bluuebunny/crossref_metadata_2025 huggingface dataset:
Survey of Intermediate Filament Proteins in Optic Nerve and Spinal Cord: Evidence for Differential Expression
Authors: Quitschke Wolfgang , Jones Paul S. , Schechter Nisson
Published: October 2006

Abstract: The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two‐dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145–150K range and in the 70–85K range. In addition, predominant neurofilament proteins in the 58–62K molecular‐weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low‐molecular‐weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.
“Peripheral‐Type”Binding Sites for Benzodiazepines in Brain: Relationship to the Convulsant Actions of Ro 5–4864
Authors: Weissman B. A. , Cott J. , Jackson J. A. , Bolger G. T. , Weber K. H. , Horst W. D. , Paul S. M. , Skolnick P.
Published: October 2006

Abstract: Previous studies have shown that Ro 5–4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5–4864 were not mediated by “brain‐type”benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5–4864 for their capacities to displace [3H]Ro 5–4864 from “peripheral‐type”binding sites and their potencies as convulsants (or as antagonists of Ro 5‐4864‐induced convulsions). It was observed that compounds such as KW 3600 (the N‐desmethyl analog of Ro 5–4864), which have very low affinities for “peripheral‐type”sites, are convulsants with a potency nearly equal to that of Ro 5–4864. In contrast, compounds such as Ro 5–6900 and PK 11195, which bind with very high affinities to “peripheral‐type”binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5–4864. Within a series of compounds that are structurally related to Ro 5–4864 there is a good correlation (r = 0.93; p < 0.01) between their potencies as convulsants and their capacities to displace [35S]t‐butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of “peripheral‐type”binding sites by high‐affinity ligands may not be directly involved in the convulsant actions of Ro 5–4864 and related compounds.
Fe2+‐Induced Lysis and Lipid Peroxidation of Chromaffin Granules
Authors: Spears Ronald M. , Holz Ronald W.
Published: October 2006

Abstract: Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10−9–10−7M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 μM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 μM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+‐dependent reaction involving the chromaffin granule.
Solubilization and Characterization of Rat Brain α2‐Adrenergic Receptor
Authors: Matsui Hiroaki , Asakura Mikio , Tsukamoto Tohru , Imafuku Jun , Ino Miyuki , Saitoh Noriko , Miyamura Satoko , Hasegawa Kazuo
Published: October 2006

Abstract: α2‐Adrenergic receptors labelled by [3H]clonidine (α2‐agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3‐[3‐(cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO‐bound complex was performed by the vacuum filtration method using polyethylenimine‐treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membranebound α2‐adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized α2‐receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio‐Gel A‐1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6–5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl‐reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain α2‐adrenergic receptor system at the molecular level.
Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum
Authors: Ravikumar B. V. , Sastry P. S.
Published: October 2006

Abstract: The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([3H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations, one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the high‐affinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmol/mg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60–70 fmol/mg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life.
Epidermal Growth Factor in Synaptosomal Fractions of Mouse Cerebral Cortex
Authors: Lakshmanan J. , Weichsel M. E. , Fisher D. A.
Published: October 2006

Abstract: Using a specific and sensitive epidermal growth factor radioimmunoassay (EGF‐RIA) we measured EGF concentrations in whole brain, cerebral cortex, and cerebral cortical synaptosomal (pinched‐off presynaptic nerve terminals) fractions of 26‐day‐old mouse brain. The relative EGF concentration in synaptosomal fractions was significantly greater than the growth factor concentrations in whole brain or cerebral cortex. Intracerebral injection, in an amount of EGF, several‐fold greater than whole brain EGF content, did not appreciably increase synaptosomal EGF concentration, suggesting that no artifact was involved. The high synaptosomal EGF content suggests a neurotransmitter or a neuromodulator role for EGF in the CNS.
Delineation of a Particulate Thyrotropin‐Releasing Hormone‐Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase
Authors: Friedman Theodore C. , Wilk Sherwin
Published: October 2006

Abstract: The degradation of thyrotropin‐releasing hormone in rat brain homogenates was studied in the presence of N‐benzyloxycarbonyl‐prolyl‐prolinal and pyroglutamyl diazomethyl ketone, specific and potent active‐site‐directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin‐releasing hormone‐degrading enzyme(s). Reports of a thyrotropin‐releasing hormone‐degrading enzyme with narrow specificity that cleaves the pGlu‐His bond of this tripeptide led us to develop a coupled assay using pGlu‐His‐Pro‐2NA as the substrate to measure this activity. Cleavage of the pGlu‐His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl‐peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o‐phenanthroline. The particulate pyroglutamyl‐peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin‐releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu‐His bond of thyrotropin‐releasing hormone but is distinct from pyroglutamyl peptide hydrolase.
Molecular Characteristics and Peptide Specificity of Vasoactive Intestinal Peptide Receptors from Rat Cerebral Cortex
Authors: Couvineau Alain , Gammeltoft Steen , Laburthe Marc
Published: October 2006

Abstract Vasoactive intestinal peptide (VIP) receptors have been identified in CNS by their chemical specificity and molecular size. Using synaptosomes isolated from rat cerebral cortex, it was shown that central VIP receptors discriminated among natural and synthetic VIP‐related peptides, because half‐maximal inhibition of [125I]VIP binding to synaptosomes was obtained for 0.6 nM VIP, 9 nM peptide histidine isoleucineamide (PHI), 50 nM VIP 2–28, 70 nM secretin, 100 nM rat growth hormone‐releasing factor (GRF), and 350 nM human GRF. Other peptides of the VIP family, such as glucagon and gastric inhibitory polypeptide, did not interact with cortical VIP receptors. The molecular components of VIP receptors in rat cerebral cortex were identified after [125I]VIP cross‐linking to synaptosomes using the cross‐linker dithiobis(succinimidyl propionate). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of synaptosomal proteins revealed two major [125I]VIP‐pro‐tein complexes of Mr 49,000 and 18,000. The labeling of the Mr49,000 component was specific, because it was abolished by native VIP, whereas the labeling of the Mr 18,000 component was not. Natural VIP agonists reduced the labeling of the Mr49,000 component with the following order of potency: VIP > PHI > secretin ∼ rat GRF. In contrast, glucagon and octapeptide of cholecystokinin were without effect, a result indicating its peptide specificity. Densitometric scanning of autoradiographs showed that the labeling of the Mr 49,000 component was inhibited by low VIP concentrations between 10−‐10 and 10−‐6M (IC50= 0.8 nM), a result indicating the component's high affinity for VIP. Labeling was also reduced by GTP in the concentration range between 10−‐7 and 10−‐3M (ED50= 5 μM). Assuming that one molecule of [125I]VIP is bound per molecule of synaptosomal protein, a major protein of Mr 46,000 was identified as a component of the VIP receptor in rat cerebral cortex.
Solubilization and Characterization of D2‐Dopamine Receptors in an Estrone‐Induced, Prolactin‐Secreting Rat Pituitary Adenoma
Authors: Bouvier C. , Potier M. , Beauregard G. , Lafond J. , Amlaiky N. , Caron M. G. , Collu R.
Published: October 2006

Abstract D2‐dopamine (3,4‐dihydroxyphenylethylamine) receptors were successfully solubilized with 3‐[(3‐cholami‐dopropyl)‐dimethylammonio]‐l‐propane sulfonate from an estrone‐induced rat pituitary adenoma. Forty‐five percent of initial protein and 48% of initial [3H]spiroperidol binding sites were solubilized. The high affinity as well as the stereoselectivity of the sites was preserved. The order of potency of dopaminergic agonists was found to be typical of D2 receptors. Target size analysis by radiation inactivation indicated a molecular weight of 143,000 ± 3,000 and of 106,000 ± 4,000 daltons for membrane‐bound and solubilized receptors, respectively. This suggests the loss of a 37,000‐dalton subunit during solubilization without significant modification of binding characteristics. Sodium do‐decyl sulfate‐polyacrylamide gel electrophoresis of receptor protein preparation photolabeled with N‐(p‐azido‐w[125I]‐iodophenethyl)spiroperidol confirmed the existence of a 94,000‐dalton peptide which probably constitutes the li‐gand binding site of the receptor. Thus, our data indicate that chronic estrogen treatment of rats, although inducing a pituitary adenoma, does not modify the pharmacological characteristics of D2 receptors. These data suggest therefore that these adenoma may represent an ideal source of material for further biochemical characterization of D2 receptors.
Ethanol Administration in the Rat Decreases Prostacyclin Production by Isolated Brain Microvessels
Authors: Pediconi M. , Colombo C. , Galli C.
Published: October 2006

Abstract: The effects of short‐ and long‐term ethanol administration to rats on basal levels and formation of prostacyclin (PGI2) measured as 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), and on lipid class content and fatty acid composition of isolated brain microvessels (BMV) were studied. After acute treatment (2 h, at the peak of plasma ethanol concentration) basal 6‐keto‐PGF1α levels in BMV and release on incubation were reduced to 50% of control values. After chronic administration (15 days), PGI2 release was reduced to about 40% of control values, without changes in basal levels. Total lipid, phospholipid, and cholesterol levels in BMV, measured after prolonged administration of alcohol, were not modified. Also, only minor changes in the fatty acid composition of individual phospholipid classes were detected. The observed reduction of PGI2 synthesis in BMV thus could not be related to changes of the fatty acid precursor pool in the preparation. Precursor release and/or the biosynthetic pathways may be affected by ethanol administration.
Significance of Testosterone in Regulating Hypothalamic Content and In Vitro Release of β‐Endorphin and Dynorphin
Authors: Almeida O. F. X. , Nikolarakis K. E. , Herz A.
Published: October 2006

Abstract: The effects of castration and testosterone replacement on hypothalamic pools of β‐endorphin and dynorphin and on the basal and corticotropin‐releasing factor (CRF)‐stimulated release of these peptides from hypothalamic slices in vitro were studied. The experiments were done in adult male rats. The hypothalamic content of both peptides increased significantly within 1 week of castration, and levels remained elevated for up to 4 weeks. Testosterone treatment, begun at the time of castration, prevented these increases. In addition, testosterone replacement 6 weeks after castration reversed peptide levels to normal. Basal in vitro release rates of β‐endorphin and dynorphin were significantly lower from hypothalamic slices derived from 1‐week castrated animals than from intact males, and when testosterone was administered in various doses in vivo, basal release rates in vitro increased in a dose‐related manner. Hypothalami from rats that had been castrated for 4 weeks, however, showed basal release rates similar to those in tissues from intact controls, a finding indicating that castration initially alters both opioid peptide synthesis and release; later, release is normalized, whereas synthesis remains elevated. CRF was found to stimulate β‐endorphin and dynorphin release from hypothalami from intact and from 1‐ and 4‐week‐castrated rats, a result indicating that castration does not alter the response of β‐endorphin and dynorphin neurons to this stimulus.
Changes in Composition of Endoneurial and Perineurial Fatty Acids During Glycerol‐Induced Wallerian Degeneration and Regeneration in the Sciatic Nerve of the Adult Rat
Authors: Fressinaud C. , Vallat J. M. , Durand J. , Archambeaud‐Mouveroux F. , Rigaud M.
Published: October 2006

Abstract: Intraneural injection of pure glycerol induces Wallerian degeneration with subsequent regeneration. In agreement with other reports, we observed an increase in endoneurial polyunsaturated fatty acids 8 days after the glycerol injection. Levels then fell until day 30. After a period of 5 months, there was an increase in C18:2(n‐6) in the intrafascicular tissue, concomitant with a marked fall in this fatty acid in the remaining extrafascicular perineurium. The rise in C18:2(n‐6) in endoneurium correlated with infiltration of this tissue by perineurial cells. Interactions between perineurium and endoneurium during nerve regeneration are discussed.
Sulfoglucuronyl‐Neolacto Series of Glycolipids in Peripheral Nerves Reacting with HNK‐1 Antibody
Authors: Chou Denise K. H. , Schwarting Gerald A. , Evans James E. , Jungalwala Firoze B.
Published: October 2006

Abstract: Novel sulfoglucuronyl‐neolacto series of glycolipids were detected in peripheral nerves of various species by TLC followed by immunostaining with HNK‐1 antibody. The major antigenic glycolipid, sulfoglucuronylneolactote‐traosylceramide, previously described in human nerves, was shown to be also present in the sciatic nerves of various species including rodents. A second slower migrating antigenic glycolipid present in the sciatic nerves of human and dog was isolated and purified. It was characterized by chemical and enzymatic degradation, sugar analysis after permethylation, and gas liquid chromatography‐mass spectrometry techniques as well as by fast atom bombardment‐mass spectrometry, as 3‐sulfogIucuronylneolactohexaosylceramide. During postnatal development of the rat sciatic and trigeminal nerves the concentration of these antigenic glycolipids increased with age.
Calcium Binding Protein in Squid Brain: Biochemical Similarity to the 28,000‐Mr Vitamin D‐Dependent Calcium Binding Protein (Calbindin‐D28k)
Authors: Christakos Sylvia , Malkowitz Lorraine , Sori Alan , Sperduto Andrea , Feldman Susan C.
Published: October 2006

Abstract: A calcium binding protein that is biochemically similar to vertebrate 28,000‐Mr vitamin D‐dependent calcium binding protein (calbindin‐D28k) has been purified from squid brain. Squid brain calbindin was found to have an isoelectric point of 5.0, was heat stable up to 60°C, and showed increased electrophoretic mobility in the presence of chelator Amino acid analysis revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, and tyrosine, a finding similar but not identical to the composition of vertebrate calbindin‐D28k. The molecular weight of the squid protein, determined by Ferguson plot analysis of data obtained from sodium dodecyl sulfategel electrophoresis, was calculated to be 25,700, as compared with 27,800 for rat renal calbindin. Immunocyto‐chemical analysis demonstrated immunoreactive protein in a selected population of neurons and fibers in several areas of the molluscan nervous system. This study represents the first purification from an invertebrate of a calcium binding protein that is biochemically similar to vitamin D‐dependent calcium binding protein. These results demonstrate that calbindin, although not identical in vertebrates and cephalopods, may be phylogenetically conserved in structure. The restricted distribution of immunoreactive calbindin in both the cephalopod and mammalian brain suggests that the function of neuronal calbindin may also be conserved in evolution.).
Cholera Toxin and Dibutyryl Cyclic AMP Inhibit the Expression of Neurofilament Protein Induced by Nerve Growth Factor in Cultures of Naive and Primed PC12 Cells
Authors: Doherty Patrick , Mann Derek A. , Walsh Frank S
Published: October 2006

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neuro‐filament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme‐linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30‐fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B‐cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.
Myelin‐Associated Glycoprotein and Related Glycoconjugates in Developing Cat Peripheral Nerve: A Correlative Biochemical and Morphometric Study
Authors: Willison Hugh J. , Ilyas Amjad I. , O'Shannessy Daniel J. , Pulley Mike , D. Trapp, Bruce , Quarles Richard H.
Published: October 2006

The expression and accumulation of the myelin‐associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK‐1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60‐kilodalton (kD) glycoprotein and a group of 20–26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate‐3‐glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20–26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.
[3H]GBR‐12935 Binding to the Dopamine Transporter Is Decreased in the Caudate Nucleus in Parkinson's Disease
Authors: Janowsky Aaron , Vocci Frank , Berger Paul , Angel Itzchak , Zelnik Nethanel , Kleinman Joel E. , Skolnick Phil , Paul Steven M.
Published: October 2006

The specific binding of [3H]GBR‐12935 to membranes prepared from human caudate nucleus is saturable (Bmax 1.36 ± 0.18 pmol/mg protein), sodium dependent, and of high affinity (KD 2.34 ± 0.18 nM). Freezing of tissue from rat brain, or refrigeration followed by freezing, results in a small but significant (20%) decrease in specific [3H]GBR‐12935 binding when compared to the binding observed in fresh (nonfrozen) tissue, and this decrease may account, in part, for the differences in specific binding between rat and human brain membranes. Despite small differences in binding site density between fresh and frozen tissue there is a good correlation (r= 0.98; p < 0.01) between the potencies of a series of drugs in displacing specific [3H]GBR‐12935 binding to human caudate membranes and rat striatum as well as in inhibiting dopamine uptake in rat striatal synaptosomes (r= 0.96; p < 0.01). The specific binding of [3H]GBR‐12935 to membranes prepared from the caudate nuclei of patients with Parkinson's disease is decreased compared to membranes prepared from age‐and sex‐matched controls. These data suggest that [3H]GBR‐12935 binds in a sodium‐dependent fashion to the dopamine transport complex in human brain and that specific binding is decreased by a pathological degeneration of dopaminergic neurons to the caudate nucleus.
Thyrotropin‐Releasing Hormone Reduces myo‐Inositol Content in Rat Cerebellum Pretreated with Lithium
Authors: Miyamoto Yasufumi , Kikkawa Ryuichi , Hatanaka Ikuo , Yasuda Hitoshi , Terada Masahiko , Yamashita Makio , Shigeta Yukio
Published: October 2006

Abstract: The effect of thyrotropin‐releasing hormone (TRH) and lithium on myo‐inositol metabolism has been assessed in rat cerebral cortex, cerebellar cortex, and sciatic nerves. Sprague‐Dawley male rats were injected subcutane‐ously with 10 mEq/kg of LiCl and intraperitoneally with 10 mg/kg of TRH‐tartrate, alone or in combination. Either lithium or TRH alone had little effect on the myo‐inositol concentration in cerebellar cortex, whereas the combination of lithium and TRH significantly lowered the level. The myo‐inositol level of cerebellar cortex reached its nadir (70% of values in untreated control rats) 30 min after addition of TRH and then returned to the control level at 90 min. In cerebral cortex, both lithium alone and lithium plus TRH significantly reduced the myo‐inositol level. No effect was seen on the myo‐inositol concentration in sciatic nerves with these regimens. These results suggested that the pharmacological dose of TRH activated phosphatidylinositol turnover in rat cerebellar cortex and subsequently reduced the myo‐inositol level in the presence of lithium.
Endogenous Dopamine Functionally Activates D‐1 and D‐2 Receptors in Striatum
Authors: Kelly Eamonn , Nahorski Stefan R.
Published: October 2006

Abstract: Rat striatal slices incubated with the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine at 1 mM were exposed to different concentrations (1–100 μM) of the cate‐cholamine‐releasing drug amphetamine. This produced both a concentration‐dependent release of endogenous dopamine and accumulation of cyclic AMP in the slices. The cyclic AMP accumulation due to amphetamine was greatly increased when slices were coincubated with the selective dopamine D‐2 antagonist (−)‐sulpiride (30 μM), but the amphetamine‐induced release of dopamine from the slices was the same in the presence or absence of (−)‐sulpiride. Pretreatment of animals with reserpine (5 mg/kg s.c., 18 h before death) and in vitro incubation with a‐methyl‐p‐tyrosine (50 μM for 90 min), respectively, reduced the ability of amphetamine (1–100 μM) [in the presence of 30 μM (−)‐sulpiride] to induce release of dopamine and to elevate cyclic AMP accumulation in striatal slices. A similar reduction in amphetamine‐induced dopamine release and cyclic AMP accumulation in striatal slices was observed 7 days following unilateral 6‐OHDA lesions of the medial fore‐brain bundle of rats. These results suggest that amphetamine induces release of endogenous dopamine from the terminals of nigrostriatal dopamine neurones. Released dopamine is then able functionally and concomitantly to activate D‐1 and D‐2 receptors, seen as stimulation and inhibition of cyclic AMP accumulation, respectively.
2‐Amino‐7‐Phosphonoheptanoic Acid Inhibits Insulin‐Induced Convulsions and Striatal Aspartate Accumulation in Rats with Frontal Cortical Ablation
Authors: Chapman A. G. , Engelsen B. , Meldrum B. S.
Published: October 2006

Abstract: Pretreatment of rats with the excitatory amino acid antagonist 2‐amino‐7‐phosphonoheptanoic acid (2‐APH; 0.5 mmol/kg, i.p.) protected against insulin‐induced clonic seizures. Complete protection was observed in 38% of the rats and partial protection in an additional 50%. Le‐sioning of the corticostriatal pathway by frontal cortical ablation caused decreases in the striatal levels of aspartate (‐28%) and glutamate (‐18%), an increase in striatal glutamine level (45%), and decreased high‐affinity uptake of D‐[3H]aspartate (‐27%) in the lesioned dorsal neostriatum. Insulin‐induced hypoglycemia caused a predicted sharp increase in aspartate level (165%) and decreased glutamate (‐20%) and glutamine (‐38%) levels in the intact striatum. Pretreatment of rats with 2‐APH significantly reversed the insulin‐induced changes in striatal aspartate, glutamate, and glutamine levels, especially in the intact hemisphere. In normoglycemic control rats, the “metabolic”, i.e., concentration in the lesioned hemisphere, aspartate pool constituted 72% and the “synaptic”, i.e., the concentration difference between the intact and lesioned hemispheres, 28% of the total striatal aspartate pool. 2‐APH had no effect on the level of “metabolic” aspartate in the striata of normoglycemic rats but caused an almost complete suppression of “synaptic” aspartate. Following insulin‐induced hypoglycemia, the “metabolic” aspartate pool doubled, whereas the “synaptic” aspartate pool increased 3.5‐fold in the absence of 2‐APH. The insulin‐induced rise in “synaptic” aspartate level was almost completely blocked by 2‐APH (a 5% rise instead of a 3.5‐fold rise). The turnover of “metabolic” aspartate was also affected by 2‐APH, but to a lesser extent (a 25% rise in the presence vs. a 94% rise in the absence of 2‐APH). The striatal changes in levels of dicarboxylic acids after systemic 2‐APH administration can be most simply explained in terms of an action of 2‐APH in the cortex blocking excitatory inputs to the corticostriatal pathway.
Taurine Biosynthesis in Rat Brain: A New Specific and Sensitive Microassay of Cysteine Sulfinate Decarboxylase (CSDI) Activity Through Selective Immunotrapping and Its Use for Distribution Studies
Authors: Legay F. , Weise V. K. , Oertel W. H. , Tappaz M. L.
Published: October 2006

Abstract: Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, has been shown to exist in two forms in rat brain, respectively CSDI and CSDII, one of which (CSDII) is considered to be in fact glutamate decarboxylase (GAD). CSDI assay after immunotrapping was made possible by using an anti‐CSD antiserum raised in sheep immunized with a partially purified CSD fraction from liver. This antiserum immunoprecipitated both liver CSD and brain CSDI activities with the same affinity but did not inhibit their enzymatic activities. The immunotrapping of CSDI was selective without any contamination by GAD/CSDII activity. The immunotrapped CSD activity, which corresponded exactly to the amount of CSD not precipitated by a GAD/CSDII antiserum, was not inhibited by a specific irreversible GAD inhibitor. A quantitative, selective and sensitive assay was thus developed by measuring CSD activity on the solid phase after immunotrapping. Kinetic parameters of the immunotrapped enzyme remained unchanged. CSDI activity represented only a fraction, around 20% with saturating concentration of substrate, of the total CSD activity in rat brain homogenate. This indicates that most studies on total CSD activity dealt essentially with CSDII activity that is indeed GAD. Regional and subcellular distributions of CSDI have been determined. CSDI activity was about threefold higher in the richest (cerebellum) compared to the poorest (striatum) region without any correlation with GAD/CSDII distribution. Subcellular distribution showed a fourfold enrichment of CSDI activity in the synaptosomal fraction. The precise role of CSDI and CSDII in the biosynthesis of taurine in vivo remains to be elucidated.
Ionic Dependence of Membrane Potential and Glutamate Receptor‐Linked Responses in Synaptoneurosomes as Measured with a Cyanine Dye, DiS‐C2‐(5)
Authors: Åkerman Karl E. O. , Scott Ian G. , Heikkila Jari E. , Heinonen Erkki
Published: October 2006

Abstract: Membrane potentials of particles present in a sub‐cellular brain preparation, called synaptoneurosomes, have been monitored by measurement of changes in the absor‐bance of a cyanine dye, DiS‐C2‐5. The membrane potential of the particles seems to be dependent on both Cl− and K+ diffusion potentials, as judged from dependence of the ab‐sorbance changes on the K+ equilibrium potential across the membrane in the presence of Ba2+ or when Cl− was replaced with gluconate. The apparent high Cl− permeability of the membrane preparation was reduced in the presence of pic‐rotoxin, a finding suggesting endogenous activation of receptor‐linked Cl− channels. Glutamate and kainate caused depolarization of the membranes present in the preparation. This effect was only seen if K+ channels had been blocked in the presence of Ba2+ or 4‐aminopyridine. No responses were observed with other glutamate receptor agonists (quisqualate or N‐methyl‐d‐aspartate). The membrane potential of particles present in conventional synaptosomal preparations neither had a high Cl− permeability nor reacted to glutamate or kainate in the present conditions. The results suggest that synaptoneurosome preparations may be used for functional studies on postsynaptic neurotransmit‐ter receptor‐linked membrane potential changes with optical probes of membrane potential.
Monoclonal Antibodies to Mammalian Carnosine Synthetase
Authors: Margolis F. L. , Grillo M. , Hempstead J. , Morgan J. I.
Published: October 2006

Abstract: A set of mouse monoclonal antibodies has been generated against rabbit muscle camosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross‐reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low‐abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.
S100a0 (αα) Protein, a Calcium‐Binding Protein, Is Localized in the Slow‐Twitch Muscle Fiber
Authors: Haimoto Hajime , Kato Kanefusa
Published: October 2006

Abstract: We previously showed that, in contrast to the distribution of S100b (ββ), S100a0 (αα) is mainly present in human skeletal and heart muscles at the level of 1–2 μg/mg of soluble protein and is universally distributed at high levels in skeletal and heart muscles of various mammals. To elucidate cellular and ultrastructural localizations of the α subunit of S100 protein (S100‐α) in skeletal muscle, we used immunohistochemical and enzyme immunoassay methods. The immunohistochemical study revealed that S100‐α is mainly localized in slow‐twitch muscle fibers, whereas the β subunit of S100 protein (S100‐β) was not detected in both types of muscle fibers, an observation indicating that the predominant form of S100 protein in the slow‐twitch muscle fiber is not S100a or S100b, but S100a0. The quantitative analysis using enzyme immunoassay corroborates the immunohistochemical finding: The S100‐α concentration of mouse soleus muscle (mainly composed of slow‐twitch muscle fibers) is about threefold higher than that of mouse rectus femoris muscle (mainly composed of fast‐twitch muscle fibers). At the ultrastructural level, S100‐α is associated with polysomes, sarcoplasmic reticulum, the plasma membrane, the pellicle around lipid droplets, the outer membrane of mitochondria, and thin and thick filaments, by immunoelectron microscopy.
Opposing Roles of Dopamine D1 and D2 Receptors in Nigral γ‐[3H]Aminobutyric Acid Release?
Authors: Starr Michael
Published: October 2006

Abstract: This study examined the effects of dopamine D1and D2 receptor agonists and antagonists on the spontaneous and calcium‐dependent, K+‐induced release of γ‐[3H]‐aminobutyric acid ([3H]GABA) accumulated by slices of rat substantia nigra. SKF 38393 (D1 agonist) and dopamine (dual D1/D2 agonist) were without effect on [3H]GABA efflux by themselves (1–40 μM), or in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (0.5 μM), but potentiated evoked release in the presence of forskolin (0.5 μM), an adenylate cyclase activator. These increases in release were prevented by the D1 antagonist SCH 23390 (0.5 μM), but not by the D2 antagonist metoclopramide (0.5 μM), Higher concentrations of forskolin (10–40 μM) augmented stimulus‐evoked [3H]GABA release directly, whereas dibutyryl cyclic AMP (100–200 μM) depressed it. Apomorphine, noradrenaline, and 5‐hy‐droxytryptamine (1–40 μM) had no effect. The D2 stimulants lisuride, RU 24213, LY 171555, and bromocriptine dose‐dependently inhibited depolarisation‐induced but not basal [3H]GABA outflow. These inhibitory responses were not modified by the additional presence of SKF 38393 (10 μM) or SCH 23390 (1 μM), or by injection of 6‐hydroxydopamine into the medial forebrain bundle 42 days earlier, but were attenuated by metoclopramide (0.5 μM). Higher amounts (10 μM) of SCH 23390, metoclopramide, or other D2 antagonists (loxapine, haloperidol) reduced evoked GABA release by themselves, probably by nonspecific mechanisms. These results suggest D1 and D2 receptors may have opposing effects on nigral GABA output and could explain the variable effects of mixed D1/D2 dopaminomimetics in earlier release and electrophysiological experiments.
Characteristics of [3H]Hemicholinium‐3 Binding to Rat Striatal Membranes: Evidence for Negative Cooperative Site‐Site Interactions
Authors: Chatterjee Tapan K. , Cannon Joseph G. , Bhatnagar Ranbir K.
Published: October 2006

Abstract: The characteristics of [3H]hemicholinium‐3 ([3H]HC‐3) interactions with rat striatal membranes were investigated. Under the described assay conditions, [3H]‐HC‐3 binds with a saturable population of membrane binding sites having the following regional distribution: striatum « hippocampus ≧ cerebral cortex > cerebellum. The specific binding of [3H]HC‐3 showed an obligatory requirement for NaCl; other halide salts of sodium or KCl failed to substitute for NaCl. The Scatchard transformation of saturation isotherm data generated a curvilinear plot with high‐and low‐affinity components of binding. The dissociation of [3H]HC‐3 at infinite dilution was also multiexponential. The dissociation could, however, be accelerated if unla‐beled HC‐3 was included in the diluting buffer, and this increase in dissociation appeared to be dependent on the concentrations of unlabeled HC‐3 used, with the maximal increase demonstrable at 100 nM The dissociation was also dependent on the fractional saturation of binding sites with labeled HC‐3, such that, at higher fractional saturation of binding sites, the overall dissociation was faster and the difference in the dissociation observed between „dilution only” and „dilution + unlabeled HC‐3” was reduced. This occupancy‐dependent change in dissociation could also be influenced by temperature and pH. Based on the results of these kinetic studies, the steady‐state [3H]HC‐3 binding data were analyzed for a homogeneous population of binding sites undergoing site‐site interactions of the negative cooperative type. Such an analysis yielded a KD of 9.3 nM for the high‐affinity state and a KD of 22.8 nM for the low‐affinity state of binding sites, with a Bmax of 434 fmol/ mg of protein. Competitive binding studies showed that unlabeled HC‐3 was most potent in displacing [3H]HC‐3, followed by choline. Other drugs known to have little influence on the synaptosomal sodium‐dependent high‐affinity choline uptake system (SDHACU) had no significant effect on [3H]HC‐3 binding sites. Similarities in ionic dependencies, regional distributions, and pharmacological selectivi‐ties of [3H]HC‐3 binding with synaptosomal SDHACU suggest that [3H]HC‐3 selectively labels SDHACU sites located on presynaptic cholinergic neurons in rat CNS. We suggest that the two affinity states of [3H]HC‐3 binding sjtes represent the different „functional” states of the SDHACU system. The binding of HC‐3 (or choline) with the high‐affinity state of the binding sites induces negative cooperative site‐site interactions among the binding sites, resulting in the formation of a low‐affinity binding state. Because the affinities of HC‐3 and choline for this low‐affinity state of the [3H]HC‐3 binding sites correspond to the affinities of these agents for the SDHACU system, we also suggest that the low‐affinity binding state represents the „functional” form for SDHACU.
Effect of Dexamethasone on Transport of α‐Aminoisobutyric Acid and Sucrose Across the Blood‐Brain Barrier
Authors: Ziylan Y. Z. , LeFauconnier J. M. , Bernard G. , Bourre J. M.
Published: October 2006

Abstract: The effect of glucocorticoids on the blood‐brain barrier (BBB) was studied in rats following a single injection or 3 days of dexamethasone administration. Tracers with a low permeability across the intact endothelium, [14C]sucrose and α‐[3H]aminoisobutyric acid ([3H]AIB), were simultaneously injected intravenously in untreated rats or in rats treated with dexamethasone. Unidirectional blood‐to‐brain transfer constants (Ki) in 14 regions of the rat brain were determined. In regions of control brain, average Ki values for AIB and sucrose were ∼0.0020 and 0.00060 ml g−1 min−1, respectively. The lowest transfer constants were found in caudate nucleus, hippocampus, white matter, and cerebellum. In dexamethasone‐treated animals, Ki values for both sucrose and AIB markedly decreased by 30‐50% in almost all brain regions. These results indicate that a single injection or 3 days of treatment with dexamethasone causes an apparent reduction in the normal BBB permeability, and dexamethasone may greatly interfere with drug delivery into brain. These observations may have an importance for the administration of drugs in brain disease in the presence of steroids.
Binding of [3H]Hemicholinium‐3 to the High‐Affinity Choline Transporter in Electric Organ Synaptosomal Membranes
Authors: O'Regan S.
Published: October 2006

Abstract: Sodium‐dependent binding of [3H]hemicholinium‐3 was observed to be 10–fold higher with presynaptic membranes from the electric organ than with electroplaque membranes and this binding site copurified with synaptosomal membranes. The KD for specific [3H]hemicholinium‐3 binding was found to be 31 ± 4 nM and the Bmax, 5.0 ± 0.2 pmol/ mg protein; a Ki of 16 nM was estimated for hemicholinium‐3 as a competitive inhibitor of high‐affinity choline transport in electric organ synaptosomes. Choline and choline analogues were equally potent as inhibitors of [3H]choline uptake and [3H]hemicholinium‐3 binding. Tubocurarine and oxotremorine also inhibited uptake and binding, but carbachol was without effect in both tests. These findings suggest that [3H]hemicholinium binds to the high‐affinity choline trans porter present at the cholinergic nerve terminal membrane. A comparison of maximal velocities for choline transport and the maximal number of hemicholinium‐3 binding sites indicated that the high‐affinity choline transporter has an apparent turnover number of about 3 s−1 at 20°C under resting conditions. The high transport rates observed in electric organ synaptosomes are likely due to the high density of high‐affinity choline transporters in this tissue, estimated on the basis of [3H]hemicholinium‐3 binding to be of the order of 100/μm2 of synaptosomal membrane.
Subcellular Localization of Functionally Differentiated Microtubules in Squid Neurons: Regional Distribution of Microtubule‐Associated Proteins and β‐Tubulin Isotypes
Authors: Arai Takao , Matsumoto Gen
Published: October 2006

Abstract: The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immu‐nologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high‐molecular‐weight microtubule‐associated proteins [MAP A of ∼ 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of β‐tubulin isotypes (β1 and β2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two β‐tubulin isotypes (β1 and β2); (3) axolinin and the β1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the β1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the β1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonai microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.