bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potassium‐Induced Release of Endogenous Amino Acids in the Guinea Pig Cochlea
Authors: Jenison G. L. , Bobbin R. P. , Thalmann R. 
Published: October 2006

Abstract:Guinea pig cochleae were perfused with highpotassium solutions to depolarize hair cells artificially and induce the release of afferent neurotransmitter. Sequential injections of artificial perilymph containing 5 mM KCl, then 50 mM KCl, and finally 5 mM KCl were made into the scala tympani. This injection sequence was conducted under either normal divalent‐cation conditions (2.0 mM CaCl2, 1.0 mM MgCl2) or calcium‐deficient conditions intended to antagonize evoked transmitter release (0.1 mM CaCl2, 20.0 mM MgCl2). The levels of 21 endogenous primary amines in effluent collected from the scala vestibuli were determined by gradient‐elution, reverse‐phase HPLC using o‐phthaldialdehyde‐thiol adducts with fluorescence detection. Analyses indicated effluent concentrations of glutamate, taurine, and a coeluting taurine‐γ‐aminobutyrate (GABA) fraction (but not GABA alone) increased significantly after exposure to 50 mM KCl and returned to baseline levels after reintroduction of 5 mM KCl under normal divalent‐cation conditions. Correspondent changes in the release of these constituents were significantly attenuated under calcium‐deficient conditions. This was not the case for potassium‐induced changes in the release of arginine, aspartate, and isoleucine. These data are consistent with the hypothesis that the receptoneuronal transmitter is glutamate and further suggest a calcium‐dependent mechanism involving taurine.
Expression of Glial Fibrillary Acidic Protein in Rat C6 Glioma Relates to Vimentin and Is Independent of Cell‐Cell Contact
Authors: Backhovens H. , Gheuens J. , Siegers H. 
Published: October 2006

AbstractGlial fibrillary acidic protein (GFAP) was induced in rat C6 glioma cells grown in M199 and HAM F10 media by addition of 1 mM dibutyryl cyclic AMP. The amount of GFAP per cell increased 7‐and 33‐fold in M199 and HAM F10 media, respectively. GFAP could be induced in each phase of the cell culture except for the lag phase, where GFAP synthesis was delayed until the onset of the logarithmic growth. The induction took place under conditions where the total protein content of the cell decreased. Mear surement of the amount of vimentin indicated that GFAP was induced under conditions of low vimentin concentration. Our results do not support the hypothesis that GFAP induction depends on cell‐cell contact or cell proliferation. They indicate a shift from vimentin to GFAP synthesis by an as yet unknown mechanism.
Thiamine Triphosphate and Membrane‐Associated Thiamine Phosphatases in the Electric Organ of Electrophorus electricus
Authors: Bettendorff Lucien , Michel‐Cahay Colette , Grandfils Christian , Rycker Cécile De , Schoffeniels Ernest 
Published: October 2006

AbstractThe main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri‐, pyro‐, and monophos‐phate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane‐associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.
Nature and Immunochemical Characteristics of a Ca2+/Calmodulin Kinase Activity Endowed in a Highly Insoluble Protein Purified from Adult Rat Brain
Authors: Bezamahouta C. , Zanetta J. P. , Revel M. O. , Zwiller J. , Meyer A. , Malviya A. N. , Vincendon G. 
Published: October 2006

AbstractUsing sequential extraction procedure of proteins from adult rat forebrain, a protein of Mr 52,000, insoluble in neutral detergents, capable of binding calmodulin in the presence of Ca2+, was isolated. Antibodies to this antigen had the capacity to inhibit the Ca2+/calmodulin‐dependent kinase activity associated with this protein. This protein (52K) (in many respects identical to the major protein of postsynaptic densities) shares by itself the Ca2+/calmodulin‐dependent kinase activity, thus differing from soluble Ca2+/calmodulin‐dependent kinases isolated by others. Despite its insolubility in most detergents, the 52K protein is not particularly rich in hydrophobic amino acids. Its richness in cysteine and proline residues suggests that the active conformation of the enzyme is sustained by numerous disulfide bridges.
Mechanism of Kainate Toxicity to Cerebellar Neurons In Vitro Is Analogous to Reperfusion Tissue Injury
Authors: Dykens James A. , Stern Arnold , Trenkner Ekkhart 
Published: October 2006

Abstract:The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate‐induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine ox‐idase, a cellular source of cytotoxic superoxide radicals (O2‐). Moreover, neurons are also protected from excitotoxin‐induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin‐induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.
Evidence for Disulfide Bonds in Membrane‐Bound and Solubilized Opioid Receptors
Authors: Kamikubo Keita , Murase Hiroshi , Murayama Masanori , Matsuda Masafumi , Miura Kiyoshi 
Published: October 2006

AbstractThe effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane‐bound and digitonin‐solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D‐Ala2‐D‐Leu5‐enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin‐solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2, [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.
Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells
Authors: Marriott Deborah , Adams Maria , Boarder Michael R. 
Published: October 2006

AbstractTreatment of adrenal chromaffin cells with forskolin (0.1–10 μM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20–17–24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine‐induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine‐stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium‐ and nicotine‐stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura‐2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 μM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.
Hypoxanthine Transport and Metabolism in the Central Nervous System
Authors: Spector Reynold 
Published: October 2006

AbstractThe mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypo‐xanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [I4C]hypoxanthine; however, l4C was readily released from choroid plexus principally as [I4C]‐hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid‐soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [l4C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C‐nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.Key Words: Blood‐brain barrier—Cerebrospinal fluid—Choroid plexus. Spector R. Hypoxanthine transport and metabolism in the central nervous system.
A Putative M3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK‐N‐SH Neuroblastoma Cells
Authors: Fisher Stephen K. , Heacock Anne M. 
Published: October 2006

AbstractThe M1‐selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N‐[3H]methylscopolamine ([3H]NMS) and [3H]qui‐nuclidinylbenzilate from intact human SK‐N‐SH neuroblastoma cells with a low affinity (Ki= 869–1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF‐DX 116 {11–2[[2‐[(diethylamino)methyl]‐1‐piperidinyl]‐acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepin‐6‐one} bound to the mAChRs with a very low affinity (Ki= 6.0 μM), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki= 7.2 nM). 4‐DAMP was also 1,000‐fold more effective than AF‐DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilylcholine mustard) suggest that the size of the SK‐N‐SH mAChR (Mr= 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr=66,000), an M1‐enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.
Polyamines, Ornithine Decarboxylase, and Diamine Oxidase in the Substantia Nigra and Striatum of the Male Rat After Hemitransection
Authors: Desiderio Maria Alfonsina , Zini Isabella , Davalli Pierpaola , Zoli Michele , Corti Arnaldo , Fuxe Kjell , Agnati Luigi Francesco 
Published: October 2006

AbstractPartial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosyn‐thetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.
Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N‐Methyl‐d‐Aspartate Receptor Ligands
Authors: Ambar Ifat , Kloog Yoel , Sokolovsky Mordechai 
Published: October 2006

AbstractPhencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20–30% of the high‐affinity PCP binding sites were solubilized. The high affinity toward PCP‐like drugs, the stereo‐selectivity of the sites, and the sensitivity to N‐methyl‐d‐aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N‐[3H][l‐(2‐thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD= 35 nM), and PCP‐like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane‐bound receptors; the most potent inhibitors were TCP (Ki= 31 nM) and the anticonvulsant MK‐801 (Ki= 50 nM). The NMDA receptor antagonist 2‐amino‐5‐phosphonovaleric acid inhibited‐binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose‐dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.
Late‐Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108‐15 Cells
Authors: Hatanaka Michiyo , Yumoto Noboru , Miwa Naoto , Morii Hiroshi , Tanemura Mihoko , Ueno Ryuji , Watanabe Yasuyoshi , Hayaishi Osamu 
Published: October 2006

Abstract: The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108‐15 cells preincubated with myo‐[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15‐60 min) of incubation in the cells. PGD2 and PGF2adid not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP1) formation at a late phase of incubation. The maximum stimulation was obtained at <10−7M concentrations of these PGs. the levels being three‐and twofold for IP2 and IP1 respectively. 9α, 11 β‐PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2α have no effect up to 10−6M. The effects of PGD2 and PGF2α were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation.Inositol 1‐monophosphate was mainly identified in the stimulation by 10−5 M PGD2 and 10−5MPGF2a, whereas both inositol 1‐monophosphate and inositol 4‐monophosphate were produced in the stimulation by 10−5M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2α and late‐phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late‐phase effects. These results suggest that PGD2 and PGF2α specifically stimulate hydrolysis of inositol phospholipids.
Occurrence in Brain Lysosomes of a Sialidase Active on Ganglioside
Authors: Fiorilli Amelia , Venerando Bruno , Siniscalco Carola , Monti Eugenio , Bresciani Roberto , Caimi Luigi , Preti Augusto , Tettamanti Guido 
Published: October 2006

AbstractA lysosomal preparation, obtained from brain ho‐mogenate of 17‐day‐old C57BL mice by centrifugation on a self‐generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40–120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GDla and fluorimetrically with 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid (MUB‐NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane‐bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma Membrane‐bound enzyme. The apparent Km values for GDl a and MUB‐NeuAc were 1.5 × 10‐5 and 4.2 × 10‐5M, respectively, for the lysosomal enzyme and 2.7 × 10‐4 and 6.3 × 10‐5M for the plasma membrane‐bound one. Triton ×‐ 100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane‐bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane‐bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane‐bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers. It is thus evident that the sialidase present in the lysosomal fraction is an authentic lysosomal enzyme distinct and different from the sialidase contained in the plasma membrane. The lysosomal sialidase affected other ganglio‐sides, like GDlb and GM3. These data constitute the first direct evidence for the presence in brain lysosomes of a sialidase activity on gangliosides and contribute to a better knowledge of ganglioside breakdown and turnover in the brain.
Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate‐Enzyme Interactions
Authors: Bettendorff Lucien , Grandfils Christian , Wins Pierre , Schoffeniels Ernest 
Published: October 2006

AbstractThe main eJectric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4′‐diisothiocyanostilbene‐2.2′‐disul‐fonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimium is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Am (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3 (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3 increases the Vmax 24‐fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10‐5‐10‐6M) substrate concentrations. Lineweaver‐Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [γ‐32P]TTP. Our data suggest the existence of a high‐affinity binding site (KD of ˜0.5 μM) for the Mg‐TTP complex. The membrane‐bound thiamine triphosphatase is insensitive to low concentrations of vanadate and is not activated by protonophores. These and other features suggest that this enzyme is quite distinct from most of the known membrane ATPases and that it may have a specific function in excitable cell membranes, possibly in relation to their permeability to certain anions.
Postnatal Development of Cholecystokinin‐Like Immunoreactivity and Its mRNA Level in Rat Brain Regions
Authors: Takeda Katsumichi , Koshimoto Hiroyuki , Uchiumi Fumiaki , Haun Randy S. , Dixon Jack E. , Kato Takeshi 
Published: October 2006

AbstractDevelopmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin‐like immunoreactivity (CCK‐LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK‐LI levels in all regions examined were Very low at birth. Excluding the cerebellum, the levels in these Regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK‐LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK‐LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK‐LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK‐LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK‐LI is supplied from extrastriatal regions. These results suggest that (a) CCK gene expression, numbers of CCK neurons, or both may increase from 7 to 14 days of age and (b) differential regulation of CCK gene expression and posttranscriptional regulatory mechanisms may exist in the various brain regions during development.