bluuebunny/crossref_metadata_2025_split huggingface dataset:
[3H]Spiroxatrine: A 5‐HT1A Radioligand with Agonist Binding Properties
Authors: Herrick‐Davis Katharine , Titeler Milt 
Published: October 2006

AbstractSpiroxatrine has been reported to be a 5‐HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5‐HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5‐HT1A antagonist radioligand. [3H]8‐Hydroxydipropylaminotetralin ([3H]8‐OH‐DPAT), a well‐characterized 5‐HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8‐OH‐DPAT binding produced KD values of 0.9 nAf and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r= 0.98; p < 0.001) exists between K1 values obtained for a series of drugs in competing for [3H]‐spiroxatrine and [3H]8‐OH‐DPAT binding. Of special interest was the observation that 5‐HT1A agonists such as serotonin, 8‐OH‐DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8‐OH‐DPAT‐labelled 5‐HT1A receptors. [3H]Spiroxatrine and [3H]8‐OH‐DPAT binding to 5‐HT1A receptors was inhibited by guanosine 5′‐(β,γ‐imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration‐dependent manner whereas adenosine 5′‐(β,γ‐imido)triphosphate (a non‐hydrolyzable analog of ATP) had no effect. The similarities in the 5‐HT1A receptor radiolabelling properties of [3H]‐spiroxatrine and [3H]8‐OH‐DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has “agonist‐like” binding properties in its interaction with the 5‐HT1A receptor.
Effects of Dorsal Bilateral Rhizotomy Treatment on Transmitter Systems in the Spinal Cord of Normal and Spastic Dogs
Authors: McBride W. J. , Shapiro S. , Chernet E. , Sartorius C. , Hall P. V. 
Published: October 2006

AbstractThe high‐affinity uptakes of [3H]serotonin, [3H]‐glutamate, and γ‐[3H]aminobutyric acid were studied using a myelin‐free crude synaptosomal fraction prepared from the spinal cords of normal dogs and spastic dogs following sham treatment or dorsal bilateral rhizotomy surgery. Compared to sham‐operated controls, rhizotomy surgery of normal dogs produced, after 1 week, a 30% reduction in the Vmax value of [3H]glutamate, but did not alter the uptake of γ‐[3H]aminobutyric acid. This treatment also produced a 60% decrease in the Vmax value of [3H]serotonin. Comparison of the effect of rhizotomy surgery on normal and spastic dogs revealed that the spastic group had 60% higher Vmax values for uptakes of [3H]glutamate and γ‐[3H]aminobutyric acid. Comparison of sham‐operated spastic dogs and rhizotomy‐treated spastic animals showed that there was a 25% decrease in the uptake of both amino acids in the rhizotomy‐treated spastic group. Overall, the data (a) support the hypothesis that glutamate is the neurotransmitter from some of the primary afferents, and (b) suggest that sprouting of interneuronal amino acid transmitter systems may occur in the spinal cords of spastic dogs.
Transient and Persistent Depolarization‐Induced Changes of Protein Phosphorylation in a Molluscan Nervous System
Authors: Naito Shigetaka , Bank Barry , Alkon Daniel L. 
Published: October 2006

AbstractPhosphoproteins in the CNS of the nudibranch mollusc, Hermissenda crassicornis, were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography. After preincubation in artificial sea‐water containing 32P, nervous systems were exposed to elevation of external K+ (100 or 300 mM) for a period (e.g., 30 min) approximating a period of depolarization which occurs during classical conditioning. Elevated external K+ was found to change the state of phosphorylation of three distinct proteins (Mr 56,000, 25,000, and 20,000) in three distinct ways without consistently changing that of any other proteins. Phosphorylation of an Mr 56,000 protein was increased by high K+ about twofold only in the presence of external Ca2+ ([Ca2+]o). Phosphorylation of Mr 25,000 protein, on the other hand, was decreased up to 10‐fold by high K+, irrespective of the level of [Ca2+]o. The effect of depolarization on Mr 25,000 protein phosphorylation most likely represents dephosphorylation rather than proteolysis. This interpretation is consistent with the observations that (a) reappearance of the Mr 25,000 protein occurred in the presence of the protein synthesis inhibitors cycloheximide, puromycin, or anisomycin, and (b) the Hermissenda nervous system apparently contains a NaF and EDTA‐sensitive protein phosphatase capable of de‐phosphorylating Mr 25,000 protein. High K+ also reduced Mr 20,000 protein phosphorylation which was dependent on [Ca2+]o even in normal low K+ (10 mM) medium. Removal of [Ca2+]o enhanced reduction of Mr 20,000 phosphorylation due to the high K+ treatment. Interestingly, reduction of the Mr 25,000 protein phosphorylation was long‐lasting, i.e., its phosphorylation did not fully recover to a control level for at least 30 min after the high K+ conditions had been removed. On the other hand, changes of Mr 56,000 and Mr 20,000 phosphorylation did not persist after depolarization had been terminated. A23187, a Ca2+ ionophore, partially reduced Mr 25,000 phosphorylation. These results suggested that depolarization‐induced modification of protein phosphatase and/or protein kinase activity which regulates Mr 25,000 phosphorylation may be involved in biochemical pathways important for memory storage.
Comparative Study of Glial Marker Proteins in the Hypoplastic Cerebellum of Jaundiced Gunn Rats
Authors: Aono Sachiko , Sato Hiroshi , Semba Reiji , Kashiwamata Shigeo , Kato Kanefusa , Eng Lawrence F. 
Published: October 2006

AbstractThe behavior of marker proteins of glial cells [α‐enolase, β‐S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of eerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, γ‐enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of β‐S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in α‐ and γ‐enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of β‐S100 and α‐enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of γ‐enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that β‐S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of eerebellar damage, respectively, but that a‐enolase is less available.
pH Dependence of Histidine Affinity for Blood‐Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids
Authors: Oldendorf William H. , Crane Paul D. , Braun Leon D. , Gosschalk Eddy A. , Diamond Jared M. 
Published: October 2006

AbstractThe effects of pH (3.5–7.5) on the brain uptake of histidine by the blood‐brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single‐pass carotid injection technique. Cationic amino acid ([14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ([14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.
Tissue Content of Opioid Peptides in the Myenteric Plexus‐Longitudinal Muscle of Guinea‐Pig Small Intestine
Authors: Corbett Alistair D. , McKnight Alexander T. , Kosterlitz Hans W. 
Published: October 2006

AbstractWe have developed a method that is based on two HPLC systems and permits the separation of endogenous opioid peptides in tissue extracts. The individual peptides are bioassayed on the mouse isolated vas deferens; nalox‐one (100 nM) ensures opioid specificity. In the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig small intestine, the tissue content of prodynorphin‐derived peptides is lower than those of proenkephalin‐derived peptides. No β‐endorphin was detected. Of the prodynorphin fragments, α‐neoendorphin, β‐neoendorphin, dynorphin A(1–8), and dynorphin B are present in equimolar concentrations (12–15 pmol/g) whereas the tissue content of dynorphin A is only 0.8 pmol/g. Processing of proenkephalin leads to at least six opioid peptides. The tissue contents of [Leu5)enkephalin, [Met5]enkephalyl‐Arg‐Gly‐Leu, and [Met5]enkephalyl‐Arg‐Phe are 90–100 pmol/g and the content of [Met5]enkephalin is 405 pmol/g. BAM‐18 and [Met5]enkephalyl‐Arg‐Arg‐Val‐NH2 are present in much lower concentrations, 24 and 5 pmol/g, respectively. Although present in low amounts, BAM‐18 and [Met5]‐enkephalyl‐Arg‐Arg‐Val‐NH2 have high affinity for the μ‐opioid binding site and to a lesser extent for the k‐site; this binding profile differs from that of the other proenkephalin fragments all of which have high affinities for the μ‐ and δ‐sites.
Identification of a Trypsin‐Like Site Associated with Acetylcholinesterase by Affinity Labelling with [3H]Diisopropyl Fluorophosphate
Authors: Small David H. , Chubb Ian W. 
Published: October 2006

AbstractIn addition to its ability to hydrolyze acetylcho‐line, purified eel acetylcholinesterase possesses a trypsin‐like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor Nα‐p‐tosyl‐l‐lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion‐exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate‐polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide‐containing cells, acetylcholinesterase could act as a neuro‐peptide processing enzyme in these cells.
Evidence that Alterations in γ‐Aminobutyric Acid and Acetylcholine in Rat Striata and Cerebella Are Not Related to Soman‐Induced Convulsions
Authors: Liu Duan Duan , Ueno Eiko , Ho Ing K. , Hoskins Beth 
Published: October 2006

AbstractMany reports have suggested that γ‐aminobutyric acid (GABA) may play a role in organophosphate‐in‐duced convulsions. The balance between GABA and acetylcholine (ACh) in the brain also has been suggested by some investigators to be related to brain excitability. We examined these questions by studying the levels of GABA and ACh and the ratios of GABA to ACh in rat striata and cerebella (two major motor control areas in the CNS) after the administration of soman, an organophosphate acetylcholinesterase inhibitor also known as nerve gas. Male Sprague‐Dawley rats weighing 250–300 g were injected subcutaneously with three different doses of soman: a subconvulsive dose of 40 μg/kg (approximately 30% of the ED50 for convulsions in rats), a convulsive dose of 120 μg/kg (approximately one ED50 for convulsions), and a higher convulsive dose of 150 μg/kg (approximately 120% of the ED50 for convulsions). The incidence and severity of convulsions were monitored in individual rats until they were sacrificed by focused microwave irradiation of the head at the following time points after soman administration: 4 min, a time prior to the onset of convulsions; 10 min, the time of onset of convulsions; 1 h, the time of peak convulsive activity; and 6 h, a time at which rats were recovering from convulsions. Results showed that in rat striata and cerebella, neither changes in levels of GABA and ACh nor changes in ratios of GABA to ACh were related to soman‐induced convulsions, i.e., none of the changes in either levels or ratios of these two neurotransmitters were related to the initiation of, maintenance of, or recovery from soman‐induced convulsions.
Bovine Chromaffin Cells Have Insulin‐Like Growth Factor‐I (IGF‐I) Receptors: IGF‐I Enhances Catecholamine Secretion
Authors: Dahmer Mary K. , Perlman Robert L. 
Published: October 2006

AbstractThe binding of 125I‐insulin‐like growth factor‐I (125I‐IGF‐I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 ± 40,000 IGF‐I binding sites/cell. These sites bound IGF‐I with a KD of 1.1 ± 0.3 nM and had a much lower affinity for insulin. Cross‐linking studies showed that 125I‐IGF‐I bound to a protein that had an Mr of ∼ 125,000, similar to the Mr of the α subunit of the IGF‐I receptor in other tissues. Cells cultured with IGF‐I (10 nM) for 4 days exhibited an almost twofold increase in high K+‐evoked catecholamine secretion. Insulin was much less potent than IGF‐I in enhancing catecholamine secretion. These data indicate that binding of IGF‐I to its receptors on chromaffin cells can modulate the function of these cells.
Regulatory Properties of Calcium/Calmodulin‐Dependent Protein Kinase II in Rat Brain Postsynaptic Densities
Authors: Rich Devra P. , Cdlbran Roger J. , Schworer Charles M. , Soderling Thomas R. 
Published: October 2006

AbstractCalcium/calmodulin (CaM)‐dependent protein kinase II (CaM‐kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+‐indepen‐dent following initial activation by Ca2+/CaM. Generation of this Ca2+‐independent species was dependent upon auto‐phosphorylation of both subunits of the enzynme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 ± 5% of the total activity within 1–2 min. Subsequent to the generation of this partially Ca2+‐independent form of PSD CaM‐kinase II, addition of EGTA to the autophos‐phorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+‐dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the α‐ and β‐subunits, respectively, may be responsible for the transition of PSD CaM‐kinase II to the Ca2+‐independent species. A synthetic peptide 281–309 corresponding to a portion of the regulatory domain (residues 281–314) of the soluble kinase inhibited syntide‐2 phosphorylation by the Ca2+‐independent form of PSD CaM‐kinase II (IC50= 3.6 ± 0.8 μM). Binding of Ca2+/CaM to peptide 281–309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281–309 also decreased its inhibitory potency. These data suggest that CaM‐kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM‐kinase II.
Detection of Synenkephalin, the Amino‐Terminal Portion of Proenkephalin, by Antisera Directed Against Its Carboxyl Terminus
Authors: Stell William K. , Chaminade Michel , Metters Kathleen M. , Rougeot Catherine , Dray Fernand , Rossier Jean 
Published: October 2006

Abstract:Synenkephalin (SYN), the nonopioid amino‐terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63–70)‐octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63–70)‐octapeptide as standard, and 125I‐[Tyr63]SYN(63–70)‐octapeptide as tracer, the IC50 was ∼51 fmol/100‐μl sample at equilibrium or 12 fmol/100 μl in disequilibrium, and the sensitivity was ∼3 fmol/100 μl. Cross‐reactivity of the assay was 100% with [Cys63]SYN(63–70)‐octapeptide and with bovine adrenal 8.6‐kilodalton peptide digested with trypsin and carboxypeptidase B, but < 0.1% with transforming growth factor‐alpha, 2 × 10‐6 with Leu‐Leu‐Ala [SYN(68–70)‐tripeptide], and ≪ 10‐6 with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63–70)‐octapeptide, we refer to the RIA as an assay for SYN(63–70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris‐CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63–70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63–70) in striatum by a factor of 1.5–2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63–70) (after sequential digestion) was ∼6:1. At least 90% of the immunoreactive SYN(63–70) in extracts of bovine caudate nucleus eluted from Sephadex G‐100 with an apparent molecular weight equal to that of bovine PRO(1–77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C‐terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.
Binding of Progesterone to Nerve Cell Membranes of Rat Brain Using Progesterone Conjugated to 125I‐Bovine Serum Albumin as a Ligand
Authors: Ke Ferng‐Chun , Ramirez Victor D. 
Published: October 2006

Abstract:Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11α‐hydroxyprogesterone‐11‐hemisuccinate‐125I‐bovine serum albumin conjugate reached saturation after 30 min of incubation at 5°C. Several bovine serum albumin‐conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 ± 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17β‐estradiol 6‐(O‐carboxy‐methyl)oxime‐bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma‐enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone‐125I‐bovine serum albumin conjugate can be used as a ligand to study progesterone‐membrane receptor interactions.
2′‐Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery
Authors: Padua R. , Geiger J. D. , Dambock S. , Nagy J. I. 
Published: October 2006

Abstract:2′‐Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 μM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 μg/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the >95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5–50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68–78% of control values in brain regions with normally high levels of activity and by 44–59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.