, although its synthesis is increased, apo‐A‐I does not accumulate in avian optic nerves nor does apo‐E in rat optic nerves (two nonregenerative systems).
Species Distribution of Paraoxon‐Resistant Brain Polypeptides Radiolabeled with Diisopropyl Phosphorofluoridate ([3H]DiPF): Electrophoretic Assay for the Aged Polypeptide of [3H]DiPF‐Labelled Neuropathy Target Esterase
Authors: Meredith Clive , Johnson Martin K.
Published: October 2006

Abstract Brain neuropathy target esterase is identified as a paraoxon‐resistant, mipafox‐sensitive esterase that can be labelled with [3H]diisopropyl phosphorofluoridate. During “aging” of the labelled (inhibited) esterase, half the label (one isopropyl group) is transferred to a site (of the same molecular weight in sodium dodecyl sulphate) whence it may be released in volatile form by treatment with alkali. Our previously published procedure for complete extraction in a form suitable for scintillation counting of tritium‐labelled proteins from polyacrylamide gels includes treatment of part‐solubilised gels with alkali. Particles from brain of the hen, pig, sheep, guinea‐pig, and rat were preincubated with paraoxon with or without mipafox, treated with [3H]diisopropyl phosphorofluoridate, and solubilised in sodium dodecyl sulphate. Labelled polypeptides (except from the rat) were separated by electrophoresis. Both mipafox‐sensitive labelling and “volatilisable counts” were located principally in the 155‐kilodalton region, with the residues dispersed throughout the gels. The quantities of paraoxon‐resistant, mipafox‐sensitive labelling sites and of “volatilisable counts” (in pmol/particles from 1 g) were, respectively, 12.2 and 8.65 in hen brain, 9.80 and 6.82 in pig, 8.48 and 5.46 in sheep, 4.46 and 4.01 in guinea‐pig, and 4.91 and 2.08 in rat. The “volatilisable count” assay seems more specific for neuropathy target esterase and is easier and more precise than assays based on differences in labelling of two samples, each subjected to much processing. Hydrolytic activity of particles taken before labelling was measured against phenyl valerate. By comparison with the “volatilisable counts,” the catalytic centre activity of neuropathy target esterase of brain from these species was calculated to be (in the same order) 1.95, 1.95, 2.34, 1.82, and 2.78 ± 105 mol min‐1, respectively.
Effect of Isozyme‐Selective Inhibitors of Phosphodiesterase on Histamine‐Stimulated Cyclic AMP Accumulation in Guinea‐Pig Hippocampus
Authors: Stanley Clare , Brown Anthony M. , Hill Stephen J.
Published: October 2006

Abstract: Addition of histamine (0.1 mM) to guinea‐pig hippocampal slices causes a 20‐ to 30‐fold increase in the accumulation of cyclic AMP compared with basal levels. This accumulation represents a balance between cyclic AMP production by adenylate cyclase and cyclic AMP breakdown mediated by phosphodiesterase (PDE). However, brain tissues are known to contain several different PDE isozymes. To determine which are involved in this response to histamine, the effect of isozyme‐specific PDE inhibitors on cyclic AMP accumulation was examined in the hippocampus. MB 22948 (0.1 mM), an inhibitor of PDEs I and II, had no significant effect on the response to either 1 μM or 0.1 mM histamine. SKF 94120 (0.1 mM), a PDE III inhibitor, was also without effect in the presence of 1 μM histamine, although with 0.1 mM histamine, it caused a weak (1.25‐fold compared with control), but statistically significant, enhancement of cyclic AMP accumulation. However, both rolipram (0.1 mM), a PDE IV inhibitor, and 3‐isobutyl‐l‐methylxanthine (0.1 or 1 mM), an inhibitor of all forms of PDE, significantly increased cyclic AMP accumulation (2.8‐ to 6.5‐fold compared with controls), and the relative size of this effect decreased with increasing histamine concentration. It is concluded that PDE IV is the main PDE isozyme involved in cyclic AMP turnover in guinea‐pig hippocampal slices responding to histamine.
Comparison of the Properties of γ‐Aminobutyric Acid and L‐Glutamate Uptake into Synaptic Vesicles Isolated from Rat Brain
Authors: Fykse Else M. , Christensen Hege , Fonnum Frode
Published: October 2006

Abstract: Rat brain synaptic vesicles exhibit ATP‐dependent uptake of γ‐[3H]amino‐n‐butyric acid ([3H]GABA) and l‐[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both l‐glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of l‐glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondrial uncoupler carbonyl cyanide m‐chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K+ carrier valinomycin. N, N′‐Dicyclohexyl‐carbodiimide and N‐ethylmaleimide, Mg2+‐ATPase inhibitors, inhibited the GABA and L‐glutamate uptakes similarly. Low concentrations of CI− stimulated the vesicular uptake of l‐glutamate but not that of GABA. The uptakes of both l‐glutamate and GABA were inhibited by high concentrations of CI−. These results indicate that the vesicular GABA and l‐glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+‐ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems.
Hippocampal Membranes Contain a Neurotrophic Activity That Stimulates Cholinergic Properties of Fetal Rat Septal Neurons Cultured Under Serum‐Free Conditions
Authors: Emerit Michel B. , Segovia Jose , Alho Hannu , Mastrangelo Michael J. , Wise Bradley C.
Published: October 2006

Abstract: Primary cultures of fetal rat septal neurons were used to identify a membrane‐associated cholinergic neurotrophic activity. Under serum‐free culture conditions, ∼98% of the septal cells are neurons, and ∼6% of the neurons are cholinergic as determined immunocytochemically. Crude membranes prepared from rat hippocampal homogenates stimulate choline acetyltransferase (ChAT) activity in treated septal neurons. The membrane‐associated trophic activity is apparent at lower protein concentrations than activity present in the soluble fraction and is unevenly distributed in various brain regions; it is highest in hippocampus and striatum and negligible in cerebellum. Membrane trophic activity is developmentally regulated, is heat and trypsin sensitive, and increases the rate of expression of ChAT in septal neurons. Upon gel nitration chromatography of a high‐salt membrane extract, trophic activity elutes as a broad peak in the 500 kilodalton (kD) molecular mass range. Stimulation of septal neuronal ChAT activity by either crude membranes or partially purified preparations is not inhibited by antibodies against nerve growth factor (NGF), and its maximal activity is additive to maximally active doses of NGF. The results indicate that hippocampal membranes contain cholinergic neurotrophic activity which may be important for the development of septal cholinergic neurons.
Isolation and Characterization of Secretory Granules Storing a Vasoactive Intestinal Polypeptide‐Like Peptide in Torpedo Cholinergic Electromotor Neurones
Authors: Agoston Denes V. , Dowe Gordon H. C. , Whittaker Victor P.
Published: October 2006

Abstract: Previous immunocytochemical work showed that the cholinergic electromotor neurones of Torpedo marmorata contain a vasoactive intestinal polypeptide‐like immuno‐reactivity (VIPLI) that is conveyed to the terminals by axonal transport from the cell bodies where it is presumably synthesized. In extension of this work, we have now succeeded in isolating the VIPLI storage granules from both the terminals and the axons of these neurones and characterizing them morphologically and biochemically. They were readily separated from synaptic vesicles but contained several components in common that had previously been regarded as specific for synaptic vesicles. Among these were a heparan sulphate type of proteoglycan, synaptophysin, and a Mg2+‐dependent ATPase. The VIPLI concentration in lobe tissue and the amount of tissue available were both insufficient to permit the isolation of granules from the electromotor cell bodies by the same technique but it was possible to establish the presence of such granules by particle‐exclusion chromatography, using the stable markers mentioned above. In contrast to the VIPLI‐containing granules, axonal synaptic vesicles differed from their terminal counterparts in having a very low acetylcholine content relative to stable vesicle markers: they presumably fill up on reaching the terminal where they are exposed to higher concentrations of cytoplasmic acetylcholine.
Solubilization of Peripheral‐Type Benzodiazepine Binding Sites from Cat Cerebral Cortex
Authors: Awad M. , Gavish M.
Published: October 2006

Abstract: The present study demonstrates for the first time the solubilization of peripheral‐type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested {digitonin, 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X‐100 in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane‐bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 ± 0.2 and 1.9 ± 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 ± 150 and 980 ± 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 ± 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5–4864, diazepam, flunitrazepam, clonazepam, methyl‐β‐carboline‐3‐carboxylate, and Ro 15–1788) in displacing [3H]PK 11195 specific binding from membrane‐bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60°C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein‐binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4°C, which will enable future application of purification procedures without major concern for stability of the material.
Presence of Proteolipid Protein in Coelacanth Brain Myelin Demonstrates Tetrapod Affinities and Questions a Chondrichthyan Association
Authors: Waehneldt T. V. , Malotka J.
Published: October 2006

Abstract: The protein and glycoprotein compositions of CNS myelin from the living coelacanth (Latimeria chalumnae) were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. An unglycosylated component of 25 kilodaltons showed substantially stronger immunoblot reactivity with antibodies against mammalian proteolipid protein (PLP) than lungfish glycosylated PLP. DM‐20 (intermediate protein) was not detectable in either fish. The presence of unglycosylated PLP in CNS myelin of the actinistian coelacanth contradicts an association with cartilaginous fishes but supports tetrapod affinities closer than those of lungfish.
Regional Dissociation of Cyclic AMP and Inositol Phosphate Formation in Response to Thyrotropin‐Releasing Hormone in the Rat Brain
Authors: Iriuchijima Tokuji , Mori Masatomo
Published: October 2006

Abstract: The present study was undertaken to define effects of thyrotropin‐releasing hormone (TRH) on formation of cyclic AMP (cAMP) and inositol phosphates (IPs) in rat brain regions. The brain of male Wistar rats was dissected into seven discrete regions, and each region was sliced. The slices were incubated in Krebs‐Henseleit glucose buffer containing varying doses of TRH. TRH caused a significant and consistent increase in cAMP level, but not in formation of IPs, in the hypothalamus, striatum, and midbrain. TRH stimulated formation of IPs in the cerebellum, where the tripeptide did not change the cAMP level. In contrast, formation of neither cAMP nor IPs was affected by TRH in the cortex, hippocampus, or pons‐medulla. These data suggest that TRH possesses two distinct types of brain intracellular signaling systems, which vary with brain regions.
p‐Chloromercuribenzoate Causes Ca2+‐Dependent Exocytotic Catecholamine Secretion from Cultured Bovine Adrenal Medullary Cells
Authors: Tachikawa Eiichi , Takahashi Saburo , Kashimoto Takeshi
Published: October 2006

Abstract: Incubation of cultured bovine adrenal medullary cells with p‐chloromercuribenzoate (50–500 μM), a sulfhy‐dryl‐reacting agent, caused an increase in the secretion of catecholamines. p‐Chloromercuriphenyl sulfonate, a p‐chlo‐romercuribenzoate analogue that poorly penetrates the cell membrane, caused a similar increase in catecholamine secretion. In both cases, catecholamine secretion was dependent on extracellular Ca2+. Furthermore, p‐chloromercuribenzoate caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to p‐chloromercuribenzoate concentration. The time courses of the increased secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration by p‐chloromercuri‐benzoate were also quite similar. The stimulation of cate‐holamine secretion by p‐chloromercuribenzoate was reversed by washing the cells with dithiothreitol‐containing medium, but not by dithiothreitol‐free medium. When the cells were treated with p‐chloromercuribenzoate, dopamineβ‐hydroxylase, an enzyme present in the chromaffin granules along with catecholamines, was also released. However, p‐chloromercuribenzoate did not cause release of phenyletha‐nolamine‐JV‐methyltransferase, an enzyme present in the cytoplasm. These results indicate that catecholamine secretion due to p‐chloromercuribenzoate occurs by Ca2+‐dependent exocytosis.
Phosphorylated Thiamine Derivatives and Cortical Activity in the Baboon Papio papio: Effect of Intermittent Light Stimulation
Authors: Bettendorff L. , Schoffeniels E. , Naquet R. , Silva‐Barrat C. , Riche D. , Ménini C.
Published: October 2006

Abstract: The effect of intermittent light stimulation (ILS) on the distribution of thiamine derivatives in three brain areas (occipital, motor, and premotor) was compared in photosensitive and nonphotosensitive baboons. ILS induces paroxysmal discharges in the motor and premotor areas of photosensitive animals only. In baboons submitted to ILS, thiamine triphosphate (TTP) decreases in both photosensitive and nonphotosensitive animals; thiamine monophosphate (TMP) increases in photosensitive animals, which present ILS‐induced paroxysmal discharges, whereas it is unaffected in nonphotosensitive animals. The variations are the most significant in the occipital (visual) cortex. A consumption of TTP may result from electrical activity induced by light stimulation in the occipital area. No correlation between ILS‐induced paroxysmal activity and a decrease in TTP contents was found. However, photosensitive animals are affected differently from nonphotosensitive animals, as their content of TMP in the cerebral cortex increases on stimulation. However, as long as the exact role of thiamine compounds in relation to membrane excitability in the nervous system remains unknown, it is impossible to conclude whether the differences observed in the metabolism of thiamine compounds are the cause or the consequence of the photosensitivity in the baboon Papio papio.
Purification and Characterization of Prosomatostatin from Rat Brain
Authors: Strieder Georg , Sperk Giinther
Published: October 2006

Abstract: A high molecular weight somatostatin‐immuno‐reactive polypeptide, presumably prosomatostatin, was purified from rat brain and characterized. Purification steps included extraction with 2 M acetic acid, precipitation of contaminating proteins at pH 6.5, Sephadex G‐50 chromatography, immunoaffinity chromatography, and HPLC steps (size exclusion and reversed‐phase HPLC). The protein was purified more than 30,000‐fold. It is heat stable. Sodium do‐decyl sulfate‐gel electrophoresis and immunoblotting revealed one major immunoreactive band of ∼ 13,000 molecular weight which roughly corresponds to the size of prosomatostatin as derived from its DNA sequence. Isoelectric focusing and two‐dimensional sodium dodecyl sulfate‐gel electrophoresis gave a single immunoreactive spot at a pI of 5.4. The polypeptide did not bind to concanavalin A or to wheat germ lectin columns, suggesting lack of N‐glycosylation in the molecule. Regional distribution of prosomatostatin varied between 6%, 10%, and 18% of total immunoreactivity in the brainstem, cortical areas, and striatum, respectively.
Selective Visualization of Rodent Locus Ceruleus by a Radiolabeled N‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Analog
Authors: Efange S. M. N. , Mash D. , Hefti F. , Kung H. F. , Billings J.
Published: October 2006

Abstract: The neurotoxic metabolite of N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), 1 ‐methyl‐4‐phenylpyr‐idinium, selectively accumulates in dopaminergic neurons via the dopamine reuptake system. Consequently, nontoxic radiolabeled MPTP analogs may be potentially useful for visualizing catecholaminergic neurons in vivo. N‐Methyl‐4‐(4 ‐ hydroxy ‐ 3 ‐ [l25I]iodobenzyl) ‐1,2,3,6 ‐ tetrahydropyridine ([l25I]MHTP), an analog of the nontoxic N‐methyl‐4‐benzyl‐1,2,3,6‐tetrahydropyridine, has been studied in rats and mice. After intravenous administration of [125I]MHTP to rodents, the initial accumulation of radioactivity within the brain was found to be comparable to that of radiolabeled MPTP. Following intravenous administration of [125I]MHTP, in vivo autoradiographic visualization of the rodent brain revealed selective accumulation of [I25I]MHTP‐derived radioactivity within the locus ceruleus; there was no accumulation of the radiotracer within dopaminergic fibers and cell bodies. The accumulation of radioactivity within the locus ceruleus was blocked by pretreatment with pargyline, a result suggesting that an MHTP metabolite formed by monoamine oxidase was responsible for the localization of the radiotracer within this structure. The anatomical distribution of the radiolabel demonstrates selective accumulation of this metabolite within noradrenergic cell bodies and those fibers making up the locus ceruleus. These findings further suggest that nontoxic metabolites of MPTP may become useful for in vivo labeling of selected populations of catecholaminergic neurons.
Subcellular Distribution of 65,000 Calmodulin‐Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla
Authors: Fournier S. , Novas M. L. , Trifaró J.‐M.
Published: October 2006

Abstract: Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense‐core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin‐binding protein (65‐CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65‐CMBP and synaptophysin). Subsequent im‐munoblotting and 125I‐Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65‐CMBP was present in fractions corresponding t o granule membranes and intact chromaffin granules. Similar immunoblotting and 125I‐Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti‐synaptophysin and high 125I‐Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65‐CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65‐CMBP and synaptophysin in the chromaffin cell.
Antibodies to a Synthetic Peptide Corresponding to a Ser‐40‐Containing Segment of Tyrosine Hydroxylase: Activation and Immunohistochemical Localization of Tyrosine Hydroxylase
Authors: Lee K. Y. , Lew J. Y. , Tang D. , Schlesinger D. H. , Deutch A. Y. , Goldstein M.
Published: October 2006

Abstract: A peptide corresponding to position 32‐47 in tyrosine hydroxylase was synthesized (TH‐16) and polyclonal antibodies against this peptide were raised in rabbits (anti‐TH‐16). The effects of anti‐TH‐16 on modulation of tyrosine hydroxylase activity were investigated. Anti‐TH‐16 enhanced the enzymatic activity in a concentration‐dependent manner, and the antigen TH‐16 inhibited the stimulatory activity of the antiserum in a concentration‐dependent manner. The activated enzyme had a lower Kmapp for the cofactor 2‐amino‐4‐hydroxy‐6‐methyl‐5,6,7,8‐tetrahydropterin and a higher Vmax app than the nonactivated enzyme. Anti‐TH‐16 was characterized further by its ability to immunoprecipitate the enzyme activity by labeling tyrosine hydroxylase after Western blotting and by immunohistochemical labeling of cate‐cholaminergic neurons. Anti‐TH‐16 did not block activation of tyrosine hydroxylase by phosphorylation catalyzed by cyclic AMP‐dependent protein kinase. Exposure of the enzyme to anti‐TH‐16 and subsequent phosphorylation of the enzyme resulted in a greater activation of the enzyme than the sum of activation produced by these two treatments separately. However, the activation was less than additive when the enzyme was first phosphorylated and subsequently exposed to anti‐TH‐16. The present study demonstrates the utility of anti‐TH‐16 in investigating the molecular aspects of the enzyme activation.
Isolation and Partial Characterization of an 80,000‐Dalton Protein Kinase from the Micro vessels of the Porcine Brain
Authors: Dechert Ute , Weber Marion , Weber‐Schaeuffelen Martin , Wollny Eric
Published: October 2006

Abstract: A novel serine/threonine‐specific protein kinase was isolated from the microvessels of porcine brains‐ The molecular mass of the protein is 80,000 daltons, as judged by gel electrophoresis under denaturing conditions, or J 22,000 daltons. on high‐resolution gel permeation chromatography in the native state. The activity of this enzyme is stimulated by various histones or polyamines, like spermine or spermidine, but not by any of the common second messengers. The amino‐terminal sequence data show no homologies to any of the published kinases, but rather to a heat‐shock protein of unknown function.
Postmortem Degradation Alters Fluorographic Labeling Patterns and Affinities of Benzodiazepine Binding Proteins
Authors: Reichelt Ralf , Schmitz Elke , Hebebrand Johannes
Published: October 2006

Abstract: To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degra dation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.
pros‐Methylimidazoleacetic Acid in Rat Brain: Its Regional Distribution and Relationship to Metabolic Pathways of Histamine
Authors: Prell George D. , Khandelwal Jai K. , Hough Lindsay B. , Green Jack Peter
Published: October 2006

Abstract: pros‐Methylimidazoleacetic acid (p‐MIAA; 1‐methylimidazole‐5‐acetic acid), an isomer of the histamine metabolite, tele‐methylimidazoleacetic acid (t‐MIAA), is present in brain and CSF. Its relationship to histamine synthesis and catabolism was assessed in brains of rats. p‐MIAA distribution in brain regions was heterogeneous although the concentrations in regions with the highest (hypothalamus) and the lowest (medulla‐pons) levels differed less than fourfold. There was no significant correlation between the regional distributions of p‐MIAA with those of histamine or its metabolites. pros‐Methylhistidine (1 g/kg, i.p.) produced a 20‐fold increase in mean levels of p‐MIAA and up to a 50‐fold increase in levels of pros‐methylhistamine (p‐MH), a putative intermediate; levels of histamine and its metabolites were unaltered. l‐Histidine (1 g/kg, i.p.) or α‐fluoromethylhistidine (100 mg/kg, i.p.), the irreversible inhibitor of histamine synthesis, did not alter the levels of p‐MIAA in brain. Like the levels of t‐MIAA, the levels of p‐MIAA were unaltered after probenecid administration. Contrary to its effects in lowering t‐MIAA levels, pargyline (75 mg/kg, i.p.) produced a slight rise in levels of p‐MIAA in all regions. These findings suggest that, in brain, the metabolic pathways of histamine are independent of pathways that generate p‐MIAA. Further, since brain is capable of p‐MH formation, its use as an internal standard in analytical methods merits caution.
Cerebrospinal Fluid Parameters in Healthy Volunteers During Serial Lumbar Punctures
Authors: Menachem E. Ben , Persson L. , Schechter P. J. , Haegele K. D. , Huebert N. , Hardenberg J.
Published: October 2006

Abstract: Lumbar punctures were performed on four occasions over a 5‐day period (8:30 a.m. on days 1, 3, and 5; 2: 30 p.m. on day 2) on 10 normal volunteers (five of each sex; mean age, 27.7 years) to assess, with repeated sampling, the day‐to‐day variation of selected CSF parameters. Two subjects abstained from the lumbar puncture on day 5 due to headache after the third puncture. Lumbar CSF was analyzed for concentrations of free and total γ‐aminobutyric acid (GABA), homocarnosine, homovanillic acid (HVA), 5‐hydroxyindoleacetic acid (5‐HIAA), total protein, albumin, and immunoglobulin (Ig)G. No significant concentration differences were found between the afternoon and next morning samples. No differences were found in concentrations of free GABA, total GABA, homocarnosine, 5‐HIAA, or albumin across the study. In contrast, HVA concentrations significantly increased by day 5, whereas total protein and IgG decreased during the study. The most likely explanation for these changes involves the known concentration gradients in the CSF column.
Alterations in Protective Enzymes Against Peroxidation in the Central and Peripheral Nervous System of Control and Dysmyelinating Mutant Mice
Authors: Cloëz Isabelle , Tayarani Ivan , Morel Frédérique , Bourre Jean‐Marie
Published: October 2006

Abstract: The activities of peroxide‐detoxifying enzymes such as superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase were measured in the nervous system of neurological dysmyelinating mutants: quaking (Qk), shiverer (Shi), and trembler (Tr) mice. Cu/Zn–SOD activity was higher in the cerebellum of Qk and Shi mice (by 53% and 106%, respectively) in comparison with controls, but it was the same in the cerebellum of Tr mice and their corresponding controls. In contrast, there was no difference in the level of Cu/Zn–SOD activity in the cerebrum of Qk, Shi, and Tr mice and their respective controls. Mn–SOD activity was the same among all the mutants compared to control animals in both cerebrum and cerebellum. In Shi cerebellum, glutathione peroxidase and glutathione reductase activities were slightly decreased (a 21.6% and a 13.2% diminution, respectively), whereas catalase activity in cerebrum and cerebellum was the same among mutants and control mice. In the sciatic nerve from Tr mice, all the enzymatic activities were enhanced: sixfold increase for total SOD, and 2.4‐fold, 3.5‐fold, and 1.8‐fold increase for glutathione peroxidase, glutathione reductase, and catalase, respectively.
Studies on the Formation of 6‐Hydroxydopamine in Mouse Brain After Administration of 2,4,5‐Trihydroxyphenylalanine (6‐HydroxyDOPA)
Authors: Evans Jacqueline M. , Cohen Gerald
Published: October 2006

Abstract: 2,4,5‐Trihydroxyphenylalanine (6‐OH‐DOPA) destroys central and peripheral noradrenergic neurons, while sparing dopaminergic neurons. Previous studies indicate that 6‐OH‐DOPA toxicity is mediated by the formation of 6‐hydroxydopamine. However, levels of 6‐hydroxydopamine in brain following peripheral administration of 6‐OH‐DOPA have not been documented. In the current study, 6‐OH‐DOPA and 6‐hydroxydopamine were measured in brain by HPLC with electrochemical detection after intraperitoneal injection of 6‐OH‐DOPA. When mice were injected with 100 mg 6‐OH‐DOPA/kg, 6‐hydroxydopamine levels in the striatum were highest (1.9 μg/g) at 15 min and fell slowly to 24% of the peak value at 4 h. Experiments with reserpine indicated that the relative stability of 6‐hydroxydopamine was largely dependent upon storage in synaptic vesicles. Reserpine (10 mg/kg) lowered striatal 6‐hydroxydopamine levels to 21.6% of control (non‐reserpine‐treated) values at 1 h, and to 8.9% of control values at 4 h. Levels of 6‐hydroxydopamine in the striatum at 1 h were increased 113% by pargyline (100 mg/kg), 145% by α‐methyldopahydrazine (carbidopa; 25 mg/kg), and 261% by pargyline and carbidopa together. Levels of dopamine in the striatum were unchanged at 2.5 h after 200 mg 6‐OH‐DOPA/kg (with pargyline and 50 mg carbidopa/kg), whereas levels of norepinephrine in the frontal cortex fell by 77%. At the same time, 6‐hydroxydopamine levels were 8.8‐fold higher in the striatum (5.54 μ/g) than in the cortex (0.63 μg/g). The latter results show that the striatum is resistant to 6‐OH‐DOPA‐mediated depletion of catechol‐amines even though the concentrations of 6‐hydroxydopamine approach those of endogenous dopamine.
Construction of a Physiologically Active Photoaffinity Probe Based on the Structure of Bradykinin: Labelling of Angiotensin Converting Enzyme but Not Candidate Bradykinin Receptors on NG108–15 Cells
Authors: de Vries Johannes G. , Phillips Elsa , Snell Christopher R. , Snell Penelope H. , Webb Michael
Published: October 2006

Abstract: The peptides bradykinin and kallidin are released in response to noxious stimuli and mediate various physiological effects, including a direct stimulation of nociceptive afferent neurones. The nature of the receptor molecules through which these ligands act is presently unknown. We synthesised an iodinatable photoaffinity probe, N′‐4‐azido‐salicylylkallidin, and used it in an attempt to identify candidate bradykinin receptors on the NG108–15 neuroblastoma X glioma hybrid cell line. The ligand bound in subdued light to a particulate fraction of NG108–15 tumours and could be displaced by bradykinin with an IC50 of 0.33 nM. In a physiological assay, it behaved as an agonist equipotent with bradykinin. Gel analysis of the labelled products after photolysis of the iodinated ligand in the presence of NG108–15 cells or tumour membranes revealed bradykinin‐blockable labelling of a glycoprotein with an Mr of 166,000. The probe was also able to label purified commercial angiotensin converting enzyme. The band labelled in NG108–15 cells was immunoprecipitable with a polyclonal antiserum to angiotensin converting enzyme, an enzyme shown to be present in low amounts in these preparations by direct binding using the iodinatable specific ligand MK351A.
Decrease of Atrial Natriuretic Peptide Content in Rat Superior Cervical Sympathetic Ganglion After Denervation and Axotomy
Authors: Nagata Yutaka , Ebisu Hiroshi , Tamaru Masao , Fujita Kimikazu , Koide Tadashi
Published: October 2006

Abstract: We found atrial natriuretic peptide (ANP), known as a humoral factor in regulating body fluid volume and blood pressure, in considerable quantities in rat superior cervical sympathetic ganglion (SCG) by radioimmunoassay after separation with reverse‐phase HPLC. Although the ANP content of the immature rat 1 week after birth was low, it doubled at 2 weeks and then increased gradually, until it reached the adult level. Denervation caused a rapid decrease in the ANP content to half of the intact SCG level after 3 h, which then fell to 10% of the control value on day 2 after operation. The time course of ANP content reduction after denervation was similar but rather faster than that of activity of the acetyl‐choline‐synthesizing enzyme, choline acetyltransferase, an observation suggesting that ANP may partly contribute to cholinergic synaptic transmission. On the other hand, axotomy produced a rather slower decrease in the ANP content than did denervation. Enucleation and sialoadenectomy also caused a considerable reduction of the ANP content. Thus, part of the ANP found in the ganglion is apparently transported from sympathetically innervated extraganglionic organs via retrograde axoplasmic flow.
Confirmation of the Cholinergic Specificity of the Chol‐1 Gangliosides in Mammalian Brain Using Affinity‐Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus
Authors: Derrington E. A. , Masco D. , Whittaker V. P.
Published: October 2006

Abstract: An antiserum raised to Torpedo electromotor synaptosomal membranes (anti‐TSM antiserum) induces a cho‐linergic‐specific immune lysis of mammalian brain synap‐tosomes and recognizes a group of minor gangliosides in mammalian brain. These minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol‐1. To confirm the cholinergic specificity of the Chol‐1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol‐1 gangliosides inhibit the cholinergic‐specific immune lysis induced by the anti‐TSM antiserum, but also it can be used to affinity‐purify a subpopulation of immunoglobulins from the anti‐TSM antiserum that also induce a cholinergic‐specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol‐1 gangliosides concomitant with that shown by choline acetyl transferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol‐1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol‐1 gangliosides