bluuebunny/crossref_metadata_2025 huggingface dataset:
A Hydroxyproline‐Containing Protein from Shark Brain That Is Related to Myelin Basic Protein
Authors: Wood D. D. , McLaurin J. , Moscarello M. A.
Published: October 2006

Abstract: Myelin basic protein (MBP) from shark (Chondricthyes) consists of a simpler mixture of charge isomers than human MBP. About two‐thirds of the total amount applied to a CM‐52 cellulose cation‐exchange column was recovered in the unbound fraction of the column; the remaining one‐third bound to column and was eluted as a single OD280 peak. This bound material did not show the usual pattern of charge microheterogeneity found with human or bovine MBP. The unbound fraction was composed of a high molecular weight protein (55–60 kDa), which constituted most of this protein fraction and a low molecular weight protein (∼ 18 kDa). The amino acid composition of our unbound fraction was similar to that reported earlier. The Glx (glutamic acid + glutamine) was increased about threefold whereas the Arg content was only about 25% of that of the 18.5 kDa variant of bovine or human origin. The presence of hydroxyproline (1.2 residues/100) in this protein was noteworthy, identification of which was achieved by amino acid analysis in two different systems and by mass spectrometry. In the precolumn derivatization method, hydroxyproline eluted at 2.7 min; in the postcolumn derivatization method it eluted at 12.2 min. Identification of hydroxyproline was completed by fast atom bombardment‐mass spectral analysis. The effect of hydroxyproline on the secondary structure of this protein is being studied. Verification that this high molecular weight protein contained MBP sequences within its primary structure was confirmed by immunological methods. Good reactivity, comparable to that found with human MBP was observed in the enzyme‐linked immunosorbent assay with an antibody (S‐53) raised in rats against the synthetic peptide GSLPQKAQRPQDEN. The fraction that bound to the CM‐52 cellulose column and was eluted with a salt gradient corresponded in net charge to component 5 of the bovine or human MBPs as deduced by alkaline gels.
Phosphorylation of Charge Isomers (Components) of Human Myelin Basic Protein: Identification of Phosphorylated Sites
Authors: Ramwani J. , Moscarello M. A.
Published: October 2006

Abstract: Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM‐52 column chromatography. Two of the components, C‐1 and C‐4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N‐terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5‐I3 was ∼10 times greater than that of any of the other peptides derived from C‐1, while the rate of phosphorylation of peptide 5‐I3 derived from C‐4 was 10‐20 times greater than that of any of the other peptides derived from C‐4. In addition, peptide 5‐13, which contained a major phosphorylation site in both C‐1 and C‐4, was phosphorylated at a faster rate in C‐4 (460 cpm/nM/min) compared with C‐1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of p‐structure in a previous study. In that study, C‐1, which contained about 13% & structure before phosphorylation, increased to −40% after phosphorylation. Construction of a model peptide of this N‐terminal region, which included the phosphorylation site at Ser7, demonstmted that the β structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidy groups of Arg5 and Arg9. On the other hand, phosphorylation of C‐4, which was already −34% P‐structure, was twice as fast at Ser7 as for C‐I, suggesting that the preexistence of 8‐structure in the molecule facilitated phosphorylation at this site. In previous studies phosphorylation of C‐I was shown to decrease vesicle aggregation, which we concluded to be the result of electrostatic repulsion between the phosphate on the protein and that of the lipid. The present data suggest that phosphorylation of some specific site may be playing an important role in the conformation of the protein.
Glutamate‐Stimulated, Guanine Nucleotide‐Mediated Phosphoinositide Turnover in Astrocytes Is Inhibited by Cyclic AMP
Authors: Robertson Patricia L. , Bruno George R. , Datta Subhash C.
Published: October 2006

Abstract: The potential for cross‐talk between the adenyl cy‐clase and phosphoinositide (PPI) lipid second messenger system was investigated in astrocytes cultured from neonatal rat brain. Glutamate‐stimulated PPI turnover, measured by the formation of total inositol phosphates from myo‐[3H]inositoI‐labeled lipids, was inhibited in a concentration‐dependent manner by the elevation of intracellular cyclic AMP levels produced either by stimulation of the isoproter‐enol receptor linked to adenyl cyclase or by its direct activation by forskolin. N6,2′‐O‐Dibutyryl cyclic AMP, an analogue that can also activate cyclic AMP‐dependent kinase, inhibited glutamate‐stimulated PPI turnover in a concentration‐dependent manner as well, a result suggesting that cyclic AMP‐dependent kinase is involved in mediating the inhibition. Inclusion of an inhibitor of cyclic AMP‐dependent kinase, l‐(5‐isoquinolinesulfonyl)‐2 methylpiperazine dihy‐drochloride or N‐(2‐guanidinoethyl)‐5‐isoquinolinesulfon‐amide hydrochloride, blocked the cyclic AMP‐mediated inhibition in a concentration‐dependent manner, a finding further supporting this hypothesis. The site of inhibition of the phosphoinositol lipid pathway by cyclic AMP was probed using a digitonin‐permeabilized cell system. Guanosine 5′‐O‐(3‐thiotriphosphate), a nonhydrolyzable analogue of GTP, stimulated PPI turnover and potentiated glutamate‐stimulated PPI turnover, and guanosine 5′‐O‐(3‐thiodiphosphate) inhibited glutamate‐stimulated PPI turnover in these cells, results providing evidence that glutamate receptors are coupled to phospholipase C by a guanine nucleotide binding protein in astrocytes. N6,2′‐O‐Dibutyryl cyclic AMP and agents that elevate cyclic AMP levels inhibited the PPI turnover stimulated by guanosine 5′‐O‐(3‐thiotriphosphate), as well as that potentiated by guanosine 5′‐O‐(3‐thiotriphos‐phate) in the presence of glutamate, results suggesting that the cyclic AMP‐dependent inhibition occurs at or distal to the putative guanine nucleotide binding protein. Because basal PPI turnover was not altered by elevation of cyclic AMP levels, direct inhibition of phospholipase C is unlikely.
Dynamics of Phosphorylation and Assembly of the High Molecular Weight Neurofilament Subunit in NB2a/d1 Neuroblastoma
Authors: Shea Thomas B. , Sihag Ram K. , Nixon Ralph A.
Published: October 2006

Abstract: In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton‐insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/dl neuroblastoma cells also contain Triton‐soluble, extensively phosphorylated 200‐kDa high molecular weight neurofilament subunits (NF‐H). High‐speed centrifugation (100,000 g) of the Triton‐soluble fraction for 1 h sedimented some, but not all, soluble NF‐H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF‐H subunits in this fraction are assembled into (Triton‐soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton‐soluble 200‐kDa NF‐H variants. Because only extensively phosphorylated NF‐H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse‐chase analyses, radiolabeled 200‐kDa NF‐H migrated into the 100,000 g particulate fraction of Triton‐soluble extracts before its arrival in the Triton‐insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton‐insoluble, extensively phosphorylated NF‐H, contain a sizeable pool of Triton‐soluble extensively phosphorylated NF‐H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF‐H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF‐H into neurofilaments and integration into the Triton‐insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides. Because NF‐H can be extensively phosphorylated in perikarya, additional mechanisms besides differential localization of the responsible kinase systems must account for the segregation of Triton‐insoluble NF‐H in NB2a/d1 neurites.
Novel Glutamate Receptor Antagonists Selectively Protect Against Kainic Acid Neurotoxicity in Cultured Cerebral Cortex Neurons
Authors: Frandsen Aase , Krogsgaard‐Larsen Povl , Schousboe Arne
Published: October 2006

Abstract: The effect on excitatory amino acid (EAA)‐induced toxicity of two novel non‐N‐methyl‐d‐aspartate (non‐NMDA) antagonists 2‐amino‐3‐[3‐(carboxymethoxy)‐5‐methylisoxazol‐4‐yl]propionic acid (AMOA) and 2‐amino‐3‐[2‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)methyl‐5‐methyl‐3‐oxoisoxazolin‐4‐yl]propionic acid (AMNH) was tested in primary cultures of cerebral cortex neurons. Such cultures provide a useful model for the investigation of the toxicity of EAAs and a convenient screening system for potential neuroprotective activity of pharmacological agents. It was demonstrated that AMNH and AMOA abolished neurotoxicity induced by kainic acid with IC50 values of 62 ± 10 and 120 ± 19 μM, respectively. No effect on neuronal damage induced by NMDA or AMPA could be detected.
Some Characteristics of a Peptidyl Dipeptidase (Kininase II) from Rat CSF: Differential Effects of NaC1 on the Sequential Degradation Steps of Bradykinin
Authors: Yoshida Takeshi , Nosaka Shoichiro
Published: October 2006

Abstract: Incubation of various authentic peptides with rat CSF in vitro and analysis of their products by HPLC demonstrated the presence in CSF of a peptidyl dipeptidase [peptidyl dipeptide hydrolase; angiotensin I converting enzyme (ACE); kininase II; EC 3.4.15.1] which sequentially degraded bradykinin (BK) by liberating the carboxy‐terminal dipeptides and converted angiotensin I to angiotensin II. This CSF enzyme was gel‐chromatographed by means of HPLC., and the molecular weight was estimated. The susceptibility to various peptidase inhibitors of the rat CSF enzyme, as well as the effect of NaC1 on the degradation of BK and Hip‐His‐Leu catalyzed by it, was also determined. These properties were compared with those of ACE or kininase II from brain or other tissues, as described in the literature. NaC1 was shown to exert specific and concentration‐dependent effects on each step of the sequential degradation of BK, via BK(1–7) to BK(l–5), catalyzed by the enzyme. In addition, the enzyme system for metabolism of BK appears to differ between rat CSF and blood, the former containing exclusively kininase II, whereas the latter contains both kininase I (carboxypeptidase N; EC 3.4.12.7) and kininase II.
Alterations in the Production of 14CO2 and [14C]Acetylcholine from [U‐14C]Glucose in Brain Subregions Following Transient Forebrain Ischemia in the Rat
Authors: Zaidan Emad , Sims Neil R.
Published: October 2006

Abstract: The production of 14CO2 and [14C]acetylcholine from [U‐14C]glucose was determined in vitro using tissue prisms prepared from the dorsolateral striatum (a region developing extensive neuronal loss following ischemia) and the paramedian neocortex (an ischemia‐resistant region) following 30 min of forebrain ischemia and recirculation up to 24 h. Measurements were determined under basal conditions (5 mMK+) and following K+ depolarization (31 mM K+). The production of 14CO2 by the dorsolateral striatum was significantly reduced following 30 min of ischemia for measurements in either 5 or 31 mM K+ but recovered toward preischemic control values during the first hour of recirculation. Further recirculation resulted in 14CO2 production again being reduced relative to control values but with larger differences (20–27% reductions) detectable under depolarized conditions at recirculation times up to 6 h. Samples from the paramedian neocortex showed no significant changes from control values at all time points examined. [14C]Acetylcholine synthesis, a marker of cholinergic terminals that is sensitive to changes in glucose metabolism in these structures, was again significantly reduced only in the dorsolateral striatum. However, even in this tissue, only small (nonstatistically significant) differences were seen during the first 6 h of recirculation, a finding suggesting that changes in glucose oxidation during this period were not uniform within all tissue components. The results of this study provide evidence that in a region susceptible to ischemic damage there were specific changes during early recirculation in the metabolic response to depolarization. This apparent inability to respond appropriately to an increased need for energy production could contribute to the further deterioration of cell function in vivo and ultimately to the death of some cells.
Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP‐Induced Differentiation
Authors: Mullaney Ian , Milligan Graeme
Published: October 2006

Abstract: Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the α‐subunit of the (pertussis toxin‐sensitive) guanine nucleotide binding protein Go were used in two‐dimensional immunoblots of membranes of neuroblastoma × glioma (NG108–15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108–15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8‐bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of Goα, as has previously been noted in onedimensional immunoblots. Two‐dimensional analysis demonstrated that the cAMP‐induced increases in levels of Goα were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin‐catalysed ADP‐ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of Goα could be achieved in one‐dimensional sodium dodecyl sulphate‐polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of Go from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of Go from the cells. In agreement with the data from two‐dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP‐induced differentiation of NG108–15 cells. Of these two forms of “Go,” the acidic species is equivalent to Go from brain, but the basic form is not identical with Go, which has been purified from bovine brain.
Rapid Rise in Transcription Factor mRNAs in Rat Brain After Electroshock‐Induced Seizures
Authors: Cole Andrew J. , Abu‐Shakra Sawsan , Saffen David W. , Baraban Jay M. , Worley Paul F.
Published: October 2006

Abstract: Recent studies have demonstrated that several transcription factor genes are rapidly activated by neuronal stimulation. For example, we have found that prolonged and repeated seizure activity produced by administration of chemical convulsants induces a rapid and transient increase in mRNA levels of four immediate early genes in rat brain. These genes, zif/268, c‐fos, c‐jun, andjun‐B, encode sequence specific DNA binding proteins thought to act as transcription regulatory factors. To ascertain whether a brief electrically induced seizure discharge of the type utilized in clinical electroconvulsive treatment is sufficient to induce a similar genomic response, we have examined the response of these mRNAs in rat brain following single and repeated electroshock‐induced seizures. After electroshock, mRNA levels of each of these genes increase within 15 min, and all except cjun return to near baseline levels within 4 h. Although this response is most prominent in granule cell neurons of the hippocampus, increases are also apparent in neocortex and pyriform cortex. The rapid mRNA response persists in animals receiving a chronic electroshock protocol similar to that used in clinical electroconvulsive therapy. Intrahippocampal infusion of the sodium channel antagonist tetrodotoxin blocks hippocampal mRNA responses without blocking seizures, indicating a role for electrical excitation in the electroshockinduced mRNA response. By contrast, pretreatment with anticonvulsants or selective NMDA antagonists, which reduce seizure intensity and block hindlimb extension, fails to alter mRNA responses, suggesting that seizure induction, rather than spread, is linked to these mRNA responses. Because electroshock induces robust, highly reproducible mRNA responses, it may be useful to study the neuronal genomic response to stimulation.
In Vivo Measurement of Extracellular Cyclic AMP in the Brain: Use in Studies of β‐Adrenoceptor Function in Nonanesthetized Rats
Authors: Stone Eric A. , John Safy M.
Published: October 2006

Abstract: Microdialysis measurement of extracellular cyclic AMP (cAMP) in the cerebral cortex of conscious rats was evaluated as a method for assessing central β‐adrenoceptor function in vivo. Extracellular levels of the nucleotide were found to average 3 pmol/ml under baseline conditions. Local infusion of the β‐agonists norepinephrine (NE) and isoproterenol produced rapid (3 min) and marked (three‐ to sevenfold) dose‐dependent increases in extracellular cAMP. which were potentiated by the phosphodiesterase inhibitor rolipram, and blocked by the β‐antagonist timolol. Responses to both catecholamines underwent rapid desensitization (6–9 min) and recovered within several hours. Time‐course studies revealed that the baseline cAMP level underwent a gradual increase and then a decrease over the course of a single 8‐h run, and peaked at 24 h postimplantation. Responses to NE were stable for the first 24 h after implantation, then increased at 48 and 120 h. The causes of the latter changes may include reactions to novelty, local inflammatory responses, and/or reactions of adjacent glial cells to implantation. Overall, the results indicate that the microdialysiscAMP method can be extended to nonanesthetized animals and may be a useful tool for studying neurotransmission at central adenylate cyclase‐coupled membrane receptors during various behavioral states.
Purification and Characterization of a Novel Brain‐Specific 14‐kDa Protein
Authors: Nakajo Shigeo , Omata Kumiko , Aiuchi Toshihiro , Shibayama Toshiko , Okahashi Ikuko , Ochiai Hidehiko , Nakai Yasumitsu , Nakaya Kazuyasu , Nakamura Yasuharu
Published: October 2006

Abstract: A new acidic protein specifically present in the brain was purified to homogeneity from bovine brain. The apparent molecular mass was estimated to be 14 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and 57 kDa by gel filtration, a finding suggesting that it exists as a tetramer under physiological conditions. The protein had a high content of Glu and Pro, and its pI was 4.3. The first six amino acid residues of the protein were Met‐Asp‐Val‐Phe‐Met‐Lys, and the amino terminal was blocked. The distribution of the protein examined by Ouchterlony gel immunodiffusion indicates that it is present specifically in brain, including rat, human, and bovine, but could not be detected in 10 other rat tissues examined. The protein was absent in Purkinje cell bodies, as examined by electron microscopic immunocyto‐chemistry, but was present in nerve terminals that make synapse‐like contacts with Purkinje cells and in neurons with dark granules in the globus pallidus of the rat.
Developmental Study of Proteolipids in Bovine Brain: A Novel Proteolipid and DM‐20 Appear Before Proteolipid Protein (PLP) During Myelination
Authors: Schindler Patrick , Luu Bang , Sorokine Odile , Trifilieff Elisabeth , Van Dorsselaer Alain
Published: October 2006

Abstract: In a developmental study, we have shown that DM‐20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27–30 weeks of gestation), the amount of DM‐20 drastically increases. DM‐20 remains the major proteolipid until birth. PLP is detected only 2–4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM‐20. The early appearance of DM‐20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM‐20 remains quantitatively important because the PLP/DM‐20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM‐20 and is not detected in the adult brain. It is structurally related to PLP and DM‐20 because the first 31 N‐terminal amino acid residues are the same. However, in immunoblot. it did not react either with the antitridecapeptide 117–129 antiserum of PLP or with the anti‐C‐terminal hexapeptide antiserum of PLP.
Uptake of Glutamate and Cystine in C‐6 Glioma Cells and in Cultured Astrocytes
Authors: Cho Yukie , Bannai Shiro
Published: October 2006

Abstract: The uptake of glutamate in rat glioma C‐6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na+‐dependent and a Na+‐independent system. The Na+‐dependent system was inhibited by aspartate and was consistent with the commonly occurring system designated system X−ag. The Na+‐independent system was inhibited by cystine and was consistent with system x−c described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na+‐independent glutamate uptake. In C‐6 cells, the glutamate uptake via systems X−ag and x−c accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes, the glutamate uptake via system X−ag was very potent, whereas the uptake via system x−c was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C‐6 cells and astrocytes, system x−c was necessary for the uptake of cystine, another substrate of system x−c. Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration.
Nerve Growth Factor Enhances the Expression of the Cholinergic‐Specific Ganglioside Chol‐1 in Cocultures of Rat Septum and Hippocampus
Authors: Derrington Edmund A. , Gähwiler Beat H. , Whittaker Victor P.
Published: October 2006

Abstract: Cocultures of septal and hippocampal tissues taken from 6‐ to 7‐day‐old rats were maintained in culture for up to 30 days in the presence and absence of nerve growth factor (NGF). and their Chol‐1 contents determined at varying time intervals by a modified enzyme‐linked immunosorption assay (ELISA). The major brain gangliosides were determined densitometrically after spraying chromatograms with resorcinol reagent. There was little change in the contribution of the major gangliosides to the total ganglioside content of the explants either with time or the presence or absence of NGF, the only exception being an NGF‐insensitive fall in the contribution of GM1 to about 60% of its initial value at 20 and 30 days. By contrast, the concentration of Chol‐1 expressed either per unit weight of ganglioside sialic acid or protein increased considerably in culture and this increase was enhanced by NGF. The effect of NGF resembles that on other cholinergic markers, choline acetyltransferase and acetylcholinesterase, and may be attributed to an NGF‐stimulated hippocampal ingrowth of cholinergic fibres and enhanced survival of cholinergic septal neurons. The Chol‐1 concentration finally attained in the presence of NGF and the time course of its increase parallel those previously found in vivo and indicate the potential usefulness of scptal‐hippocampal cocultures for investigating the function of Chol‐1.
Cannabinoid Receptors and Modulation of Cyclic AMP Accumulation in the Rat Brain
Authors: Bidaut‐Russell Michelle , Devane William A. , Howlett Allyn C.
Published: October 2006

Abstract: The mechanism by which cannabinoid compounds produce their effects in the rat brain was evaluated in this investigation. Cannabinoid receptors, quantitated by [3H]CP‐55,940 binding, were found in greatest abundance in the rat cortex, cerebellum, hippocampus, and striatum, with smaller but significant binding also found in the hypothalamus, brainstem, and spinal cord. Using rat brain slice preparations, we evaluated the effect of desacetyllevonantradol on basal and forskolin‐stimulated cyclic AMP accumulation in the regions exhibiting the greatest cannabinoid receptor density. Desacetyllevonantradol (10 μM) reduced cyclic AMP levels in the hippocampus, frontal cortex, and striatum. In the cerebellum, however, the response to desacetyllevonantradol was biphasic with cyclic AMP accumulation being decreased at lower and increased at higher concentrations. Desacetyllevonantradol reduced cyclic AMP accumulation in isoproterenol‐stimulated slices in the cortex and cerebellum, but not in the hippocampus. Cells that responded to vasoactive intestinal peptide with an increase in cyclic AMP accumulation in the hippocampus and cortex also responded to desacetyllevonantradol. The modulation of cyclic AMP accumulation by desacetyllevonantradol could be attenuated following stereotaxic implantation of pertussis toxin, supporting the involvement of a G protein in the cannabinoid response in the brain. However, other actions of cannabinoid compounds may also affect the cyclic AMP levels in brain slice preparations.
Competitive Inhibition of N‐Acetylated‐α‐Linked Acidic Dipeptidase Activity by N‐Acetyl‐L‐Aspartyl‐β‐Linked L‐Glutamate
Authors: Serval V. , Barbeito L. , Pittaluga A. , Cheramy A. , Lavielle S. , Glowinski J.
Published: October 2006

Abstract: The endogenous neuropeptide N‐acetyl‐L‐aspartyl‐L‐glutamate (NAAG) fulfills several criteria required to be accepted as a neurotransmitter. NAAG inactivation may proceed through enzymatic hydrolysis into N‐acetyl‐L‐aspartate and glutamate by an N‐acetylated‐α‐linked acidic dipeptidase (NAALADase). Therefore, some properties of NAA‐LADase activity were investigated using crude membranes from the rat forebrain. Kinetic parameters of the hydrolysis of [Glu‐3H]NAAG were determined first (Km= 0.40 ± 0.05 μM; Vmax= 155 ± 20 pmol/min/mg of protein). The enzymatic activity, i.e., NAALADase, was inhibited noncompetitively by the glutamatergic agonist quisqualate (Ki= 1.9 ± 0.3 μM), and competitively by N‐acetyl‐L‐aspartyl‐β‐linked L‐glutamate (β‐NAAG; Ki= 0.70 ± 0.05 μM). To determine whether glutamate‐containing dipeptides, such as NAAG, β‐NAAG, N‐acetyl‐L‐aspartyl‐D‐glutamate, L‐aspartyl‐L‐glutamate, L‐alanyl‐L‐glutamate, L‐glutamyl‐L‐glutamate, and L‐glutamyl‐γ‐linked L‐glutamate, were substrates of NAA‐LADase, rat brain membranes were immobilized on a C‐8 column. Thus, endogenous trapped glutamate was washed away and formation of unlabelled glutamate could be estimated using an o‐phthaldialdehyde/reverse‐phase HPLC detection procedure. β‐NAAG was shown to be a nonhydrolyzable competitive inhibitor of NAALADase. L‐Aspartyl‐L‐glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety is not essential for NAALADase specificity. Rat brain membranes also contained nonspecific peptidase activities (insensitive to both quisqualate and β‐NAAG), which, in the case of L‐alanyl‐L‐glutamate, for instance, accounted for all observed hydrolysis.
Modulation of Histamine Release in the Rat Brain by K‐Opioid Receptors
Authors: Gulat‐Marnay Christiane , Lafitte Andrée , Arrang Jean‐Michel , Schwartz Jean‐Charles
Published: October 2006

Abstract: The opioid modulation of histamine release was studied in rat brain slices labeled with L‐[3H]histidine. The K+‐induced [3H]histamine release from cortical slices was progressively inhibited by the preferential k‐agonists ketocyclazocine, dynorphin A (1–13), Cambridge 20, spiradoline, U50,488H, and U69,593 in increasing concentrations. In contrast, the μ‐agonists morphine, morphiceptin, and Tyr‐D‐Ala‐Gly(NMe)Phe‐Gly‐ol (DAGO) were ineffective as were the preferential δ‐agonists [D‐Ala2,D‐Leu5]enkephalin (DADLE) and [D‐Pen2,D‐Pen5]enkephalin (DPDPE). Nor‐binaltorphimine (nor‐BNI) and MR 2266, two preferential k‐antagonists, reversed the inhibitory effect of the various k‐agonists more potently than did naloxone, with mean Ki values of 4 nM and 25 nM, respectively. The effects of ketocyclazocine and naloxone also were seen in slices of rat striatum, another brain region known to contain histaminergic nerve endings. We conclude that k‐opioid receptors, presumably located on histaminergic axons, control histamine release in the brain. However, nor‐BNI and naloxone failed, when added alone, to enhance significantly [3H]histamine release from cerebral cortex or striatum, and bestatin, an aminopeptidase inhibitor, failed to decrease K+‐evoked [3H]histamine release. These two findings suggest that under basal conditions these K‐opioid receptors are not tonically activated by endogenous dynorphin peptides. The inhibition of cerebral histamine release by K‐agonists may mediate the sedative actions of these agents in vivo.
Characterization of β‐Amyloid Precursor Proteins With or Without the Protease‐Inhibitor Domain Using Anti‐Peptide Antibodies
Authors: Shelton Earl R. , Cohn Ronald , Fish Linda , Obernolte Rena , Tahilramani Ram , Nestor John J. , Chan Hardy W.
Published: October 2006

Abstract: Alternative splicing of the transcript encoding the β‐amyloid precursor protein (BAPP) of Alzheimer's disease produces multiple mRNA species. Translation of these mRNAs predicts protein products of 770, 751, and 695 amino acids. The difference arises from the inclusion in BAPP‐770/ 751 of a 56‐residue insert region which is homologous to Kunitz‐type protease inhibitors. We have prepared and affinity‐purified anti‐peptide antibodies that react specifically with either BAPP‐770/751 (inssert‐specific) or BAPP‐695 (junction‐specific). A detectable level of the mRNA corresponding to the BAPP‐770/751 protein was found in all cell lines tested. Immunoprecipitation of 35S‐labeled proteins from these cell lines showed them to contain one or two Mr 105,000 bands reactive with the insert‐specific serum, i‐291. In contrast, only cos‐7 cells and the human neuroblastoma cell line, IMR‐32, contained mRNA species that encode the BAPP 695 protein, as shown by Northern analysis with a junction spanning oligonucleotide probe. A band of Mr 95,000 was immunoprecipitated specifically from these two cell lines using the junction‐specific serum, J‐284. Indirect immunofluorescence labeling of cells corroborated these findings. All cells reacted with the insert‐specific antibodies, i‐291 and i 324. Only cos‐7 and IMR‐32 cells reacted with the junction specific antibody, J‐284. These results demonstrate the use fulness of anti‐peptide antibodies for the differential detection of the BAPP‐695 and BAPP‐770/751 proteins.
Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster
Authors: Trapido‐Rosenthal Henry G. , Carr William E. S. , Gleeson Richard A.
Published: October 2006

Abstract: The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5′‐ectonucleotidase, ecto‐ADPase, and ecto‐ATPase activities of this olfactory system showed that each activity was characterized by Michaelis–Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 μM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5′‐ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto‐ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.
Tetrahydrobiopterin Synthesis Rate and Turnover Time in Neuronal Cultures from Embryonic Rat Mesencephalon and Hypothalamus
Authors: Kapatos Gregory
Published: October 2006

Abstract: 6‐(R)‐(L‐erythro‐1′,2′‐Dihydroxypropyl)‐2‐amino‐4‐hydroxy‐5,6,7,8‐tetrahydropteridine (tetrahydrobiopterin, BH4) synthesis rate and turnover time were estimated in cultures derived from the embryonic rat mesencephalon (MES) and hypothalamus (HYP) by following the decline in BH4 levels after blockade of BH4 biosynthesis by N‐acetylserotonin (NAS) or 2,4‐diamino‐6‐hydroxypyrimidine (DAHP). BH4 content of both culture systems decreased by 75% following an 8‐h incubation with maximally effective concentrations of NAS (200 μM) or DAHP (10 mM). Parameters describing BH4 metabolism were calculated from steady‐state levels of BH4 and first‐order rate constants determined by a nonlinear regression analysis of the exponential BH4 decline. These parameters were confirmed using an alternative procedure that examined the first‐order rate of recovery of BH4 following termination of BH4 synthesis inhibition. Steady‐state levels of BH4 in HYP cultures (70.3 ± 9.4 pg/culture) were significantly greater than that for MES (46.5 ± 2.8 pg/culture). The average fractional rate constants of BH4 loss for MES (0.153 ± 0.015/h) and HYP (0.159 ± 0.014/h) were equivalent. The calculated rate of BH4 synthesis was significantly greater for HYP (11.29 ± 2.13 pg/culture/h) than for MES (7.11 ± 0.85 pg/culture/h), owing to the greater steady‐state concentration of BH4. BH4 turnover time for MES (6.68 ± 0.67 h) and HYP (6.40 ± 0.62 h) and half‐life for MES (4.63 ± 0.46 h) and HYP (4.44 ± 0.43 h) did not differ. The turnover of the cofactor is thus rapid enough that alterations in its synthesis or degradation could acutely modify the rate of monoamine biosynthesis. These data also indicate that BH4 metabolism may be different between populations of dopamine‐containing neurons.
Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate
Authors: Yudkoff Marc , Pleasure David , Cregar Lynn , Lin Zhi‐Ping , Nissim Ilana , Stern Janet , Nissim Itzhak
Published: October 2006

Abstract: The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20–40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady‐state incubation medium with 0.05 mM L‐cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.
Effect of Extracellular Potassium on Ouabain‐Sensitive Consumption of High‐Energy Phosphate by Crayfish Giant Axons: A Study of the Energy Requirement for Transport in the Steady State
Authors: Lieberman Edward M. , Pascarella Jonathan , Brunder Donald , Hargittai Pa'l T.
Published: October 2006

Abstract: Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport “coupling ratio”and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady‐state consumption of high‐energy phosphate (∼P) [ATP and phospho‐L‐arginine (Arg‐P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total ∼P consumption (Q̇∼p) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q̇∼p averaged 52.8 ± 4.7%/h (n = 44) of the initial [ATP] + [Arg‐P]. Unlike total Q̇∼, the ouabain‐sensitive portion of Q̇∼p was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q̇∼p. to 8%/h, a result indicating that 85% of the total Q̇∼p was ouabain sensitive. For 1.35 mM K+o, the ouabain‐sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mMK+o, 41%. There was a small but significant increase in the ouabain‐sensitive Q̇∼p at 21.6 mM K+o, compared with Q̇∼p at 5.4 mM K+o. The pattern of effect of [K+]o on Q̇∼p was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/∼P stoichiometry of 3, an actual ratio of JNa/∼P stoichiometry of ∼33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero‐membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non‐steady‐state conditions.
Cyclic AMP and Butyrate Modulate Melatonin Synthesis in Y79 Human Retinoblastoma Cells
Authors: Wiechmann Allan F. , Kyritsis Athanassios P. , Fletcher R. Theodore , Chader Gerald J.
Published: October 2006

Abstract: Melatonin is synthesized by cultured Y79 human retinoblastoma cells and is secreted into the medium. Activity of the two key enzymes involved in the synthesis of melatonin, N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyl‐transferase (HIOMT), are present in retinoblastoma cells. The activity of these enzymes and the resulting synthesis and release of melatonin are modulated by the addition of a cyclic AMP analogue and butyrate to the culture medium. Melatonin levels increase dramatically over control levels after the addition of dibutyryl cyclic AMP (dbcAMP), whereas melatonin levels decrease after butyrate treatment. HIOMT activity is inhibited by both dbcAMP and butyrate, and NAT activity is stimulated by both of these differentiating agents, suggesting that the rise in melatonin levels in response to dbcAMP is the result of increased activity of NAT, whereas the decline in melatonin levels in response to butyrate may be due to a drop in HIOMT activity. Melatonin synthesis is dose‐ and time‐dependent, and the effect of dbcAMP is readily reversible, whereas the effect of butyrate does not appear to be reversible. These effects probably reflect basic differences in the regulatory mechanisms of the inducing agents.
Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase‐Dependent Opening of L‐Type Calcium Channels
Authors: Boess Frank G. , Balasubramanian M. K. , Brammer Michael J. , Campbell Iain C.
Published: October 2006

Abstract: In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura‐2, Incubation of fura‐2‐loaded synaptosomes with carbachol increased [Ca2+]i in a dose‐dependent manner (1–1,000 μM), with a maximum response of 22 × 2% at approximately 100 μM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 μM The effect of carbachol (100 μM) on [Ca2+], was antagonised by atropine, but not by hexamethonium (10 μM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L‐type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase‐dependent activation of 1,4‐dihydropyridinesensitive calcium channels.
Interaction of Strychnine‐Insensitive Glycine Binding with MK‐801 Binding in Brain Synaptic Membranes
Authors: Yoneda Yukio , Ogita Kiyokazu , Suzuki Takeo
Published: October 2006

Abstract: Strychnine‐insensitive [3H]glycine binding was detected in brain synaptic membranes treated with Triton X‐100 using a filtration assay method. The binding was a time‐dependent, inversely temperature‐dependent, and reversible process with a relatively high affinity for the neuroactive amino acid. Scatchard analysis revealed that Triton treatment doubled both the affinity and density of the binding sites, which consisted of a single component. The binding was not only displaced by structurally‐related amino acids such as D‐serine and D‐alanine, but also inhibited by some peptides containing glycine, including glycine methylester and N‐methylglycine. These ligands invariably potentiated the binding of [3H](+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]‐cyclohepten‐5,10‐imine ([3H]MK‐801), a noncompetitive antagonist for the N‐methyl‐D‐aspartate‐sensitive subclass of the central excitatory amino acid receptors, in a concentration‐dependent manner. Among various endogenous tryptophan metabolites, kynurenic acid significantly inhibited the strychnine‐insensitive [3H]glycine binding. The Triton treatment did not affect the pharmacological profile of [3H]MK‐801 binding sites. These results suggest that brain synaptic membranes treated with Triton X‐100 are useful in evaluating the strychnine‐insensitive and kynurenate‐sensitive binding sites of glycine, which are functionally linked to N‐methyl‐D‐aspartate‐sensitive receptor channels.
Reduction in β‐Adrenoceptor Density in Cultured Rat Glioma C6 Cells After Incubation with Antidepressants Is Dependent upon the Culturing Conditions Used
Authors: Fowler C. J. , Brännström G.
Published: October 2006

Abstract: The hydrophilic β‐adrenoceptor ligand (−)‐[3H]CGP‐12177 binds to intact C6 cells with a high affinity (KD∼ 0.1 nM) and with a high degree of specificity. The binding was inhibited by DL‐propranolol (Ki∼1 nM). Treatment of cells cultured in Dulbecco's modified Eagle medium (DMEM) without fetal calf serum for 4 days with desipramine reduced the (−)‐[3H]CGP‐12177 specific binding in a concentration‐dependent manner, a reduction from 127 to 102 fmol/mg of protein being found at a ligand concentration of 1 nM after treatment with 10 μM desipramine. Lesser effects were seen after treatment for 1 day. A similar result was found with maprotiline, and reductions in specific binding were seen after 4 days of treatment with amitriptyline, iprindole, and citalopram. The reduction in binding‐site density (measured per milligram of protein to compensate for variability in cell density per well), however, was paralleled in all cases by a reduction in the rate of cell proliferation. When C6 glioma cells were cultured in Ham's medium without fetal calf serum during the antidepressant treatment period, a higher specific binding was observed than for the DMEM‐cultured cells, and 10 μM desipramine was without effect on either the (−)‐[3H]CGP‐12177 specific binding or cell proliferation. It is concluded that the effects of the antidepressants tested upon the density of (−)‐[3H]CGP‐12177 specific binding sites in intact C6 cells may be secondary to the toxicity of the compounds under the conditions used.
[3H]N‐Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture
Authors: Alonso R. , Didier M. , Soubrie P.
Published: October 2006

Abstract: Saturation experiments with the muscarinic antagonist [3H]N‐methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax= 1.85 ± 0.01 pmol/mg of protein at 10 days in culture; KD= 0.128 ± 0.01 nM The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki= 273 ± 13 nM), whereas the M2/M3 muscarinic antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H)= 31 ± 5 nM; Ki(L)= 2,620 ± 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N‐methyl‐D‐aspartate (100 μM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.
Autopsy Samples of Alzheimer's Cortex Show Increased Peroxidation In Vitro
Authors: Subbarao Kala V. , Richardson J. Steven , Ang Lee C.
Published: October 2006

Abstract: The formation of thiobarbituric acid‐reactive products was measured as an index of peroxidation by oxygen free radicals in homogenates of frontal cortex and cerebellum from brains taken at autopsy and verified histologically as being Alzheimer's (n = 6) or normal (n = 6). Compared with controls, basal peroxidation is significantly higher in Alzheimer's cortex, and this difference is also evident in the presence of exogenous iron. Peroxidation in cerebellum and levels of total glutathione, RNA, and DNA in cortex and cerebellum do not differ significantly between Alzheimer's brain and controls. Iron‐induced peroxidation in cortex is reduced by the lazaroid U‐74500A, with calculated IC50 values that are significantly higher in Alzheimer's samples (10 μM) than in controls (2.5 μM). These observations suggest that cerebral cortex from Alzheimer's patients differs from controls with respect to in vitro peroxidation.