bluuebunny/crossref_metadata_2025 huggingface dataset:
Opsonization with Antimyelin Antibody Increases the Uptake and Intracellular Metabolism of Myelin in Inflammatory Macrophages
Authors: Trotter Jacqueline , DeJong Linda J. , Smith Marion Edmonds
Published: July 2010

Abstract: In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody‐treated myelin as in cultures fed preimmune serum‐treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label: therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin‐associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum‐ and preimmune serum‐treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin‐specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage‐mediated demyelination.
Benzodiazepine Receptor Subunits in Avian Brain
Authors: Hebebrand Johannes , Friedl Waltraut , Unverzagt Birgit , Propping Peter
Published: July 2010

Abstract: Using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography of the photoaffinity‐labeled benzodiazepine receptor in duck brain, two equally intensive bands of relative molecular masses of 53,000 and 54,000 were observed, thus being identical to the subunit pattern previously described for chicken brain. An attempt was made to chacterize further the avian subunits. Comparison of the fluorographic subunit pattern in cerebellum and telencephalon revealed a pronounced quantitative heterogeneity. Addition of ethyl β‐carboline‐3‐carboxylate resulted in the selective inhibition of the 53,000 subunit. These results are compared with the mammalian subunit pattern in the corresponding brain regions. The findings are discussed in view of the apparent contradiction between the existence of regional heterogeneity when photoaffinity labeling is applied and its lack when monoclonal antibodies are used.
Proteolytic Inactivation of Substance P and Neurokinin A in the Longitudinal Muscle Layer of Guinea Pig Small Intestine
Authors: Nau R. , Schäfer G. , Deacon C. F. , Cole T. , Agoston D. V. , Conlon J. M.
Published: July 2010

Abstract: Membrane vesicles, showing a 21 ± 2‐fold enrichment in the activity of 5′‐nucleotidase and a 11 ± 4‐fold enrichment in the activity of angiotensin‐converting enzyme relative to homogenate, were prepared from the myenteric plexus‐containing longitudinal muscle layer of guinea pig ileum. Incubation of the vesicles with substance P and neurokinin A led to degradation of the peptides, and metabolites were isolated by reverse‐phase HPLC and identified by amino acid composition. Cleavages of substance P between Glu6‐Phe7, Phe7‐Phe8, and Gly9‐Leu10 and of neurokinin A between Gly8‐Leu9 were observed and could be inhibited in a dose‐dependent manner by phosphoramidon, an inhibitor of neutral endopeptidase 24.11. Formation of these metabolites was not completely inhibited by this agent, indicating that a phosphoramidon‐insensitive form of endopeptidase 24.11 was present in the gut. Substance P was resistant to degradation by aminopeptidases, but neurokinin A was a substrate for bestatin‐sensitive aminopeptidase(s), so that the neurokinin A (3–10) fragment represented the predominant metabolite in the chromatograms. The rate of formation of all the metabolites was not inhibited by ena‐lapril and not enhanced by an increased Cl− concentration, indicating that angiotensin‐converting enzyme was unimportant in the degradation process. Degradation of neurokinin A by the vesicles (Km 30 μM; Vmax 7.2 ±0.8 nmol min−1 mg of protein−1) was more rapid than degradation of substance P (Km 25 μM; Vmax 4.4 ± 0.4 nmol min−1 mg of protein−1).
Comparison of Benzodiazepine Receptors in Cerebellum and Inferior Colliculus
Authors: Sieghart W.
Published: July 2010

Abstract: Reversible and irreversible binding of [3H]flunitrazepam was investigated in membranes from cerebellum and inferior colliculus of young and adult rats. Results indicate that in adult animals predominantly BZ1 receptors are enriched in both brain regions. In the brains of newborn animals, however, additional benzodiazepine receptor subtypes seem to exist in cerebellum as well as in inferior colliculus.
Characterization of Messenger RNA from the Cerebral Cortex of Control and Alzheimer‐Afflicted Brain
Authors: Guillemette J. G. , Wong L. , Crapper McLachlan D. R. , Lewis P. N.
Published: July 2010

Abstract: A detailed comparative study of RNA transcripts isolated from the neocortex of control and Alzheimer postmortem brains was made to determine whether morphological changes in the chromatin of Alzheimer neurons and glia, which we reported earlier, are accompanied by changes in the products of transcription. A number of parameters were determined including the yields of total and mRNA per gram of tissue, the relative proportions of polyadenylated [poly(A) +] mRNA in the total RNA, the size distribution of the transcripts and the length of their poly(A) tails, and the nature of their in vitro translation products. The levels of endogenous RNase activity were also measured. The effect of the agonal process on the transcript complement was examined by Northern blotting of a cloned human heat‐shock cDNA to total human brain RNA. Our results reveal that the yields of total RNA, unadenylated mRNA, and poly(A) tail lengths from Alzheimer neocortex samples do not differ significantly from those of control and non Alzheimer dementia neocortex. On the other hand we find a significant reduction in the levels and proportion of poly(A)+ mRNA in the Alzheimer samples as compared to control brain samples. Quantitative rather than qualitative differences were observed in the in vitro translation products when programmed with control and Alzheimer mRNA. No differences were found in the levels of RNase activity between control and Alzheimer simples. Heatshock mRNA transcripts were detected in brain samples from patients in whom fever was associated with death. The direct correlation of reduced poly(A)+ mRNA and chromatin condensation in Alzheimer neocortex suggests a cause‐and‐effect relationship. Whether all transcribed genes are affected or only a specific subset has yet to be determined.
Quantitative Electroimmunoblotting Study of the Calcium‐Activated Neutral Protease in Human Myelin
Authors: de Rosbo N. Kerlero , Carnegie P. R. , Bernard C. C. A.
Published: July 2010

Abstract Degradation of myelin basic protein (MBP) in human myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca2+‐dependent protease acting on MBP was active at endogenous levels of Ca2+ (∼300 μg/g myelin) and was inhibited in the presence of Ca2+ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca2+, reaching a plateau after a 1 h incubation (80–85% degradation). The proteolytic activity was not enhanced in the presence of 2‐mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca2+ to the protease may lead to the regulation of Ca2+‐activated myelinolysis.
Synaptosomal Transport of Radiolabel from N‐Acetyl‐Aspartyl‐[3H]Glutamate Suggests a Mechanism of Inactivation of an Excitatory Neuropeptide
Authors: Blakely Randy D. , Ory‐Lavollee Laure , Thompson Reid C. , Coyle Joseph T.
Published: July 2010

Abstract This study was undertaken to explore in synaptosomal preparations the disposition of N‐acetyl‐aspartyl‐glutamate (NAAG), an endogenous acidic dipeptide neurotransmitter candidate. Radiolabel from N‐acetyl‐aspartyl‐[3H]glutamate was taken up rapidly into an osmotically sensitive compartment by rat brain synaptosomal preparations in a sodium‐, temperature‐, and time‐dependent manner. HPLC analysis of the accumulated radiolabel indicated that the bulk of the tritium cochromatographed with glutamic acid and not with NAAG. In contrast, [14C]NAAG, labeled on the N‐terminal acetate, was not taken up by the synaptosomal preparation. All effective inhibitors of synaptosomal, Na+‐dependent [3H]glutamate uptake were found to exhibit similar potency in inhibiting uptake of tritium derived from [3H]NAAG. However, certain α‐linked acidic dipeptides, structurally similar to NAAG, as well as the potent convulsant quisqualic acid inhibited synaptosomal transport of [3H]NAAG but were ineffective as inhibitors of [3H]glutamate transport. Together with a demonstration of disparities between the regional accumulation of radiolabel from [3H]NAAG and high‐affinity [3H]glutamate uptake, these data suggest the presence in brain of a specific peptidase targeting carboxy‐terminal glutamate‐containing dipeptides that may be coupled to the Na+‐dependent glutamate transporter. These findings provide a possible mechanism for NAAG inactivation subsequent to its release from nerve endings.
Transport of Cysteate by Synaptosomes Isolated from Rat Brain: Evidence that It Utilizes the Same Transporter as Aspartate, Glutamate, and Cysteine Sulfinate
Authors: Wilson David F. , Pastuszko Anna
Published: July 2010

Abstract Synaptosomes isolated from rat brain accumulated cysteic acid by a high‐affinity transport system (Km= 12.3 ±2.1 μM; Vmax= 2.5 nmol mgprotein‐1 min‐1). This uptake was competitively inhibited by aspartate (Ki= 13.3 ± 1.8 μM) and cysteine sulfinate (Ki= 13.3 ± 2.3 μM). Addition of extrasynaptosomal cysteate, aspartate, or cysteine sulfinate to synaptosomes loaded with [35S]cysteate induced rapid efflux of the cysteate. This efflux occurred via stoichiometric exchange of amino acids with half‐maximal rates at 5.0 ± 1.1 μM aspartate or 8.0 ± 1.3 μM a cysteine sulfinate. Conversely, added extrasynaptosomal cysteate exchanged for endogenous aspartate and glutamate with half‐maximal rates at 5.0 ± 0.4 μM acysteate. In the steady state after maximal accumulation of cysteate, the intrasynaptosomal cysteate concentrations exceeded the extrasynaptosomal concentrations by up to 10,000‐fold. The measured concentration ratios were the same, within experimental error, as those for aspartate and glutamate. Depolarization, with either high [K+] or veratridine, of the plasma membranes of synaptosomes loaded with cysteate caused parallel release of cysteate, aspartate, and glutamate. It is concluded that neurons transport cysteate, cysteine sulfinate, aspartate, and glutamate with the same transport system. This transport system catalyzes homoexchange and heteroex‐change as well as net uptake and release of all these amino acids.
Effect of Phospholipases on Chronic Opiate Action in Neuroblastoma × Glioma NG108‐15 Hybrid Cells
Authors: Griffin Michael T. , Law P. Y. , Loh H. H.
Published: July 2010

AbstractChronic treatment of neuroblastoma × glioma NG108‐15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospho‐lipids with various lipases. Treatment with phospholipase C fromClostridium welchiiproduced an enzyme concentration‐dependent decrease of prostaglandin E1‐stimulated adenylate cyclase activity in control or etorphine‐treated (1μMfor 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of >0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C fromBacillus cereusand phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate‐ and NaF‐sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.
Effects of Prenatal Phenobarbital on Benzodiazepine Receptor Development
Authors: Garrett Kennon M. , Tabakoff Boris
Published: July 2010

Abstract Phenobarbital (PB) was administered to pregnant mice during days 9–21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the γ‐aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero.
Preparation and Specificity of 11 Monoclonal Antibodies to GM1 Ganglioside
Authors: Mahadik Sahebarao P. , Laev Heljo , Rapport Maurice M.
Published: July 2010

Abstract Eleven monoclonal antibodies to GM1 ganglioside were prepared from hybridoma clones obtained by fusion of spleen cells from mice immunized with GM1 with mouse myeloma cells. When the reactivities of these 11 monoclonal antibodies were determined by enzyme‐linked immunosorbent assay with six glycosphingolipids (GM1, GD1a, GD1b, GT1b, GM2, and asialo‐GM1), they showed different degrees of specificity. From their reactivity patterns, they could be divided into three groups: Group 1, those that react only with GM1 (C3 and D3); Group 2, those that react predominantly with GM1 (C6, B6, D1, e1, g1, g9, and e12); and Group 3, those that show poor discrimination (h2 and A4). The clones differed in their biological activities.
Neurotensin Stimulates Inositol Phospholipid Metabolism and Calcium Mobilization in Murine Neuroblastoma Clone N1E‐115
Authors: Snider R. M. , Forray C. , Pfenning M. , Richelson E.
Published: July 2010

Abstract Murine neuroblastoma cells (clone N1E‐115) possess neurotensin receptors that mediate cyclic GMP synthesis. Because of the hypothesized relationship between phospholipid metabolism, intracellular Ca2+, and cyclic GMP synthesis, we determined with these cells the effects of neurotensin on 32P labeling of phospholipids, release of inositol phosphates, and intracellular Ca2+ (as determined with the use of Quin‐2, a fluorescent probe sensitive to free Ca2+ levels). Neurotensin stimulated incorporation of 32P into phospholipids, especially phosphatidylinositol and phosphatidate. Neurotensin also stimulated the release of [3H]‐inositol phosphates with an EC50 of about 1 nM. Mean basal Ca2+ concentration in these cells was 134 nM and this level was increased in a rapid and dose‐dependent manner by neurotensin, with an EC50 of 4 nM. Since the EC50 for neurotensin in stimulating cyclic GMP synthesis is 1.5 nM and the KD for binding of [3H]neurotensin at 0° is 11 nM, all these different effects appear to be shared proximal consequences of neurotensin receptor activation.
Immunohistochemical Localization of Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Optic Nerve: Specific Molecular Markers for Optic Nerve Structures
Authors: Jones Paul S. , Tesser Paul , Keyser Kent T. , Quitschke Wolfgang , Samadi Ramin , Karten Harvey J. , Schechter Nisson
Published: July 2010

Abstract The predominant proteins (58K) of the intermediate filament complex in the goldfish visual pathway consist of a series of isoelectric variants. Previous biochemical studies have shown that proteins ON1 and ON2 are of neuronal origin, whereas ON3 and ON4 are of nonneuronal origin. Polyclonal antibodies, purified by affinity chromatography, that are specific for ON1 and ON2 or ON3 and ON4 have been used to localize histologically the ON proteins within the normal and crushed optic nerve. Anti‐ON1/ON2 antiserum presented a pattern consistent with intraaxonal staining. A nonneuronal staining pattern was observed with anti‐ON3/ON4 antiserum. The two patterns were distinct from and complementary to each other. The data suggest that ON3 and ON4 represent a novel glial fibrillary acidic protein. The results are discussed in terms of the function of these proteins in development, plasticity, and regeneration.
Uncoupling of Cholinergic Synaptic Vesicles by the Presynaptic Toxin β‐Bungarotoxin
Authors: Anderson D. C. , Parsons Stanley M.
Published: July 2010

AbstractThe effect of the presynaptic neurotoxin β‐bungarotoxin (β‐BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ ofTorpedo californicawas studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+‐, time‐, and concentration‐dependent manner. Correlated with these effects is a 50–60% stimulation of the vesicle proton‐pumping ATPase activity. The β‐BuTx‐mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2(PLA2)‐dependent damage to the vesicle membrane caused by β‐BuTx, which results in uncoupling of the ATPase and ACh transporter systems.

The nonneurotoxicNaja najavenom PLA2causes similar effects, except that it is slightly more potent on a molar basis. About 100‐fold more β‐BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of β‐BuTx‐ but not ofN. najaPLA2‐mediated uncoupling. The observations suggest that a component of β‐BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle‐like structures and that a nucleotide‐like factor might modulate the toxicity.
Selectivity and Regulation in the Phospholipase A2‐Mediated Attack on Cholinergic Synaptic Vesicles by β‐Bungarotoxin
Authors: Noremberg Krystyna , Parsons Stanley M.
Published: July 2010

Abstract The total fatty acid composition of purified Torpedo californica electric organ synaptic vesicles was determined by GLC analysis of methyl esters. Limit amounts of fatty acids released by high concentrations of either β‐bungarotoxin (β‐BuTx) or Naja naja venom phospholipase A2 (PLA2) acting in deoxycholate are reported. The time and enzyme concentration dependence for β‐BuTx‐ and PLA2‐induced release of fatty acids from intact synaptic vesicles indicate that PLA2 is 100‐ to 1,000‐fold more active. The Ca2+ dependence for β‐BuTx‐induced release of fatty acids also was determined. ATP inhibits β‐BuTx‐ but not PLA2‐induced release of fatty acids from vesicles in a manner that can not be ascribed only to chelation of the required Ca2+. ATP, other nucleotides, and adenosine have complex effects on β‐BuTx‐induced release of fatty acids from egg yolk phosphatidylcholine dispersed in deoxycholate. The results suggest that β‐BuTx‐mediated hydrolysis of the cholinergic synaptic vesicle membrane is ∼10‐ to 100‐fold more effective at causing uncoupling of vesicles than is PLA2 and that the enzymatic activity of β‐BuTx is subject to regulation by nucleotide‐like factors.
Glyceraldehyde‐3‐Phosphate Dehydrogenase Is a Nonhistone Protein and a Possible Activator of Transcription in Neurons
Authors: Morgenegg Göpf , Winkler Gian C. , Hübscher Ulrich , Heizmann Claus W. , Mous Jan , Kuenzle Clive C.
Published: July 2010

A single‐stranded DNA‐binding protein of Mr 35,000 (35K protein) was isolated from calf cerebral cortex by affinity chromatography on immobilized double‐stranded and single‐stranded DNA. Its localization in the nuclear compartment was demonstrated by im‐munohistochemistry. Previous studies had uncovered a homologous nonhistone chromosomal protein in the nuclei of rat cerebral cortex neurons, cerebellar neurons, oligodendrocytes, and liver cells. The rat protein accumulated in the nuclear compartment of neurons in exact temporal coincidence with the arrest of cell division and the initiation of terminal differentiation. Therefore, in the present work, the 35K protein was tested for an activating role in RNA transcription. During the course of this study we became aware that the 35K protein was identical to a glycolytic enzyme, glyceraldehyde‐3‐phos‐phate dehydrogenase (GAPDH, EC 1.2.1.12). When authentic GAPDH from rabbit skeletal muscle was injected into Xenopus laevis oocytes, it greatly stimulated RNA polymerase II transcription, whereas the 35K protein from caif brain did not. This apparent discrepancy was partially resolved by the finding that rabbit muscle GAPDH could be fractionated into two components by affinity chromatography on single‐stranded DNA cellulose. Only 5% of the applied protein was retained on the column and could be eluted with a shallow salt gradient identical to the one used for the isolation of the 35K protein. This single‐stranded DNA‐binding component of rabbit muscle GAPDH did not stimulate transcription. Apparently, the 35K protein from calf brain corresponded to this single‐stranded DNA‐binding subfrac‐tion, which explained its failure to activate transcription. So far, we have not been able to isolate the activating factor from calf brain but suggest that the temporal coincidence between the accumulation of GAPDH in rat neu‐ronal nuclei during differentiation and the concomitant increase in transcriptional activity may not be fortuitous.
Cyclic Nucleotide Response of the Hippocampal Formation to Septal Stimulation in Naive and Kindled Rats
Authors: Wasterlain Claude G. , Farber Debora B.
Published: July 2010

Rats were kindled through nonmagnetic electrodes stereotaxically implanted into the medial septum. Concentrations of cyclic AMP and cyclic GMP were measured by radioimmunoassay in seven brain regions after microwave fixation during the development and expression of kindled seizures. Hippocampal concentrations were similar to untreated controls (cyclic GMP level in the left and right hippocampus, 0.66 ± 0.04 and 0.68 ± 0.07 pmol/mg of protein, respectively; cyclic AMP, 9.4 ± 0.9 and 9.6 ± 0.8 pmol/mg of protein, respectively), in kindled animals that were not stimulated, and in naive animals in response to septal stimulation, in spite of the presence in the latter group of bilateral hippocampal after‐discharges. Animals that failed to develop kindling and kindled animals that failed to have a seizure in response to stimulation also showed no change in cyclic nucleotide concentrations in any brain region. Kindled animals that developed a seizure following stimulation showed significant elevations in levels of both cyclic GMP and cyclic AMP in hippocampus and in several other brain regions. A single naive animal that had a seizure in response to its first stimulation also appeared to have elevated concentrations of both cyclic nucleotides in hippocampus. These data suggest that the elevation in levels of both cyclic GMP and cyclic AMP during kindled seizures is associated with seizure development rather than with the generation of afterdischarges or with the kindling engram.
Chromogranin C: A Third Component of the Acidic Proteins in Chromaffin Granules
Authors: Fischer‐Colbrie Reiner , Hagn Claus , Kilpatrick Lynn , Winkler Hans
Published: July 2010

We characterized a group of acidic proteins of bovine chromaffin granules with an antiserum raised against a protein described by Rosa and Zanini [Eur. J. Cell Biol. 31, 94–98 (1983)] in pituitary gland. In adrenal medulla the proteins reacting with this antiserum are confined to chromaffin granules. Their largest component has a Mr of 86,000 and a pl of 5.0. In addition six proteins of lower molecular weight are recognized by this antiserum. In a cell‐free system only one protein is synthesized that can be precipitated with this antiserum. The properties of these proteins are very similar to those of the previously described chromogranins A and B; however, there is no immunological cross‐reaction between these protein groups. We suggest this third group of acidic proteins of chromaffin granules be named chromogranins C.
Insulin Binding to the Blood‐Brain Barrier in the Streptozotocin Diabetic Rat
Authors: Frank H. J. L. , Pardridge W. M. , Jankovic‐Vokes T. , Vinters H. V. , Morris W. L.
Published: July 2010

Abstract: 125I‐Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin‐treated diabetic rats was measured. The binding was highest in the control (21.1 ± 1.8%/mg capillary protein) and lowest in the diabetic (14.8 ± 1.9%, p < 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 ± 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high‐ and low‐affinity sites in the diabetic animals. To test whether the failure of up‐regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up‐ and down‐regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down‐regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low‐insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes. Furthermore, insulin levels in both plasma (33.4 ± 3.5 μU/ml in controls versus 21.9 ± 2.1 in diabetic, p < 0.02) and brain 2.6 ± 0.4 μU/g wet weight in control versus 0.32 ± 0.30 in diabetic, p < 0.02) were lower in the diabetic animals. However, the ratio of brain to plasma insulin levels was lower in the diabetic (0.029 ± 0.011) than in the control animals (0.092 ± 0.024, p < 0.02), implying that there is a selective decrease in the brain insulin relative to the plasma insulin in diabetes. These findings suggest that the brain capillaries may be involved in transport of insulin from the blood to the underlying brain cells and that a specific defect occurs in the diabetic brain capillary that can cause a further deficiency in brain insulin in poorly controlled diabetes.
Disulfide Bond Formation Between Nerve Growth Factor and the Nerve Growth Factor Receptor from Embryonic Sensory Neurons
Authors: Compito Gerard A. , Wagner Barbara J. , Stach Robert W.
Published: July 2010

Abstract: Recent studies with sympathetic neurons using radiolabeled nerve growth factor have indicated that a high‐molecular‐weight covalent complex is formed. This complex is between the nerve growth factor and the highaffinity (type I) receptor and occurs through the formation of a disulfide bond. Studies presented in the present article demonstrate a similar complex is formed on chicken embryonic sensory neurons. The formation of this complex is inhibited by the addition of unlabeled nerve growth factor, metabolic energy inhibitors (dinitrophenol and NaF), and of sulfhydryl reagents. On the other hand, formation of this complex is not inhibited by temperature, or by the addition of insulin or epidermal growth factor. The receptor involved in the covalent complex formation is the high‐affinity (type I) receptor. The molecular weight of this complex is approximately 232,000 daltons. Evidence indicates that this covalent complex may be required for the biological activity of the nerve growth factor.
Increased 5‐Hydroxytryptamine and Norepinephrine Release from Rat Brain Slices by the Red Sea Flatfish
Authors: Pardaxin Toxin , Wang Hoau Yan , Friedman Eitan
Published: July 2010

Abstract: The effect of the Red Sea flatfish toxin pardaxin was examined on K+‐evoked and on basal release of either [3H]norepinephrine or [3H]5‐hydroxytryptamine from preloaded rat cortical slices. The K+‐induced release of the neurotransmitters was stimulated in a dose‐related manner at concentrations ranging from 0.5 to 4 μg/ml. Basal release of the two transmitters was elevated to a lesser extent. Although the stimulation of evoked release was approximately equivalent for the two neurotransmitters, the response to 5‐hydroxytryptamine was reversible whereas that of norepinephrine was not washed by 20 min of superfusion. The mechanisms involved in producing these actions of pardaxin are not known; however, they may be mediated by changes in electrolyte fluxes across the neuronal membranes.
Electroconvulsive Shock and Cyclic AMP Signal Transduction: Effects Distal to the Receptor
Authors: Newman Michael E. , Solomon Haim , Lerer Bernard
Published: July 2010

Chronic electroconvulsive shock (ECS) induced a significant decrease in noradrenaline‐ and forskolin‐stimulated cyclic AMP production in rat cortical slices, whereas a single ECS had a much smaller effect. In a cortical membrane preparation, adenylate cyclase activity in response to stimulation by forskolin, guanosine‐5′‐(β,γ‐imido)triphosphate, and Mn2+ ions was significantly increased in membranes derived from rats that had received chronic ECS, but was either unchanged or reduced in membranes from rats that received a single treatment only. The results are interpreted in terms of changes occurring at components of the adenylate cyclase enzyme distal to the receptor.
Effect of Early Iron Deficiency in Rat on the γ‐Aminobutyric Acid Shunt in Brain
Authors: Taneja Veena , Mishra Kamalapati , Agarwal K. N.
Published: July 2010

Early iron deficiency in rat does not affect the weight or the protein, DNA, and RNA content but results in a slight reduction in γ‐aminobutyric acid (GABA) (13%, p < 0.01) and glutamic acid (20%, p < 0.001) content of the brain. The activities of the two GABA shunt enzymes, glutamate dehydrogenase and GABA‐transaminase, and of the NAD+‐linked isocitrate dehydrogenase (ICDH) were inhibited whereas the glutamic acid decarboxylase, mitochondrial NADP+‐linked ICDH, and succinic dehydrogenase activities remained unaltered in brain. On rehabilitation with the iron‐supplemented diet for 1 week, these decreased enzyme activities in brain attained the corresponding control values. However, the hepatic nonheme iron content increased to about 80% of the control, after rehabilitation for 2 weeks. A prolonged iron deficiency resulting in decreased levels of glutamate and GABA may lead to endocrinological, neurological, and behavioral alterations.
Acetylcholinesterase Activity in Regions of the Rat Brain Following a Convulsion
Authors: Appleyard Margaret E. , Green A. R. , Smith A. D.
Published: July 2010

The effects of electroconvulsive shock on the levels of acetylcholinesterase in several brain regions of the rat were studied. Hippocampus, mesencephalon, cortex, and striatum exhibited rapid changes in acetylcholinesterase activity during the first few minutes following the convulsion, whereas brainstem and basal forebrain levels remained unchanged. In both hippocampus and midbrain there was a sustained decrease in activity: the total acetylcholinesterase activity was decreased by up to 40% within 2 min of the convulsion and did not return to control values for another 3 h. Thirty minutes after a flurothyl‐induced convulsion there was a similar fall in acetylcholinesterase activity in both these regions, whereas a subconvulsive electric shock produced no change. It is concluded that a convulsion produces significant short‐term decreases in acetylcholinesterase activity in areas of the rat brain that are involved in the generation and propagation of seizures, and the question is raised of whether this is related to the increase in seizure threshold that follows a convulsion.
A Semiautomated Analysis Method for Catecholamines, Indoleamines, and Some Prominent Metabolites in Microdissected Regions of the Nervous System: An Isocratic HPLC Technique Employing Coulometric Detection and Minimal Sample Preparation
Authors: Kilpatrick Ian C. , Jones Martin W. , Phillipson Oliver T.
Published: July 2010

The application of a commercially available coulometric electrochemical detector to the automated HPLC analysis of some monoamines and their metabolites in microdissected areas of the rat nervous system is described. Apart from the stability and high sensitivity of the system, other appealing features of the technique are the facile sample preparation and long‐term sample storage characteristics which show minimal analyte degradation. Basal values of some regional monoamine and metabolite concentration are listed together with a brief appendix that serves as a user's guide to the operation and maintenance of the detection system.
Spontaneous Release of γ‐Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+‐Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats
Authors: Noto Tadashi , Hashimoto Hiromichi , Nakao Junji , Kamimura Hiroshi , Nakajima Teruo
Published: July 2010

The spontaneous release of [3H]γ‐aminobutyric acid ([3H]GABA) in various areas of rat brain in jected with [3H]putrescine was examined using a push pull perfusion technique. The release in a 25‐min perfusate was highest in the caudate‐putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate‐putamen. The release was enhanced by high K+ solution in a Ca2+‐dependent manner.
Stimulation of Phosphatidylinositol Hydrolysis by Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 in Rat Cerebral Cortical Neurons Developing in Culture
Authors: Widmer Hans R. , Knüsel Beat , Hefti Franz
Published: July 2010

Abstract:Phosphatidylinositol (PI) breakdown represents a powerful system participating in the transduction mechanism of some neurotransmitters and growth factors and producing two second messengers, diacylglycerol and inositol trisphosphate. The transformation of PC12 neuroblastoma cells into neuron‐like cells induced by nerve growth factor (NGF) is preceded by a rapid stimulation of PI breakdown; however, it was not known whether PI breakdown mediates actions of other members of the neurotrophin family. The present study analyzed the effects of NGF, brain‐derived neurotrophic factor (BDNF), and neurotrophin‐3 (NT‐3) on PI breakdown in primary cultures of embryonic rat brain cells. Cultures were grown for 7 days; PI was then labeled by incubating cultures withmyo‐[3H]inositol, which then were exposed acutely to growth factors. BDNF and NT‐3, but not NGF, elevated the levels of labeled inositol phosphates within 10–15 min after addition to the cultures in a dose‐dependent manner. ED50values for BDNF and NT‐3 were 12.4 and 64.5 ng/ml, respectively. Comparable effects were found in cultures of cortical, striatal, and septal cells. The actions of BDNF and NT‐3 probably reflect actions on neurons, because no effects were seen in cultures of nonneuronal cells. In contrast, basic fibroblast growth factor induced a marked stimulation of PI breakdown in cultures of nonneuronal cells. K252b, which selectively blocks neurotrophin actions by inhibitingtrk‐type receptor proteins, prevented the PI breakdown mediated by BDNF and NT‐3. The findings suggest that rapid and specific induction of PI breakdown is involved in the signal transduction of BDNF and NT‐3, and they provide evidence that cortical neurons are functionally responsive to BDNF and NT‐3 during development.
Extracellular Brain Glucose Levels Reflect Local Neuronal Activity: A Microdialysis Study in Awake, Freely Moving Rats
Authors: Fellows L. K. , Boutelle M. G. , Fillenz M.
Published: July 2010

Abstract: The relationship between brain extracellular glucose levels and neuronal activity was evaluated using microdialysis in awake, freely moving rats. The sodium channel blocker tetrodotoxin and the depolarizing agent veratridine were administered through the dialysis probe to provoke local changes in neuronal activity. The extracellular glucose content was significantly increased in the presence of tetrodotoxin and decreased sharply following veratridine application. The systemic injection of a general anaesthetic, chloral hydrate, led to a large and prolonged increase in extracellular glucose levels. The brain extracellular glucose concentration was estimated by comparing dialysate glucose efflux over a range of inlet glucose concentrations. A mean value of 0.47 mM was obtained in five animals. The results are discussed in terms of the coupling between brain glucose supply and metabolism. The changes observed in extracellular glucose levels under various conditions suggest that supply and utilization may be less tightly linked in the awake rat than has previously been postulated.
Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide‐ and Catecholamine‐Biosynthetic Enzymes
Authors: Das Banasree , Sabban Esther L. , Kilbourne Edward J. , Fricker Lloyd D.
Published: July 2010

Abstract:PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine α‐amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mMKCl. Short‐term (1‐ to 3‐h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]‐Met into CPE. Also, CPE mRNA is not altered by 2‐h treatments with KCl. In contrast, prolonged treatment (24–48 h) of PC12 cells with 50 mMKCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long‐term treatment of the cells with KCl, with increases of 35% after 5 h and 55–75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine β‐hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long‐term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin of DBH.
Specific Irreversible Monoamine Oxidase B Inhibitors Stimulate Gene Expression of Aromatic L‐Amino Acid Decarboxylase in PC12 Cells
Authors: Li Xin‐Min , Juorio Augusto V. , Paterson I. Alick , Zhu Meng‐Yang , Boulton Alan A.
Published: July 2010

Abstract: The effect of some selective monoamine oxidase (MAO) inhibitors on aromatic L‐amino acid decarboxylase (AADC) gene expression in PC12 cells has been examined. Irreversible MAO B inhibitors [(–)‐deprenyl, pargyline, and MDL 72,974A] stimulated AADC gene expression, whereas a selective irreversible MAO A inhibitor (clorgyline) and a reversible MAO B inhibitor (Ro 19‐6327) had no effect. Because there is no apparent MAO B activity in PC12 cells, it is postulated that there is a novel site of action for these MAO B inhibitors and that the pharmacological profile of this site matches that of neuroprotective MAO B inhibitors. Finally, it is suggested that the stimulation of AADC gene expression may be relevant to the antiparkinsonian effects of MAO B inhibitors.
Increase in Neurite Formation and Acetylene line Release by Transfection of Growth‐Associated Protein‐43 cDNA into NG108–15 Cells
Authors: Kumagai‐Tohda Chihiro , Tohda Michihisa , Nomura Yasuyuki
Published: July 2010

Abstract: We previously reported that growth‐associated protein‐43 (GAP‐43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP‐43. In the present study, to clarify the functional significance of GAP‐43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG‐G11 cells by transfection of GAP‐43 cDNA into NG108‐15 cells. NG‐G11 cells expressed GAP‐43 mRNA at levels approximately twice that in nontransfected or vector‐transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu‐cAMP) or 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) plus diBu‐cAMP. Neurite outgrowth after addition of diBu‐cAMP was greater in NG‐G11 than in control cells. In NG‐G11 cells, neurites elongated by treatment with diBu‐cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu‐cAMP for 24 h induced neurite outgrowth in NG‐G11 cells, although control cells required 72 h. Depolarization by 50 mM KCI induced ACh release in both NG‐G11 and control cells treated with diBu‐cAMP or TPA/diBu‐cAMP. Although removal of the drugs following diBu‐cAMP treatment reversed ACh release to nontreated levels in control cells, a high‐K+‐induced level of ACh release remained in NG‐G11 cells after removal of diBu‐cAMP. ACh release induced by TPA plus diBu‐cAMP for 24 h was further enhanced after removal of the drugs in NG‐G11 cells, but it was not seen in control cells. These results suggest that levels of GAP‐43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP‐43 may be involved in neuronal differentiation in NG108‐15 cells.
Evidence for the Corelease of Dynorphin and Glutamate from Rat Hippocampal Mossy Fiber Terminals
Authors: Conner‐Kerr Teresa A. , Simmons Duncan R. , Peterson Gary M. , Terrian David M.
Published: July 2010

Abstract: Hippocampal mossy fiber (MF) nerve endings may be isolated in a subcellular fraction (P3) that releases both prodynorphin‐derived peptides and glutamate (Glu) in a calcium‐dependent manner when depolarized. However, this isolation procedure does not yield a pure preparation of MF synaptosomes. The present study evaluates the proportion of dynorphin (Dyn) and Glu that is released from synaptosomes in the P3 fraction that are of MF origin. We have addressed this issue by determining the degree to which a selective lesion of the dentate granule cell/MF system in vivo concomitantly reduces the exocytosis of Dyn and Glu from the P3 subcellular fraction. Unilateral injections of colchicine into the dentate gyrus resulted in a substantial and selective degeneration of the granule cell/ MF pathway in the rat hippocampal formation. The overall integrated density of the Timm‐stained band, which corresponds to the position of the MF terminal field, was estimated to be reduced by 75%. After this extensive loss of MF boutons, the K+‐evoked release of Dyn and Glu from the P3 fraction was reduced by 95 and 51 %, respectively. The loss of Timm staining and evoked Dyn release indicate that colchicine effectively eliminated MF synaptosomes from the P3 fraction. Those subcellular entities that were not destroyed by colchicine comprised ∼50% of the protein and evoked Glu release measured by using the P3fraction. In addition, the present results demonstrate that the inhibitory potency of the K opioid agonist U‐50.488H was not altered by the elimination of MF boutons from this synaptosomal preparation. This finding indicates that U‐50,488H is capable of suppressing Glu exocytosis from both MF and non‐MF synaptosomes. These results are consistent with the hypothesis that Dyn peptides and Glu are coreleased from hippocampal MF terminals.
Methyl Mercury Increases Intracellular Ca2+ and Inositol Phosphate Levels in Cultured Cerebellar Granule Neurons
Authors: Sarafian Theodore A.
Published: July 2010

Abstract: In an effort to explain the previously observed methyl mecury (MeHg)‐induced stimulation of protein phosphorylation in cerebellar granule neuron cultures, the effect of MeHg on protein kinase activities in cell‐free assays and on second messenger systems in cultured neurons has been examined. Using cell‐free assays for several protein kinases, no stimulation of enzyme activity was found at any concentration of MeHg tested. After 24 h exposure, 1–5 μM MeHg was found to have no significant effect on neuronal cyclic AMP levels. In contrast, intracellular levels of Ca2+ and rates of 45Ca2+ uptake were elevated 2.2‐fold and 3.6‐fold, respectively, by 5 μM MeHg. These effects were not observed with mercuric chloride, triethyllead, or lead acetate. Measurement of inositol phosphate production in granule cell cultures revealed a sensitive, pretoxic effect of MeHg with twofold stimulation following 30‐min exposure to 5 μM MeHg and 1.6‐fold after 24‐h exposure to 3 μM MeHg. Detection of inositol phosphate production after 30 min of MeHg was largely neuron‐specific. These results suggest that second messenger‐mediated activation of select protein kinase enzymes may be the mechanism underlying MeHg‐induced stimulation of protein phosphorylation in cerebellar neuronal culture. In addition, these findings indicate a specific interference with neuronal signal transduction and suggest a basis for the selective neurotoxic action of this agent.