bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+‐Dependent and Ca2+‐Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease
Authors: Matsushima Hideyuki , Shimohama Shun , Chachin Motohiko , Taniguchi Takashi , Kimura Jun
Published: July 2010

Abstract: We examined protein kinase C (PKC) activity in Ca2+‐dependent PKC (Ca2+‐dependent PKC activities) and Ca2+‐independent PKC (Ca2+‐independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age‐matched controls. In cytosolic and membranous fractions, Ca2+‐dependent and Ca2+‐independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca2+‐independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC‐ε, was used in the assay; whereas Ca2+‐independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC‐δ, was not significantly reduced in AD. Further studies on the protein levels of Ca2+‐independent PKC‐δ, PKC‐ε, and PKC‐ζ in AD brain revealed reduction of the PKC‐ε level in both cytosolic and membranous fractions, although PKC‐δ and PKC‐ζ levels were not changed. These findings indicated that Ca2+‐dependent and Ca2+‐independent PKC are changed in AD, and that among Ca2+‐independent PKC isozymes, the alteration of PKC‐ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.
Distinct Thermodynamic Parameters of Serotonin 5‐HT3 Agonists and Antagonists to Displace [3H]Granisetron Binding
Authors: Maksay Gábor
Published: July 2010

Abstract: Specific binding of [3H]granisetron was examined to serotonin 5‐HT3 receptors in synaptosomal membranes of rat cerebral cortex between 1 and 37°C. Displacing potencies were determined for 5‐HT3 antagonists (granisetron, ondansetron, tropisetron, and d‐tubocurarine) and agonists (5‐hydroxytryptamine, 2‐methyl‐5‐hydroxytryptamine, phenylbiguanide, m‐chlorophenylbiguanide, and SR 57227A). Displacing potencies of the agonists decreased with decreasing temperature. In contrast, displacing potencies of all antagonists increased with decreasing temperature, whereas those of tropisetron and d‐tubocurarine passed a maximum. Scatchard analysis of [3H]granisetron binding resulted in KD values lower than the IC50 values of granisetron and a decreasing number of binding sites at higher temperatures. It can be reconciled with temperature‐dependent agonist and antagonist states of 5‐HT3 receptors. A semiquantitative thermodynamic analysis was based on displacing potencies. The distinct patterns for the signs of entropy, enthalpy, and heat capacity changes on binding can be reconciled with ionic interactions for agonists and hydrophobic interactions for antagonists. The distinctive differences in these thermodynamic parameters exceed those for GABAA and glycine receptor‐ionophore complexes.
Expression of Heme Oxygenase Isozyme mRNAs in the Human Brain and Induction of Heme Oxygenase‐1 by Nitric Oxide Donors
Authors: Takahashi Kazuhiro , Hara Eishi , Suzuki Hiroyuki , Sasano Hironobu , Shibahara Shigeki
Published: July 2010

Abstract: Heme oxygenase is an essential enzyme in the heme catabolism that produces carbon monoxide (CO). This study was designed to examine the expression of two heme oxygenase isozyme mRNAs in the human brain and to explore the involvement of nitric oxide (NO) and various neuropeptides in the regulation of their expression. Northern blot analysis showed the expression of heme oxygenase‐1 and heme oxygenase‐2 mRNAs in every region of the brain examined, with the highest levels found in the frontal cortex, temporal cortex, occipital cortex, and hypothalamus. In a human glioblastoma cell line, T98G, treatment with any of three types of NO donors—sodium nitroprusside, 3‐morpholinosydnonimine, and S‐nitroso‐l‐glutathione—caused a significant increase in the levels of heme oxygenase‐1 mRNA but not in the levels of heme oxygenase‐2 and heat‐shock protein 70 mRNAs. Sodium nitroprusside increased the levels of heme oxygenase‐1 protein but not the levels of heat‐shock protein 70 in T98G cells. The increase in content of heme oxygenase‐1 mRNA caused by sodium nitroprusside was completely abolished by the treatment with actinomycin D. On the other hand, the levels of heme oxygenase isozyme mRNAs were not noticeably changed in T98G cells following the treatment with 8‐bromo cyclic GMP, sodium nitrite, or various neuropeptides, such as calcitonin gene‐related peptide, endothelin‐1, and corticotropin‐releasing hormone. The present study has shown the expression profiles of heme oxygenase‐1 and ‐2 mRNAs in the human brain and the induction of heme oxygenase‐1 mRNA caused by NO donors in T98G cells. These findings raise a possibility that the CO/heme oxygenase system may function in concert with the NO/NO synthase system in the brain.
Expression of C‐Type Natriuretic Peptide in the Bovine Pineal Gland
Authors: Middendorff Ralf , Maronde Erik , Paust Hans‐Joachim , Müller Dieter , Davidoff Michail , Olcese James
Published: July 2010

Abstract: Recent studies have pointed to membrane‐bound guanylyl cyclases (GCs) type A and type B in the rat pineal gland, which augment levels of cyclic GMP (cGMP) in response to atrial natriuretic peptide (ANP), brain‐type natriuretic peptide (BNP), and C‐type natriuretic peptide (CNP). The present report demonstrates for the first time the expression of CNP in the bovine pineal gland. The CNP prohormone transcript (unlike prepro‐ANP) was found by reverse transcriptase (RT)‐PCR in bovine pineal extracts. CNP immunoreactivity (ir) was revealed in a subpopulation of pinealocytes in situ and in nearly all pinealocytes in culture. Electron microscopic immunohistochemical investigations showed the presence of CNP‐ir in cytoplasmic vesicles, providing evidence for the potential secretion of this peptide by pineal cells. Furthermore, the CNP receptor (GC‐B) and GC‐A (receptor for ANP and BNP) were identified by RT‐PCR. Although melatonin secretion was unaffected, natriuretic peptides were able to elevate markedly cGMP production in cultured bovine pinealocytes with a rank order of potency of CNP > BNP = ANP. These findings describe a tissue CNP system in the bovine pineal gland and suggest that CNP may be a local auto‐ or paracrine modulator of pineal function.
Characterization of Glutamate Binding Sites in Receptors Assembled from Transfected NMDA Receptor Subunits
Authors: Kendrick Shelley J. , Lynch David R. , Pritchett Dolan B.
Published: July 2010

Abstract: Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA‐sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using l‐[3H]glutamate and [3H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg2+ can alter [3H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [3H]CGP 39653‐labeled site between the two heteromeric combinations. Agonist inhibition of [3H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.
Increased Permeability of Superoxide Dismutase at the Blood‐Nerve and Blood‐Brain Barriers with Retained Enzymatic Activity After Covalent Modification with the Naturally Occurring Polyamine, Putrescine
Authors: Poduslo Joseph F. , Curran Geoffrey L.
Published: July 2010

Abstract: Our previous studies have demonstrated that modification of superoxide dismutase (SOD) with the naturally occurring polyamines—putrescine (PUT), spermidine, and spermine—dramatically increases the permeability‐coefficient surface area (PS) product at the blood‐brain barrier and blood‐nerve barrier after parenteral administration. Because of this increased permeability, the efficient delivery of polyamine‐modified SOD (pSOD) across these barriers may enhance its therapeutic usefulness in treating ischemic neuronal degeneration, neurodegenerative disease, or even aging as an important antioxidant therapeutic strategy. Because PUT‐SOD had the highest PS values, SOD was modified in the present experiments by activating carboxylic acid groups to the reactive ester with water‐soluble carbodiimide and then reacted with PUT as the nucleophilic reagent. Preservation of SOD enzyme activity while maximizing the permeability was accomplished by adjusting the ionization of the protein carboxylic acid with pH. Both sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated graded conversion of SOD to its polyamine‐modified derivative when performed at different pH. Although modification at pH 4.7 resulted in only 6.6% retained SOD activity and the highest PS value (43.35 ± 3.81 × 10−6 ml/g/s for the hippocampus), modification at pH 5.7 resulted in 50.1% retained activity with a PS value of 24.48 ± 1.30 × 10−6 ml/g/s for nerve endoneurium and 21.95 ± 1.62 × 10−6 ml/g/s for hippocampus. This contrasts with a PS of 1.8–3.2 × 10−6 ml/g/s for native SOD in nerve and various brain regions. Reaction conditions are therefore defined that titrate enzyme activity of PUT‐SOD with PS changes in the intact animal after intravenous administration. These studies will allow an evaluation of the therapeutic usefulness of pSOD in animal models of neuronal degeneration.
Annexins in the Human Neuroblastoma SH‐SY5Y: Demonstration of Relocation of Annexins II and V to Membranes in Response to Elevation of Intracellular Calcium by Membrane Depolarisation and by the Calcium Ionophore A23187
Authors: Blanchard S. , Barwise J. L. , Gerke V. , Goodall A. , Vaughan P. F. T. , Walker J. H.
Published: July 2010

Abstract: The human neuroblastoma SH‐SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium‐dependent membrane‐binding proteins were isolated from SH‐SY5Y and analysed by 2‐dimensional gel electrophoresis. Proteins with Mr and pl values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by K+ depolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X‐100 for their solubilisation. Homogenisation of cells in calcium‐containing buffers also resulted in the formation of EGTA‐resistant membrane‐associated annexins II and V. The results demonstrate calcium‐dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium‐dependent manner to non‐phospholipid components of SH‐SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus. Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.
Cloning and Characterization of Aplycalcin and Aplysia Neurocalcin, Two New Members of the Calmodulin Superfamily of Small Calcium‐Binding Proteins
Authors: Dyer John R. , Sossin Wayne S. , Klein Marc
Published: July 2010

Abstract: We report here the sequences of two new proteins from Aplysia, aplycalcin and Aplysia neurocalcin. These proteins belong to a family of calcium‐binding proteins, found primarily in vertebrate brain and retina, that have been proposed to play a role in calcium‐dependent regulation of enzymes in signal transduction pathways. Like other members of this family, the Aplysia proteins have consensus sequences for myristoylation, bind calcium, and translocate from cytosol to membrane when the calcium level is raised above the resting intracellular concentration. Both proteins are relatively enriched in Aplysia nervous system, but are also found to a significant degree in other tissues. The expression of mRNA for these proteins in Aplysia nervous tissue is regulated during development, roughly paralleling the reported emergence of several forms of synaptic plasticity. The messages are present at low levels in stage 11, show a large increase by late stage 12, and decline to a plateau of ∼30% of the peak value afterward. On the basis of the properties of these proteins and by analogy with proposed functions of some of the retinal homologues, we suggest that these proteins may play a role in mediating calcium‐dependent processes in neuronal function. The presence of both proteins in other tissues may suggest analogous roles for the proteins in other cell types.
Angiotensin II Regulation of Intracellular Calcium in Astroglia Cultured from Rat Hypothalamus and Brainstem
Authors: Wang Desuo , Martens Jeffrey R. , Posner Philip , Sumners Colin , Gelband Craig H.
Published: July 2010

Abstract: This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1–100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura‐2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl2), the Ang II‐stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady‐state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 µM) inhibited the Ang II‐stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 µM) did not inhibit the Ang II‐stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II‐stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca2+‐free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 µM), cyclopiazonic acid (10 µM), and ryanodine (10 µM), but not caffeine (1–10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 µM) and nifedipine (10 µM); in contrast, gadolinium (100 µM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.
Low Glutathione and High Iron Govern the Susceptibility of Oligodendroglial Precursors to Oxidative Stress
Authors: Thorburne Shawn K. , Juurlink Bernhard H. J.
Published: July 2010

Abstract: We have previously shown, using qualitative approaches, that oligodendroglial precursors are more readily damaged by free radicals than are astrocytes. In the present investigation we quantified the oxidative stress experienced by the cells using oxidation of dichlorofluorescin diacetate to dichlorofluorescein as a measure of oxidative stress; furthermore, we have delineated the physiological bases of the difference in susceptibility to oxidative stress found between oligodendroglial precursors and astrocytes. We demonstrate that (a) oligodendroglial precursors under normal culture conditions are under six times as much oxidative stress as astrocytes, (b) oxidative stress experienced by oligodendroglial precursors increases sixfold when exposed to 140 mW/m2 of blue light, whereas astrocytic oxidative stress only doubles, (c) astrocytes have a three times higher concentration of GSH than oligodendroglial precursors, (d) oligodendroglial precursors have >20 times higher iron content than do astrocytes, and (e) oxidative stress in oligodendroglial precursors can be prevented either by chelating intracellular free iron or by raising intracellular GSH levels to astrocytic values. We conclude that GSH plays a central role in preventing free radical‐mediated damage in glia.
Modulation of ζ‐Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A
Authors: Wooten Marie W. , Seibenhener M. Lamar , Matthews Laura H. , Zhou Guisheng , Coleman Elaine S.
Published: July 2010

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12‐myristate 13‐acetate (PMA)‐sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose‐dependent redistribution of ζ‐PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ‐PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ‐PKC. In contrast to the positive effect that PKA‐mediated phosphorylation had on the activity of ζ‐PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.
Cyanide‐Induced Apoptosis and Oxidative Stress in Differentiated PC12 Cells
Authors: Mills Edward M. , Gunasekar Palur G. , Pavlakovic Goran , Isom Gary E.
Published: July 2010

Abstract: Terminally differentiated PC12 cells are a useful neuron‐like model for studying programmed cell death in response to nerve growth factor (NGF) deprivation. This in vitro model was used to investigate the mechanism by which cyanide‐induced histotoxic hypoxia produces neuronal degeneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN for 24 h did not produce cell death. In contrast, treatment of differentiated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blue dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg2+‐dependent endonuclease inhibitor aurintricarboxylic acid and the transcriptional inhibitor actinomycin D partially attenuated hypoxic toxicity, suggesting roles for endonuclease activation and transcription in this model of neuronal death. Extracted DNA from cyanide‐treated neurons demonstrated cleavage into oligonucleosomal fragments on gel electrophoresis. Transmission electron microscopic analysis showed morphological changes consistent with apoptotic cell death, including membrane blebbing and convolution, as well as chromatin condensation and margination to the nuclear membrane. Addition of either ascorbate or catalase to the cultures partially attenuated the loss of cell viability induced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The ability of cyanide to elevate intracellular oxidant species was determined by microfluorescence in differentiated PC12 cells loaded with the oxidant‐sensitive dye 2′,7′‐dichlorofluorescin. Exposure of cells to 0.1 mM KCN produced a rapid generation of oxidants that was blocked ∼50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undifferentiated, PC12 cells, and that antioxidants significantly reduce the incidence of cyanide‐induced apoptosis.
Effects of Steroid Exposure on Ligand Binding and Functional Activities of Diverse Nicotinic Acetylcholine Receptor Subtypes
Authors: Ke Lei , Lukas Ronald J.
Published: July 2010

Abstract: Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand‐gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. The present study tests whether nAChR are potential targets for steroids. Acute or short‐term (5 min) preexposure to steroids such as progesterone (which acts most potently), estradiol, corticosterone, or dexamethasone inhibits function of human muscle‐type (α1β1γδ) or ganglionic (α3β4) nAChR measured using 86Rb+ efflux assays in TE671/RD clonal or SH‐SY5Y neuroblastoma cells. Absolute (high nanomolar to intermediate micromolar range) and rank‐order potencies for steroid‐mediated functional inhibition are similar across nAChR subtypes but differ for some steroid derivatives. At concentrations that produce blockade of nAChR function, steroids do not affect binding of radioligands such as 125I‐labeled α‐bungarotoxin or [3H]acetylcholine to muscle‐type or ganglionic nAChR or to neuronal toxin‐binding nAChR that contain α7 subunits (α7‐nAChR). Steroid‐mediated blockade of nAChR function is insurmountable by increasing agonist concentrations, and cell‐impermeant progesterone:bovine serum albumin conjugates have full potency as inhibitors of ganglionic or muscle‐type nAChR function. Chronic (48 h) exposure to progesterone or estradiol, but not the other steroids, also produces blockade of nAChR function, without significant effects on numbers of nAChR radioligand‐binding sites. Collectively, these results suggest that steroids act noncompetitively at extracellular sites to inhibit nAChR function with unique potencies for different steroid‐nAChR subtype combinations. Thus, nAChR could be among the targets mediating physiologically relevant effects of steroid action in the nervous system.
Changes of Activity and Isozyme Pattern of Phosphofructokinase in the Brains of Patients with Alzheimer's Disease
Authors: Bigl Marina , Bleyl Anna‐Dorothea , Zedlick Dyrk , Arendt Thomas , Bigl Volker , Eschrich Klaus
Published: July 2010

Abstract: A severe reduction of the in vivo cerebral glucose consumption rate is generally found in patients with Alzheimer's disease. In postmortem studies changes in the activities of key regulatory glycolytic enzymes, including 6‐phosphofructokinase (PFK), have been reported in Alzheimer's disease brains, but the results obtained so far are inconsistent and controversial. We reevaluated the activity of PFK in brain tissue from clinically and neuropathologically confirmed cases of Alzheimer's disease using optimized tissue disintegration and assay methods and determined the PFK isozyme pattern. PFK activity in brains from patients with Alzheimer's disease was significantly increased in frontal and temporal cortex and unchanged in the other brain areas studied when compared with control brains. All three PFK isozymes were detected in each of the brain areas studied. In brains of Alzheimer's disease patients the level of the C‐type PFK was slightly reduced at the expense of the M‐ and L‐type subunits. The data presented do not support the results of other groups, which reported up to a 90% reduction of PFK activity in Alzheimer's disease. In contrast, the data presented clearly rule out the suggestion that changes of PFK activity might be one of the causes for the reduced glucose consumption in Alzheimer's disease brains.
Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form
Authors: Mills Roland G. , Minamide Laurie S. , Yuan Aidong , Bamburg James R. , Bray John J.
Published: July 2010

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [35S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1‐cm segments and separated into Triton X‐100‐soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I‐Sepharose and by one‐ and two‐dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its “inactive” phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.
Shift of the Cellular Oxidation‐Reduction Potential in Neural Cells Expressing Bcl‐2
Authors: Ellerby Lisa M. , Ellerby H. Michael , Park Sunghi M. , Holleran Anne L. , Murphy Anne N. , Fiskum Gary , Kane Darci J. , Testa Maria P. , Kayalar Celik , Bredesen Dale E.
Published: July 2010

Abstract: Expression of the protooncogene bcl‐2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl‐2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl‐2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl‐2 transfectants in two different neural cell lines—rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1‐7. Both neural cell lines overexpressing bcl‐2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1‐7 cells overexpressing bcl‐2 were significantly reduced. In addition, the NAD+/NADH ratio of bcl‐2‐expressing PC12 and GT1‐7 cells was two‐ to threefold less than that of control cell lines. GT1‐7 cells overexpressing bcl‐2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl‐2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl‐2 were similar to those of control cells. These results indicate that the overexpression of bcl‐2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.
Repetitive and Transient Increases in Hippocampal Neural Cell Adhesion Molecule Polysialylation State Following Multitrial Spatial Training
Authors: Murphy Keith J. , O'Connell Alan W. , Regan Ciaran M.
Published: July 2010

Abstract: Polysialylated neurons, located at the inner border of the dentate granule cell layer, have been demonstrated to exhibit time‐dependent change in their frequency at 10–12 h following training in the Morris water maze, a spatial learning paradigm. Such a change was not observed in animals required to locate a visible platform or in those rendered amnesic with scopolamine. This frequency response was capable of rapid reactivation following further training stimuli in a manner that was independent of circadian influence. These learning‐associated modulations in neural cell adhesion molecule (NCAM) polysialylation state did not increase in magnitude despite improved performance, suggesting their activation is required for processing information rather than contributing to previously stored, task‐associated memory. An increase in NCAM polysialylation appears to be a universal learning response to both spatial and nonspatial paradigms as similar time‐dependent changes occurred following training in a one‐trial, step‐through, passive avoidance response subsequent to water maze training.
Susceptibility of Hippocampal and Cortical Neurons to Argon‐Mediated In Vitro Ischemia
Authors: Kusumoto M. , Dux E. , Paschen W. , Hossmann K.‐A.
Published: July 2010

Abstract: Neurons from cerebral cortex and hippocampal CA1 sector exhibit a striking difference in vulnerability to transient ischemia. To establish whether this difference is due to the inherent (pathoclitic) properties of these neurons, the ischemic susceptibility was studied in primary cortical and hippocampal cultures by using a new model of argon‐induced in vitro ischemia. Neuronal cultures were exposed at 37°C for 10–30 min to argon‐equilibrated glucose‐free medium. During argon equilibration, Po2 declined to <2.5 torr within 1 min and stabilized shortly later at ∼1.3 torr. After 30 min of in vitro ischemia, total adenylate was <45% and ATP content <15% of control in both types of culture. Cytosolic calcium activity increased from 15 to 50 nM. Reoxygenation of cultures after in vitro ischemia led to delayed neuronal death, the severity of which depended on the duration of in vitro ischemia but not on the type of neuronal cultures. Energy charge of adenylate transiently returned to ∼90% of control after 3 h, but ATP content recovered only to 40% and protein synthesis to <35%. Cytosolic calcium activity continued to rise after ischemia and reached values of ∼500 nM after 3 h. The new argon‐induced in vitro ischemia model offers major advantages over previous methods, but despite this improvement it was not possible to replicate the differences in cortical and hippocampal vulnerability observed in vivo. Our study does not support the hypothesis that selective vulnerability is due to an inherent pathoclitic hypersensitivity.
Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin‐Treated Rats
Authors: Preece Nicholas E. , Cerdán Sebastián
Published: July 2010

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by 13C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2‐13C2]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2‐13C2]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by 13C NMR. In vigabatrin‐treated animals [13C]glutamine, a common intermediate for [13C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2‐13C2]glucose or [1,2‐13C2]acetate. However, [13C]GABA accumulation was sevenfold higher during [1,2‐13C2]glucose infusions or twofold higher during [1,2‐13C2]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near‐equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [13C]GABA accumulation occurs initially in the neuronal compartment.
Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain
Authors: Larrabee Martin G.
Published: July 2010

Abstract: Chains of lumbar sympathetic ganglia from 15‐day‐old chicken embryos were incubated for 4 h at 36°C in a bicarbonate‐buffered salt solution equilibrated with 5% CO2‐95% O2. Glucose (1–10 mM), lactate (1–10 mM), [U‐14C]glucose, [1‐14C]glucose, [6‐14C]glucose, and [U‐14C]lactate were added as needed. 14CO2 output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO2 from [U‐14C]glucose, and glucose reduced that from [U‐14C]lactate. When using uniformly labeled substrates in the presence of 5.5 mM glucose, the output of CO2 from lactate exceeded that from glucose when the lactate concentration was >2 mM. The combined outputs at each concentration tested were greater than those from either substrate alone. The 14CO2 output from [1‐14C]glucose always exceeded that from [6‐14C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood‐brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.
Involvement of Leukemia Inhibitory Factor in the Increases in Galanin and Vasoactive Intestinal Peptide mRNA and the Decreases in Neuropeptide Y and Tyrosine Hydroxylase mRNA in Sympathetic Neurons After Axotomy
Authors: Sun Y. , Zigmond R. E.
Published: July 2010

Abstract: In response to axonal injury, noradrenergic sympathetic neurons of the adult superior cervical ganglion (SCG) alter their neurotransmitter phenotype. These alterations include increases in the levels of the neuropeptides, galanin, vasoactive intestinal peptide (VIP), and substance P (SP) and a decrease in the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Previous studies have indicated that after axotomy in vivo, leukemia inhibitory factor (LIF) plays an important role in increasing the contents of galanin and VIP in the SCG. In the present study, by examining the time courses of the changes in LIF and neuropeptide mRNA and by using LIF null mutant mice, we have determined that LIF alters neuropeptide content in part by increasing levels of peptide mRNA. In addition, LIF also makes a small contribution to the axotomy‐induced down‐regulation of mRNA encoding TH and neuropeptide Y, both of which are normally expressed at high levels in the SCG. Finally, by using a LIF‐blocking antiserum, this cytokine was found to regulate SP expression in an in vitro axonal injury model. Thus, after axotomy, a single factor, LIF, participates in the down‐regulation of peptides/proteins involved in normal neurotransmission and the up‐regulation of a group of neuropeptides normally not present in the SCG that may be involved in regeneration.
Increased Activity‐Regulating and Neuroprotective Efficacy of α‐Secretase‐Derived Secreted Amyloid Precursor Protein Conferred by a C‐Terminal Heparin‐Binding Domain
Authors: Furukawa Katsutoshi , Sopher Bryce L. , Rydel Russell E. , Begley James G. , Pham Dao G. , Martin George M. , Fox Michael , Mattson Mark P.
Published: July 2010

Abstract: Proteolytic cleavage of β‐amyloid precursor protein (βAPP) by α‐secretase results in release of one secreted form (sAPP) of APP (sAPPα), whereas cleavage by β‐secretase releases a C‐terminally truncated sAPP (sAPPβ) plus amyloid β‐peptide (Aβ). βAPP mutations linked to some inherited forms of Alzheimer's disease may alter its processing such that levels of sAPPα are reduced and levels of sAPPβ increased. sAPPαs may play important roles in neuronal plasticity and survival, whereas Aβ can be neurotoxic. sAPPα was ∼100‐fold more potent than sAPPβ in protecting hippocampal neurons against excitotoxicity, Aβ toxicity, and glucose deprivation. Whole‐cell patch clamp and calcium imaging analyses showed that sAPPβ was less effective than sAPPα in suppressing synaptic activity, activating K+ channels, and attenuating calcium responses to glutamate. Using various truncated sAPPα and sAPPβ APP695 products generated by eukaryotic and prokaryotic expression systems, and synthetic sAPP peptides, the activity of sAPPα was localized to amino acids 591–612 at the C‐terminus. Heparinases greatly reduced the actions of sAPPαs, indicating a role for a heparin‐binding domain at the C‐terminus of sAPPα in receptor activation. These findings indicate that alternative processing of βAPP has profound effects on the bioactivity of the resultant sAPP products and suggest that reduced levels of sAPPα could contribute to neuronal degeneration in Alzhiemer's disease.
Structure of the C‐Terminal Tail of α‐Tubulin: Increase of Heterogeneity from Newborn to Adult
Authors: Redeker Virginie , Rusconi Filippo , Mary Jean , Promé Danielle , Rossier Jean
Published: July 2010

Abstract: A combination of posttranslational modifications contributes to the high heterogeneity of brain tubulin in mammals. In this report, the structures of the detyrosinated carboxy‐terminal peptides of α‐tubulin from newborn and adult mouse brain were compared. The heterogeneity of these carboxy‐terminal peptides was observed to increase from newborn to adult brain tubulin. The major part of this increased heterogeneity is due to the posttranslational excision of Glu450, which makes α‐tubulin nontyrosinatable (Δ‐2 tubulin). The structures of the polyglutamyl side chain of the bi‐ and triglutamylated peptides were analyzed in this work. In polyglutamylation of α‐tubulin, the first glutamyl residue can only be amide‐linked to the γ‐carboxyl group of Glu445, but the additional residues may be linked either to the γ‐ or to the α‐carboxyl groups of the preceding one. By optimized reverse‐phase separations and comparison with synthetic peptides corresponding to all possible linkages for the biglutamylated (γ1α2, γ1γ2) and triglutamylated (γ1α2α3, γ1γ2γ3, γ1α2γ3, γ1γ2α3, γ1γ2α2) tubulin peptides, it was possible to conclude that the mode of linkage connecting the second and third additional glutamyl residues corresponds mostly to α‐bond structures, for both newborn and adult mice.
Investigation of Myelin/Oligodendrocyte Glycoprotein Membrane Topology
Authors: Kroepfl John F. , Viise Laura R. , Charron Audra J. , Linington Chris , Gardinier Minnetta V.
Published: July 2010

Abstract: Myelin/oligodendrocyte glycoprotein (MOG) is a CNS‐specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain‐specific antisera to ascertain the localization of these hydrophilic domains. As expected, MOG's glycosylated N‐terminal Ig‐like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG1–125 antiserum. In contrast, both MOG154–169 and MOG198–218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N‐terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C‐terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane‐associated) would be unique among Ig‐like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.
Metabolism of [U‐13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling
Authors: Sonnewald U. , Westergaard N. , Jones P. , Taylor A. , Bachelard H. S. , Schousboe A.
Published: July 2010

Abstract: Metabolism of [U‐13C5]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify 13C‐labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U‐13C5]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U‐13C5]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl‐CoA. The present work demonstrates that this pathway is active, because [4,5‐13C2]glutamate was observed in astrocytes incubated with [U‐13C5]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono‐labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using 13CO2 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.
Phospholipase A2 Activity Is Selectively Decreased in the Striatum of Chronic Cocaine Users
Authors: Ross Brian M. , Moszczynska Anna , Kalasinsky Kathryn , Kish Stephen J.
Published: July 2010

Abstract: Dopamine‐mediated stimulation of arachidonic acid metabolism, via activation of the phospholipid metabolizing enzyme phospholipase A2 (PLA2), has recently been implicated in dopamine neurotransmitter function. We examined the status of PLA2 in autopsied brain of 10 chronic users of cocaine, a dopamine reuptake inhibitor. PLA2 activity, assayed at pH 8.5 in the presence of Ca2+, was significantly (p < 0.01) decreased by 31% in the putamen of cocaine users (n = 10) compared with that in controls (n = 10), whereas activity was normal in the frontal and occipital cortices, subcortical white matter, and cerebellum. In contrast, calcium‐independent PLA2 activity, assayed at pH 7.0, was normal in all brain regions examined. Our finding of altered PLA2 activity restricted to a region of high dopamine receptor density suggests that modulation of PLA2 may be involved in mediating some of the dopamine‐related behavioral effects of cocaine and could conceivably contribute to dopamine‐related processes in the normal brain.
Pigment Epithelium‐Derived Factor Protects Cultured Cerebellar Granule Cells Against Glutamate‐Induced Neurotoxicity
Authors: Taniwaki Takayuki , Hirashima Naohide , Becerra S. Patricia , Chader Gerald J. , Etcheberrigaray René , Schwartz Joan P.
Published: July 2010

Abstract: Pigment epithelium‐derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 µM glutamate, glutamate‐induced neuronal death was significantly reduced. The protective effect of rPEDF was dose‐dependent in the range from 0.023 to 7.0 nM (1–500 ng/ml), with a half‐maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate‐induced neurotoxicity, possibly via a calcium‐related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.
Protein Kinase C Activators Work in Synergy with Specific Growth Factors to Initiate Tyrosine Hydroxylase Expression in Striatal Neurons in Culture
Authors: Du Xinyu , Iacovitti Lorraine
Published: July 2010

Abstract: Our previous studies indicate that, in the non‐catecholamine (non‐CA) neurons of the striatum, expression of the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH) can be initiated by the synergistic interaction of acidic fibroblast growth factor (aFGF) and a second partner molecule. In this study, we sought to determine whether the activators of protein kinase C (PKC) signaling pathways, either alone or in conjunction with various growth factors, is sufficient to induce TH in striatal neurons. We found that when the active β form of 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (TPA), but not the inactive α analogue, was incubated in the presence of aFGF, basic FGF, or brain‐derived neurotrophic factor, TH expression was initiated. Activation of the PKC pathways alone (in the absence of growth factors) did not mimic these effects, suggesting that multiple pathway activation is required for novel TH expression. Although other specific activators of PKC were effective growth factor partners, TPA was the most potent with an ED50 of 0.008 µM. Conversely, inhibitors of protein kinases, such as H7, H8, or H89, prevented the expression of TH by aFGF and TPA. Because pretreatment with protein (cycloheximide) or RNA synthesis (amanitin and actinomycin D) inhibitors eliminated the inductive effect of aFGF and TPA, we conclude that de novo transcription and translation are necessary for the expression of TH after convergence of both PKC and growth factor pathways.