bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Cerebral Ischemia on Regional Dopamine Release and D1 and D2 Receptors
Authors: Chang C. J. , Ishii H. , Yamamoto H. , Yamamoto T. , Spatz M.
Published: October 2006

Abstract: To expand on the nature of regional cerebral vulnerability to ischemia, the release of dopamine (DA) and dopaminergic (D1 and D2) receptors were investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (1–2 h). Extracellular cortical and striatal content of DA and its metabolites was measured by microdialysis using HPLC with electrochemical detection. The kinetic properties of D1 and/or D2 receptor binding sites were determined in cortical and striatal membranes with the use of radiolabeled ligands (125I‐SCH23982 and [3H]YM‐09151‐2, respectively). The ischemic release of DA from the striatum was greater (400‐fold over preischemic level) than that from the cortex (12‐fold over preischemic content). The affinity for the D1‐receptor ligand was lower (KD= 1.248 ± 0.047 nM) after ischemia than that for sham controls (KD= 0.928 ± 0.032 nM, p < 0.001). The number of binding sites for D2 receptors decreased in striatum (Bmax= 428 ± 18.4 fmol/mg of protein) after ischemia compared with sham controls (Bmax= 510 ± 25.2 fmol/mg of protein, p < 0.05). D1 or D2 binding sites were not changed either in the ischemic cortex or postischemic striatum and cortex. The findings strongly suggest that the ischemic release of DA from striatum is associated with early transient changes in D1‐ and D2‐mediated DA neurotransmission.
Protein Phosphorylation at a Postreceptor Site Can Block Desensitization and Induce Potentiation of Secretion in Chromaffin Cells
Authors: Lin Lih Fang , Kim Kyong‐Tai , Westhead Edward W.
Published: October 2006

Abstract: Desensitization or habituation to repeated or prolonged stimulation is a common property of secretory cells. Phosphorylation of receptors mediates some desensitization processes, but the relationship of phosphorylation to desensitization at postreceptor sites is not well understood. We have tested the effect of protein phosphorylation on desensitization in bovine chromaffin cells. To increase protein phosphorylation, we have used the protein phosphatase inhibitor okadaic acid at 12.5 nM, 100 pA4 8‐bromo‐cyclic AMP to activate protein kinase A, and 10 nM phorbol 12,13‐dibutyrate to activate protein kinase C . During repeated 6‐s stimulation at 5‐min intervals, catecholamine secretion from control cells decreases. Cells exposed to 8‐bromo‐cyclic AMP or okadaic acid alone show slightly decreased rates of desensitization. In cells pretreated with phorbol 12,13‐dibutyrate, desensitization is blocked. Okadaic acid‐treated cells stimulated in the presence of 8‐bromo‐cyclic AMP show potentiation of secretion with repeated stimulation. The protein kinase inhibitor 1 ‐(5‐iso‐quinolinylsulfonyl)‐2‐methylpiperazine (H7) increases the desensitization rate. Because these phenomena are observed during secretion evoked with elevated Kf as well as by a nicotinic agonist, the effect of phosphorylation is at a postreceptor site. In contrast to desensitization to the repeated stimulations, desensitization to prolonged stimulation with high K+ is not altered by the above protocols in chromaffin Cells.
Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage
Authors: Nakajima Kazunori , Ikenaka Kazuhiro , Kagawa Tetsushi , Aruga Jun , Nakao Junji , Nakahira Kensuke , Shiota Chiyo , Kim Seung U. , Mikoshiba Katsuhiko
Published: October 2006

Abstract: Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well‐known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.
A New Family of Transcripts of the Myelin Basic Protein Gene: Expression in Brain and in Immune System
Authors: Zelenika Diana , Grima Brigitte , Pessac Bernard
Published: October 2006

Abstract: A cDNA clone (MBP2) corresponding to a novel mouse myelin basic protein (MBP) mRNA has been isolated from an adult mouse bone marrow cDNA library. It contains the MBP exons la‐7 except exon 5. Using PCR experiments we have determined that this MBP2 mRNA belongs to a new MBP mRNA family initiated upstream from exon 1b. Their 5’end extends into exon la and/or the region O’previously described. These mRNAs are generated by alternative splicing of the primary transcript involving excision of exon la, 1b, 2, 5, or 6. Thus, these new mRNAs are produced from a promoter(s) located upstream from the major promoter 1b. They are expressed in brain (at least from embryonic day 15), in bone marrow, and in other hemolymphoppietic tissues, particularly in macrophage cells. As their expression is not restricted to myelinating cells, the function of these novel MBP mRNAs and putative proteins might not be related to myelination.
Oxidative Stress in a Clonal Cell Line of Neuronal Origin: Effects of Antioxidant Enzyme Modulation
Authors: Buckman Trent D. , Sutphin Mary S. , Mitrovic Branislava
Published: October 2006

Abstract: The effects of intracellularly generated H2O2 on cell viability, morphology, and biochemical markers of injury have been investigated in a clonal cell line of neuronal origin (140‐3, mouse neuroblastoma X rat glioma) as a cell culture model for the role of oxidative stress in the longterm loss of neurons in the brain. The H2O2 was generated from the redox cycling of menadione, or by the oxidation of serotonin catalyzed by monoamine oxidase, to simulate the effect of amine neurotransmitter turnover. Incubation with menadione at concentrations as low as 10 γM for several hours resulted in significant losses of cell viability and altered morphology. Similar effects were evident in the presence of serotonin only after incubation overnight with concentrations > 1 mM. The cytotoxicity of either agent was potentiated by preincubation with specific inhibitors of two enzymes important to cellular antioxidant defenses, 3‐amino‐1,2,4‐trazole for catalase and 1,3‐bis(chloromethyl)‐1‐nitrosourea for glutathione reductase. Activity of another antioxidant enzyme of particular importance to antioxidant defenses in brain, the selenoprotein glutathione peroxidase, was stimulated fourfold by growth of cultures in the presence of sodium selenite as a source of active‐site Se for the enzyme. The only effect of the selenite on other functionally coupled antioxidant enzymes was a decrease in activity of superoxide dismutase at concentrations >200 nM. The selenite substantially protected cells against oxidative stress induced by combinations of menadione, 3‐amino‐1,2,4‐trazole, and 1,3‐bis(chloromethyl)‐1‐nitrosourea, but was only marginally effective with serotonin as a source of oxidative stress. The monoamine oxidase inhibitor pargyline increased cell survival in the presence of serotonin, demonstrating the role of this enzyme in its cytotoxicity. DNA damage (single strand breaks), but not lipid peroxidation, correlated with the cytotoxic effects of menadione.
Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells
Authors: Miller Danette R. , Lee Greta M. , Maness Patricia F.
Published: October 2006

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady‐state levels of phosphotyrosine‐modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF‐primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/trk tyrosine kinase because the presence of NGF in cultures of NGF‐primed cells was not necessary for genistein‐stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF‐primed PC12 cells. Video‐enhanced differential interference contrast microscopy revealed that growth cones of genistein‐treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.
Aromatic L‐Amino Acid Decarboxylase Activity of Mouse Striatum Is Modulated via Dopamine Receptors
Authors: Hadjiconstantinou M. , Wemlinger T. A. , Sylvia C. P. , Hubble J. P. , Neff N. H.
Published: October 2006

Abstract: Aromatic L‐amino acid decarboxylase (AAAD) activity is enhanced in the striatum of control and MPTP‐treated mice after administration of a single dose of the dopamine receptor antagonists haloperidol, sulpiride, and SCH 23390. MPTP‐treated mice appear more sensitive to the antagonists, i.e., respond earlier and to lower doses of antagonists than control mice. The rise of AAAD activity induced by the antagonists is prevented by pretreatment with cycloheximide. The apparent Km values for L‐3,4‐dihydroxyphenylalanine (L‐DOPA) and pyridoxal 5‐phosphate appear unchanged after treatment with the antagonists. Increased AAAD activity was observed also after subchronic administration of dopamine receptor antagonists or treatment with reserpine. A single dose of a selective dopamine receptor agonists had no effect on AAAD activity. In contrast, administration of L‐DOPA, quinpirole, or SKF 23390 for 7 days lowers AAAD activity in the striatum. We conclude that AAAD is modulated in striatum via dopaminergic receptors.
Sex Differences in the Effects of Acute Swim Stress on Binding to GABAA Receptors in Mouse Brain
Authors: Akinci Muallâ K. , Johnston Graham A. R.
Published: October 2006

Abstract: Acute swim stress (3‐min swim at 32°C) in female, but not in male, mice results in substantial changes in the characteristics of GABA binding to membranes prepared from the forebrain. These changes were larger when measured in a relatively crude membrane preparation than in a well‐washed membrane preparation commonly used in GABA binding assays, consistent with the loss of endogenous modulators of GABA binding in the latter preparation. These changes may be related to stress‐induced alterations in part in the modulation of the characteristics of GABA binding by endogenous steroids, as the acute swim stress produced a larger increase in plasma corticosterone levels in female than in male mice.
Effects of Chronic Morphine Treatment on β‐Endorphin‐Related Peptides in the Caudal Medulla and Spinal Cord
Authors: Bronstein David M. , Gutstein Howard B. , Akil Huda
Published: October 2006

Abstract: The effects of chronic morphine treatment on β‐endorphin (βE)‐immunoreactive (βE‐ir) peptide levels were determined in the rat caudal medulla and different areas of the spinal cord. Seven days of morphine pelleting had no effect on total βE‐ir peptides in the caudal medulla. In contrast, it significantly increased βE‐ir peptide concentrations in the cervical and thoracic regions of the spinal cord compared with placebo‐pelleted controls, whereas in the lumbosacral region this trend did not reach statistical significance. Injections of the opiate receptor antagonist naloxone 1 h before the rats were killed had no effect on the morphine‐induced increases in the cord. Chromatographic analyses revealed that enzymatic processing of βE‐related peptides in the spinal cord seemed unaffected by the morphine and/or naloxone treatments. In light of previous data showing that morphine down‐regulates βE biosynthesis in the hypothalamus, the present results suggest that the regulation of βE‐ir peptides in the spinal cord is distinct from that found in other CNS areas. These data provide support for previous results suggesting that βE‐expressing neurons may be intrinsic to the spinal cord.
Primary Structure of the Human Platelet Serotonin Uptake Site: Identity with the Brain Serotonin Transporter
Authors: Lesch Klaus‐Peter , Wolozin Benjamin L. , Murphy Dennis L. , Riederer Peter
Published: October 2006

Abstract: A cDNA encoding the human platelet serotonin (5‐HT) uptake site was isolated and sequenced using the PCR. The cDNA represents a ˜3.1‐kb mRNA transcript and contains an open reading frame encoding a hydrophobic polypeptide of 630 amino acids with 12 membrane‐spanning segments, a calculated molecular mass of 70,320 Da, and an estimated isoelectrical point of 5.84. The human platelet 5‐HT uptake site is identical with the human brain 5‐HT transporter and ˜92% homologous to the rat protein. Hydropathicity analysis indicates 12 membrane‐spanning segments with two putative glycosylation sites within the second extracellular loop. The human platelet 5‐HT uptake site contains two intraplasmatic consensus phosphorylation sites for cyclic AMP‐dependent protein kinase recognition located in the cytoplasmatic N‐terminal region and three potential protein kinase C phosphorylation sites. The identity of the human platelet 5‐HT uptake site and the brain 5‐HT transporter indicates that both proteins are encoded by the same single‐copy gene, which has been assigned to the human chromosome 17. Our findings are likely to facilitate molecular pharmacologic and genetic investigations of the 5‐HT transporter in psychiatric disorders.
Purified Human Neuromelanin, Synthetic Dopamine Melanin as a Potential Model Pigment, and the Normal Human Substantia Nigra: Characterization by Electron Paramagnetic Resonance Spectroscopy
Authors: Enochs W. S. , Nilges M. J. , Swartz H. M.
Published: October 2006

Abstract: Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.
Role of ω‐Conotoxin GVIA‐Sensitive Ca2+ Entry in Angiotensin II‐Stimulated [3H]Phorbol 12, 13‐Dibutyrate Binding in Bovine Adrenal Medullary Cells
Authors: McMillian Michael K. , Hudson Pearlie M. , Suh Harold H. , Ye Hong , Tuominen Raimo K. , Hong John S.
Published: October 2006

Abstract: The relative contributions of Ca2+ influx and intracellular Ca2+ mobilization were examined for angiotensin II‐stimulated [3H]phorbol 12, 13‐dibutyrate binding, which reflects the level of activated protein kinase C in bovine chromaffin cells. Angiotensin II receptors activate phospholipase C in chromaffin cells, leading to a shortlived mobilization of intracellular Ca2+. Angiotensin II‐stimulated [3H]phorbol 12, 13‐dibutyrate binding was largely blocked in Ca2+‐free buffer and by pretreatment with the Ca2+‐channel blocker ω‐conotoxin GVIA. The [3H]phorbol 12, 13‐dibutyrate binding response to [Sar1]angiotensin II also appeared to be voltage sensitive, as no additivity was observed with the response to the depolarizing agent 4‐aminopyridine (3 mM). Threshold sensitivities of the extra‐and intracellular Ca2+‐mobilizing pathways to angiotensin II were similar, and all examined effects of angiotensin II in these cells were apparently mediated by losartan‐sensitive (AT1‐Iike) receptors. The dependence of angiotensin II‐stimulated [3H]phorbol 12, 13‐dibutyrate binding on extracellular Ca2+ entry, in contrast to stimulation by other phospholipase C‐linked receptor agonists (bradykinin and methacholine), suggests that angiotensin II preferentially stimulates protein kinase C translocation to the plasma membrane, rather than to internal membranes, in bovine adrenal medullary cells.
Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N‐Methyl‐d‐Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain
Authors: Kitamura Yoshihisa , Miyazaki Atsuhiro , Yamanaka Yojiro , Nomura Yasuyuki
Published: October 2006

Abstract: To clarify the regulatory mechanism of the N‐methyl‐d‐aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC‐II), endogenous Ca2+/calmodulin‐dependent protein kinase II (CaMK‐II), and purified cyclic AMP‐dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC‐II and endogenous CaMK‐II catalyzed the phosphorylation of 80–200‐kDa proteins in the PSD and l‐glutamate‐(or NMDA)‐induced increase of (+)‐5‐[3H]methyl‐10, 11‐dihydro‐5H‐dibenzo[a, d]cyclohepten‐5, 10‐imine maleate ([3H]MK‐801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC‐II‐and CaMK‐II‐catalyzed phosphorylation did not change the binding activity of l‐[3H]glutamate, cis‐4‐[3H](phospho‐nomethyl)piperidine‐2‐carboxylate ([3H]CGS‐19755; competitive NMDA receptor antagonist), [3H]glycine, α‐[3H]‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate, or [3H]‐kainate in the PSD. Pretreatment with PKC‐II‐and CaMK‐II‐catalyzed phosphorylation enhanced l‐glutamate‐induced increase of [3H]MK‐801 binding additionally, although purified cyclic AMP‐dependent protein kinase did not change l‐glutamate‐induced [3H]MK‐801 binding. From these results, it is suggested that PKC‐II and/or CaMK‐II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca2+ influx through the channel.
Barium‐Evoked Glutamate Release from Guinea‐Pig Cerebrocortical Synaptosomes
Authors: McMahon H. T. , Nicholls D. G.
Published: October 2006

Abstract: Ba2+ has multiple effects on presynaptic terminals. The ion inhibits the K+ channels responsible for stabilizing the plasma membrane potential in the same way as previously reported for dendrotoxin and 4‐aminopyridine. Secondly, the ion can substitute fully for Ca2+ in supporting KCl‐evoked release of glutamate from guinea‐pig cerebrocortical synaptosomes. In the latter case, the kinetics of glutamate release in the presence of saturating Ca2+ or Ba2+ are essentially identical. Substantially lower external concentrations of Ba2+ are required to achieve the same release kinetics as with Ca2+. The average internal free Ba2+ concentration attained during KCl depolarization is some 10‐fold higher than that for Ca2+. However, because the fura‐2 signal reflects predominantly the overflow of divalent cation after dissociation from the release trigger, it is not the valid parameter to compare effectiveness of the cations in triggering glutamate exocytosis. In view of the established inability of Ba2+ to interact with calmodulin, these results are discussed in relation to theories in which Ca2+/calmodulin‐dependent protein kinase‐mediated phosphorylation is a prerequisite for synaptic vesicle exocytosis.
Differential Effects of GM1 Ganglioside Treatment on Glial Fibrillary Acidic Protein Content in the Rat Septum and Hippocampus After Partial Interruption of Their Connections
Authors: Oderfeld‐Nowak B. , Jegliński W. , Skup M. , Skangiel‐Kramska J. , Zaremba M. , Koczyk D.
Published: October 2006

Abstract: The glial fibrillary acidic protein (GFAP) content was investigated using immunoblotting techniques in the septum and hippocampus of the rat after bilateral lateral fimbria transection. Seven days after surgery GFAP content increased significantly both in the septum (140% of control) and hippocampus (120% in dorsal, the less denervated, and 145% in the most denervated ventral part), indicating the occurrence of reactive gliosis. The GM1 treatment caused statistically significant attenuation of GFAP increment in all hippocampal parts. In contrast, GM1 treatment has no influence on the increase of GFAP content in the septum. Results suggest a differential effect of GM1 on the two gliotic reactions formed as a consequence of the lesion at the level of the source of innervation (septum) and the target (hippocampus).
Receptor‐Mediated Endocytosis of a Manganese Complex of Transferrin into Neuroblastoma (SHSY5Y) Cells in Culture
Authors: Suárez Norma , Eriksson Håkan
Published: October 2006

Abstract: Exposure to manganese compounds often occurs as the result of industrial production or mining. Although manganese appears in traces in animal and human tissue and is essential to certain biological processes, it is also toxic. In humans and animals, toxicity is mainly associated with the nervous system. The mechanism underlying behavioral and biochemical alterations observed after manganese intoxication is not fully understood. We have shown that the manganese present in serum after exposure to manganese oxide is bound to transferrin as trivalent manganic ion. In this study of manganese uptake and storage we used a clone of human neuroblastoma cells (SHSY5Y). These cells differentiate and express catechol‐aminergic properties. Saturation binding analysis of the transferrin‐manganese complex to the cells revealed a single class of binding sites, with an apparent KD of 13 ± 1 nM and a density of 11, 000 ± 2, 000 binding sites per cell. The complex was internalized in a temperature‐dependent way and reached saturation after 2 h when ∼2% of the added manganese had been internalized. About 80% of the internalized manganese was found in ferritin after 24 h of exposure. The results demonstrate that the transferrin receptor on SHSY5Y cells can bind and internalize a manganese‐transferrin complex as efficiently as an iron‐transferrin complex, although a saturation of the manganese uptake was achieved.
Cationic and Anionic Requirements for the Binding of 2β‐Carbomethoxy‐3β‐(4‐Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier
Authors: Reith Maarten E. A. , Coffey Lori L.
Published: October 2006

Abstract: The present study reports the ion dependency of 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[3H]tropane ([3H]‐ CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [3H]CFT binding to synaptosomal P2 membranes requires low concentrations of Na+ (peak binding between 20 and 50 mM Na+), is stimulated by phosphate anion or l‐, but is unaffected or only slightly affected by F‐, Cl‐, Br‐, NO3‐, or SO42‐, Concentrations of Na+ of >50 mM become inhibitory except in the presence of l‐, which shifts peak binding levels toward higher Na+ concentrations and also elevates the peak binding level. K+ strongly decreased [3H]CFT binding with a shallow inhibition curve, and Na+ could not overcome this effect. Saturation analysis of [3H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 mM; 1 mM plus 49 mM NaCIversus 10 mM plus 40 mM NaCI; or 1 mM plus 129 mM Nal versus 10 mM plus 120 mM Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.
Phosphorylation Modulates Calpain‐Mediated Proteolysis and Calmodulin Binding of the 200‐kDa and 160‐kDa Neurofilament Proteins
Authors: Greenwood Jeffrey A. , Troncoso Juan C. , Costello Anthony C. , Johnson Gail V. W.
Published: October 2006

Abstract: The effects of enzymatic dephosphorylation on neurofilament interaction with two calcium‐binding proteins, calpain and calmodulin, were examined. Dephosphorylation increased the rate and extent of 200‐kDa neurofilament protein proteolysis by calpain. In contrast, dephosphorylation of the 160‐kDa neurofilament protein did not alter the rate or extent of calpain proteolysis. However, the calpain‐induced breakdown products of native and dephosphorylated 160‐kDa neurofilament protein were different. Dephosphorylation did not change the proteolytic rate, extent, or breakdown products of the 68‐kDa neurofilament protein. Calmodulin binding to the purified individual 160‐ and 200‐kDa neurofilament proteins was increased following dephosphorylation. These results suggest that phosphorylation may regulate the metabolism and function of neurofilaments by modulating interactions with the calcium‐activated proteins calpain and calmodulin.
Reduction of HSP70 and HSC70 Heat Shock mRNA Induction by Pentobarbital After Transient Global Ischemia in Gerbil Brain
Authors: Kawagoe J. , Abe K. , Kogure K.
Published: October 2006

Abstract: The effect of pentobarbital on the induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brains was investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brains, HSP70 mRNA was scarcely present, whereas HSC70 mRNA was present in most cell populations. After a 5‐min occlusion of bilateral common carotid arteries, HSP70 and HSC70 mRNAs were induced together in several cells and were especially dense in hippocampal dentate granule cells at 3 h, but the strong hybridization of the mRNAs continued only in hippocampal CA1 cells by 2 days. At 7 days after the ischemia, CA1 neuronal cell death was apparent, and the HSP70 mRNA disappeared and HSC70 mRNA content returned to the sham level, except for in the CA1 cells. Pretreatment with pentobarbital (40 mg/kg, i.p.) greatly reduced or inhibited the induction of HSP70 and HSC70 mRNAs at both early (3‐h) and late (2‐day) phases after ischemia. The drug also prevented CA1 cell death at 7 days along with the maintenance of expression of HSC70 mRNA at the sham control level. Hypothermic effects of pentobarbital were noted at 30 and 60 min after the reperfusion, whereas at 2 h there was no statistical significance between the control and drug‐treated groups. The great reduction of HSP70 and HSC70 mRNA induction at both early and late phases after ischemia suggests that pentobarbital reduces intra‐ and/or postischemic stress and may protect CA1 cells from ischemic damage. These effects of the drug may be mainly due to its specific action rather than its hypothermic effects.
Reduced Clearance of Exogenous Dopamine in Rat Nucleus Accumbens, but Not in Dorsal Striatum, Following Cocaine Challenge in Rats Withdrawn from Repeated Cocaine Administration
Authors: Cass Wayne A. , Gerhardt Greg A. , Gillespie Kelly , Curella Pamela , Mayfield R. Dayne , Zahniser Nancy R.
Published: October 2006

Abstract: We investigated whether changes in the dopamine transporter in the nucleus accumbens or striatum are involved in cocaine‐induced behavioral sensitization by using in vivo electrochemistry to monitor the clearance of locally applied dopamine in anesthetized rats. Rats were injected with cocaine‐HCI (10 mg/kg i.p.) or saline daily for 7 consecutive days and then withdrawn for 7 days. Pressure ejection of a finite amount of dopamine at 5‐min intervals from a micropipette adjacent to the electrochemical recording electrode produced transient and reproducible dopamine signals. After a challenge injection of cocaine (10 mg/kg i.p.), the signals in the nucleus accumbens of cocaine‐treated animals became prolonged and the clearance rate of the dopamine decreased, indicating significant inhibition of the dopamine transporter. In contrast, simultaneous measurements in the dorsal striatum indicated a transient increase in both the amplitude of the signals and the clearance rate of the dopamine. The signals in both brain regions in the saline‐treated animals given the cocaine challenge were similar to those in untreated animals given an acute injection of cocaine (10 mg/ kg i.p.) or saline. Behaviorally, not all of the cocaine‐ treated animals were sensitized; however, both sensitized and nonsensitized animals displayed similar changes in dopamine clearance rate. Quantitative autoradiography with [3H]mazindol revealed that the affinity of the dopamine transporter for cocaine and the density of binding sites were similar in cocaine‐ and saline‐treated rats. The decrease in dopamine clearance rate observed in the nucleus accumbens of the cocaine‐treated rats after a challenge injection of cocaine is consistent with increased do‐ paminergic transmission, but does not appear to be sufficient in itself for producing behavioral sensitization.
Acute Neurite Retraction Elicited by Diverse Agents Is Prevented by Genistein, a Tyrosine Kinase Inhibitor
Authors: Smalheiser Neil R.
Published: October 2006

Abstract: The retraction of axonal branches is a prominent feature of nervous system development and function. Although various biological and pathological signals can elicit retraction, little is known regarding their underlying mode of action. An in vitro assay using NG108–15 cells was used to demonstrate that rapid‐onset neurites exposed acutely to trypsin, serum, lysophosphatidic acid, extracellular ATP, the phorbol ester phorbol 12‐myristate 13‐acetate, and nocodazole were all protected from retraction by the tyrosine kinase inhibitor genistein. This finding indicates that a common (genistein‐sensitive) cellular event is involved in integrating the influence of multiple extrinsic and intrinsic signals and in regulating whether or not neurites will execute a retraction response.
Nitric Oxide Synthase Activity Endogenously Modulates NMDA Receptors
Authors: Manzoni Olivier , Bockaert Joel
Published: October 2006

Abstract: We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA‐induced increase in intra‐cellular Ca2+ levels with fura‐2 ratio imaging, while nitric oxide synthase activity was either increased with l‐arginihe (the natural substrate of nitric oxide synthase) or inhibited using nitro‐l‐arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an l‐arginine (1 mM, 15 min) treatment (l‐arginine preincubation reduced the 100 μM NMDA‐induced maximal effect by 30–50%). The l‐arginine blockade of NMDA receptors was long‐lasting but could be partially reversed by hemoglobin (100 μM, 10 min), which binds nitric oxide. This was not observed when the neurons were treated with l‐arginine together with nitro‐l‐arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.
Regional Distribution and Quantitative Measurement of the Phosphoinositidase C‐Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain
Authors: Milligan Graeme
Published: October 2006

Abstract: Levels of the guanine nucleotide binding proteins G11α and Gqα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis conditions that can resolve Gqα and G11α. In all regions examined, Gqα was more highly expressed than G11α. Ratios of levels of Gqα to G11α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of Gqα and G11α in each region were obtained by comparison with known amounts of purified liver Gqα and G11α and with E. coli expressed recombinant Gqα. Areas that expressed Gqα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of Gqα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G14 is discussed.
Cerebral Cortex Gsα Protein Levels and Forskolin‐Stimulated Cyclic AMP Formation Are Increased in Bipolar Affective Disorder
Authors: Young L. Trevor , Li Peter P. , Kish Stephen J. , Siu Kin Po , Kamble Arvind , Hornykiewicz Oleh , Warsh Jerry J.
Published: October 2006

Abstract: Experimental animal and peripheral blood cell studies point to guanine nucleotide regulatory (G) protein disturbances in bipolar affective disorder. We have previously reported elevated prefrontal cortex Gsα protein in bipolar affective disorder and have now extended these preliminary observations in a larger number of subjects, assessing the brain regional specificity of these changes in greater detail, determining the functional biochemical correlates of such changes, and evaluating their diagnostic specificity. Membrane G protein (Gsα, Giα, Goα, and Gβ) immunoreactivities were estimated by western blotting in postmortem brain regions obtained from 10 patients with a DSMIII‐R diagnosis of bipolar affective disorder and 10 nonpsychiatric controls matched on the basis of age, postmortem delay, and brain pH. To examine whether there were functional correlates to the observed elevated Gsα levels, basal and GTPγS‐and forskolin‐stimulated cyclic AMP production was determined in the same brain regions. Compared with controls, Gsα (52‐kDa species) immunoreactivity was significantly (p < 0.05) elevated in prefrontal (+36%), temporal (+65%), and occipital (+96%) cortex but not in hippocampus (+28%), thalamus (‐23%), or cerebellum (+21%). In contrast, no significant differences were found in the other G protein subunits (Giα, Goα, Gβ) measured in these regions. Forskolin‐stimulated cyclic AMP production was significantly increased in temporal (+31%) and occipital (+96%) cortex but not in other regions. No significant differences were apparent in basal or GTPγS‐stimulated cyclic AMP production. A significant correlation (r= 0.60, p < 0.001) was observed between forskolin‐stimulated cyclic AMP formation and Gsα (52 kDa) immunoreactivity when examined across these cortical regions. The observed increase in Gsα may be specific to bipolar disorders as no significant differences were detected in Gsα levels in temporal cortex from patients with either schizophrenia (n = 7) or Alzheimer's disease (n = 7). In summary, the present study confirms and extends our earlier findings and supports the notion that increased Gsα levels and possibly Gsα‐adenylyl cyclase‐mediated signal transduction are relevant to the pathophysiology of bipolar affective disorder.
Effects of Chronic Basic Fibroblast Growth Factor Administration to Rats with Partial Flmbrial Transections on Presynaptic Cholinergic Parameters and Muscarinic Receptors in the Hippocampus: Comparison with Nerve Growth Factor
Authors: Araujo Dalia M. , Lapchak Paul A. , Hefti Franz
Published: October 2006

Abstract: The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [3H]‐acetylcholine ([3H]ACh) synthesis (by 59%), basal and ve‐ratridine (1 μM)‐evoked [3H]ACh release (by 44 and 57%, respectively), and [3H]vesamicol binding site densities (by 35%). In addition, [3H]AF‐DX 116/muscarinic M2 binding site densities were also modestly decreased (by 23%). In contrast, [3H]pirenzepine/muscarinic M1 and [3H]AF‐DX 384/muscarinic M2/M4 binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion‐induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF‐treated (1 μg intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion‐induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 μg (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion‐induced reduction in [3H]ACh synthesis by hippocampal slices; [3H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF‐treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT activity, but also other parameters indicative of increased function in the cholinergic terminals.
Phosphoprotein Phosphatase Activities in Alzheimer Disease Brain
Authors: Gong Cheng‐Xin , Singh Toolsee J. , Grundke‐Iqbal Inge , Iqbal Khalid
Published: October 2006

Abstract: Microtubule‐associated protein τ is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of τ to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated τ could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of τ might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used 32P‐labeled phosphorylase kinase and poly(Glu.Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/ phosphothreonyl‐protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl‐protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age‐matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of τ in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of β‐amyloid by augmenting the amyloidogenic pathway processing of β‐amyloid precursor protein.
A τ Fragment Containing a Repetitive Sequence Induces Bundling of Actin Filaments
Authors: Moraga Daniel M. , Nuñez Patricio , Garrido Jorge , Maccioni Ricardo B.
Published: October 2006

Abstract: Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule‐associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin‐binding domain on τ with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of τ protein, increased turbidity at 320 nm in a dose‐dependent fashion. A salient feature of the τ peptide‐induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the τ peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6–4.7 resulted in a higher effect of τ peptide in promoting bundles of actin filaments. A similar pH‐dependent effect was observed for τ protein‐induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of ɛ‐aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a τ repetitive sequence (also found in MAP‐2 and MAP‐4) containing a common tubulin binding motif may constitute a functional domain on τ for the dynamics of the interconnections between actin filaments and microtu‐bules.
The Balance Between τ Protein's Microtubule Growth and Nucleation Activities: Implications for the Formation of Axonal Microtubules
Authors: Brandt Roland , Lee Gloria
Published: October 2006

Abstract: The microtubule‐associated protein τ is found primarily in neuronal tissues and is highly enriched in the axon. It promotes microtubule assembly in vitro and stabilizes microtubules in cells. To study how τ protein might be involved in the unique features of axonal microtubules, we have analyzed the effect of E. coli‐synthesized τ protein using an in vitro centrosome‐mediated microtubule regrowth assay over a wide range of τ/tubulin ratios. We report that microtubule assembly promoted by τ protein exhibits characteristic changes dependent on the τ/tubulin ratio. Above a threshold level, nucleation of new microtubules is favored over growth of existing ones, τ isoform variation does not change this phase transition in microtubule assembly. We discuss how τ might participate in the elaboration of axonal morphology based on our results and present evidence that the phase transition from microtubule growth to nucleation is critical for axonal development.
Muscarine Receptors Regulating Electrically Evoked Release of Acetylcholine in Hippocampus Are Linked to Pertussis Toxin‐Sensitive G Proteins but Not to Adenylate Cyclase
Authors: Allgaier Clemens , Choi Bong Kyo , Hertting Georg
Published: October 2006

Abstract: [3H]Acetylcholine release elicited with 360 pulses/3 Hz from slices of rabbit hippocampus is facilitated in the presence of the muscarine (M) receptor antagonist atropine (indicating the existence of autoinhibition) and diminished by the M receptor agonists carbachol and oxotremorine. W‐Ethylmaleimide (30 μM) and pertussis toxin (8 μg/ml) counteracted antagonist‐induced facilitation and agonist‐induced inhibition of release, suggesting that a pertussis toxin‐sensitive GTP‐binding protein is involved in the chain of events mediating activation of M receptors to inhibition of release. Neither 8‐bromo‐cyclic AMP (300 μM), a membrane analogue of cyclic AMP, nor rolipram (10 μM), a phosphodiesterase inhibitor, affected electrically evoked release of [3H]acetylcholine. They also did not influence the oxotremorine‐induced inhibition of transmitter release. In conclusion, no evidence was found for the assumption that activation of M autoreceptors is linked to inhibition of adenylate cyclase.
Molecular Cloning of Two Additional Members of the Neural Visinin‐Like Ca2+‐Binding Protein Gene Family
Authors: Kajimoto Yasuo , Shirai Yutaka , Mukai Hideyuki , Kuno Takayoshi , Tanaka Chikako
Published: October 2006

Abstract: We have isolated a rat cDNA clone encoding a neural visinin‐like Ca2+‐binding protein (NVP), which we designate NVP‐1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP‐1 cDNA probe under low‐stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP‐2 and NVP‐3, have been isolated. The deduced amino acid sequences of NVP‐2 and NVP‐3 are 89.0% and 68.6% identical to that of NVP‐1, respectively, and contain consensus sequences for EF‐hand Ca2+‐binding sites. Northern blot analysis shows that NVP‐1, NVP‐2, and NVP‐3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP‐2 and NVP‐3 are additional members of the NVP gene family.
Analysis of γ‐Aminobutyric AcidA Receptor Subunits in the Mouse Cochlea by Means of the Polymerase Chain Reaction
Authors: Drescher Dennis G. , Green Glenn E. , Khan Khalid M. , Hajela Kavita , Beisel Kirk W. , Morley Barbara J. , Gupta Anil K.
Published: October 2006

Abstract: Thirteen GABAA receptor subunits were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 14–18‐day‐old animals, and mRNA was isolated using oligo‐dT cellulose. After reverse transcription, resulting cDNA was amplified by PCR with primers specific for nucleotide sequences representing GABAA subunits. PCR products corresponding to subunits α1‐αB, β1,‐β3, and γ2 were amplified, whereas those representing γ1, γ3, and δ were not amplified above background. These results provide the most direct evidence to date that GABAA receptors composed of the detected subunits are expressed in the mammalian cochlea, lending new support to previous studies implicating GABA as a co‐chlear transmitter. The pronounced expression of α2 and α6 sub‐units suggests type II and “cerebellar‐type” benzodiazepine pharmacology in the cochlea.