bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Ganglioside Composition and Synthesis Is Different in Developing Chick Retinal Pigment Epithelium and Neural Retina
Authors: Daniotti J. L. , Landa C. A. , Maccioni H. J. F.
Published: July 2010

Abstract: We examined the immunocytochemical expression of GM3 and QD3 in 3‐day‐old chick embryo retinal pigment epithelium (RPE) and neural retina (NR). We also compared the composition of gangliosides and the activities of key ganglioside glycosyltransferases of the RPE and NR of 8‐, 12‐, and 15‐day old embryos. The immunocytochemical studies in 3‐day‐old embryos showed heavy expression of GM3 and GD3 at the inner and outer layers of the optic vesicle that are the precursors of the RPE and NR, respectively. The compositional and enzymatic studies showed pronounced differences between RPE and NR of 8‐day and older embryos. HPTLC showed that at 8 days the major species were GM3 and GD3 in RPE and GD3 and GT3 in NR. As development proceeded, GD3 decreased in both tissues, GM3 became the major ganglioside in RPE, and ganglio‐series gangliosides (mainly GD1a) became the major species in NR. At 15 days the major species were GD1 a in NR and GM3 in RPE. Enzyme determinations showed that whereas in RPE from 12‐day‐old embryos GM2 synthase was under the limit of detection and GD3 synthase activity was about sixfold lower than GM3 synthase, in NR the activities of GM3 and GD3 synthases were similar and both six‐to ninefold lower than GM2 synthase. These results evidence a markedly different modulation of the ganglioside glycosylating system in cells of a common origin that through distinct differentiation pathways originate two closely related tissues of the optic system. In addition, they reinforce the relevance of the relative activities of key transferases in determining the pattern of gangliosides in different cell types.
Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes
Authors: Collins Roger M. , Zielke H. Ronald , Woody Robert C.
Published: July 2010

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l‐[U‐14C]glutamate, label incorporation into [14C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l‐[U‐14C]glutamate into [14C]aspartate also decreased with increasing concentrations of valproate. Following a 4‐day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs‐cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.
Studies with Differentially Labeled [11C]Cocaine, [11C]Norcocaine, [11C]Benzoylecgonine, and [11C]‐and 4′‐[18F]Fluorococaine to Probe the Extent to Which [11C]Cocaine Metabolites Contribute to PET Images of the Baboon Brain
Authors: Gatley S. John , Yu Ding‐Wei , Fowler Joanna S. , MacGregor Robert R. , Schlyer David J. , Dewey Stephen L. , Wolf Alfred P. , Martin Thomas , Shea Colleen E. , Volkow Nora D.
Published: July 2010

Abstract:The psychostimulant drug of abuse, cocaine (benzoylecgonine methyl ester), is rapidly metabolized by cleavage of its two ester groups, to give benzoylecgonine (BE) and ecgonine methyl ester, and byN‐demethylation, to giveN‐norcocaine (NC). The recent use of [N‐methyl‐11CH3]cocaine to image brain cocaine binding sites with positron emission tomography (PET) raises the question of whether PET images partially reflect the distribution and kinetics of labeled cocaine metabolites. We prepared [O‐metty/‐11CH3]cocaine by methylation of the sodium salt of BE with [11C]CH3l, and showed that PET baboon brain scans, as well as regional brain kinetics and plasma time‐activity curves corrected for the presence of labeled metabolites, are nearly identical to those seen with [N‐methyl‐11CH3]cocaine. This strongly suggests that11C metabolites do not significantly affect PET images, because the metabolite pattern is different for the two labeled forms of cocaine. In particular, nearly half the11C in blood plasma at 30 min was [11C]CO2when [N‐methy/‐11CH3]cocaine was administered, whereas [11C]CO2was not formed from [O‐methy/‐11CH3]cocaine. Only a trace of [11C]NC was detected in plasma after [O‐methyl‐11CH3]cocaine administration. Nearly identical brain PET data were also obtained when 4′‐[N‐methy/‐11CH3]fluorococaine and 4′‐[18F]fluoro‐cocaine (prepared by nucleophilic aromatic substitution from [18F]fluoride‐and 4′‐nitrococaine) were compared with [N‐methy/‐11CH3]cocaine. In vitro assays with rat brain membranes showed that cocaine and 4′‐fluoroco‐caine were equipotent at the dopamine reuptake site, but that 4′‐fluorococaine was about 100 times more potent at the 5‐hydroxytryptamine reuptake site. The studies with positron‐emitting 4′‐fluorococaines thus support the lack of significance of labeled metabolites or of binding to 5‐hydroxytryptamine reuptake sites to PET images taken with [N‐methy/‐11CH3]cocaine. [11C]NC prepared byO‐methylation of norbenzoylecgonine gave PET images with preferential uptake in striatum, but slower clearance from all brain regions than [O‐methy/‐11CH3]cocaine. [11C]BE prepared byN‐methylation of norbenzoylecgonine did not show brain uptake.
Regional Specificity of the Long‐Term Variation of Tyrosine Hydroxylase Protein in Rat Catecholaminergic Cell Groups After Chronic Heat Exposure
Authors: Garcia Christine , Schmitt Pascal , D'Aléo Philippe , Bittel Jacques , Curé Michel , Pujol Jean‐François
Published: July 2010

Abstract: The present study was carried out to investigate the effect of chronic heat exposure on tyrosine hydroxy‐lase (TH) protein content in catecholaminergic rat brain‐stem areas such as the anterior (LCA) and posterior (LCP) locus coeruleus, the substantia nigra (SN), the ventral tegmental area, and the dorsomedial (DMM) and the ventrolateral medulla and in the adrenal gland (AG). Male Sprague‐Dawley rats were exposed to 34°C during 3, 7, or 14 days. Controls were kept at 25°C for the same period. In the LCA, TH content was decreased on day 7 (‐34%) and 14 (‐37%) of heat exposure. In the SN, TH protein content was decreased on day 7 (‐25%) and 14 (‐20%) after 34°C. In the DMM cell group, 14 days at 34°C produced a decrease (‐20%) of TH content. In all of these structures, TH content variations were correlated with body temperature variations. In the AG, TH content increased progressively to peak (+31%) after 14 days of chronic heat exposure. This increase was also associated with body temperature modification. The selective and body temperature‐related response of long‐term TH protein content variations following chronic heat exposure observed in the LCA, SN, DMM, and AG could represent an adaptive physiological response of these catecholaminergic cells.
Purification and Characterization of p68/70, Regeneration‐Associated Proteins from Goldfish Brain
Authors: Leski Michael L. , Agranoff Bernard W.
Published: July 2010

Abstract: Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887‐fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE‐Sephacel, Cu2+‐charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5 2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high‐resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a hetero‐dimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydro‐phobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton 114. Analysis of hydrolytic products indicated p68/70 to be a glyco‐protein, containing 11% carbohydrate.
The Quantity of Calcium that Appears to Induce Neuronal Death
Authors: Eimerl Sara , Schramm Michael
Published: July 2010

Abstract:Excessive entry of Ca2+through the NMDA receptor is thought to be the major cause of glutamate toxicity in brain neurons. However, actual quantitation of the calcium overload has not been achieved. Here we show that the absolute amount of45Ca2+taken up via the NMDA receptor correlates quantitatively with the amount of acute cell death in cultured cerebellar granule cells of the rat. Analysis of 9‐and 16‐day cultures reveals that the NMDA‐induced Ca2+uptake is about the same at these ages, whereas the Ca‐dependent lethal process is more developed in the older neurons. The calculated lethal concentration of45Ca taken up exceeds by ∼10,000 times the maximal concentration of [Ca2+], that can be measured by fluorescence imaging. It is suggested that the Ca2+taken up induces the lethal process in a subcellular structure in which it has been segregated.
Alzheimer Aβ Amyloid Forms an Inhibitory Neuronal Substrate
Authors: Eraser P. E. , Lévesque L. , McLachlan D. R.
Published: July 2010

Abstract: Alzheimer's disease (AD) is identified by the accumulation of amyloid plaques, neurofibrillary degeneration, and the accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the amyloid β(Aβ) protein, which is a proteo‐lytic fragment of the membrane‐associated amyloid precursor protein. To examine the effects of amyloid on neuron growth, a hybrid mouse motoneuron cell line (NSC34) exhibiting spontaneous process formation was exposed to artificial “plaques” created from aggregated synthetic Aβ peptides. These correspond to full‐length Aβ residues 1–40 (Aβ1–40), an internal β‐sheet region comprising residues 11–28 (Aβ11–28), and a proposed toxic fragment comprising residues 25–35 (Aβ25–35). Fibers were immobilized onto culture dishes, and addition of cells to these in vitro plaques revealed that Aβ was not a permissive substrate for cell adhesion. Neurites in close contact with these deposits displayed abnormal swelling and a tendency to avoid contact with the Aβ fibers. In contrast, Aβ did not affect the adhesion or growth of rat astrocytes, implicating a specific Aβ‐neuron relationship. The inhibitory effects were also unique to Aβ as no response was observed to deposits of pancreatic islet amyloid poly‐peptide fibers. Considering the importance of cell adhesion in neurite elongation and axonal guidance, the antiadhesive properties of Aβ amyloid plaques found in vivo may contribute to the neuronal loss responsible for the clinical manifestations of AD.
Regulation of Parathyroid Hormone‐Related Peptide Gene Expression by Estrogen in GH4C1 Rat Pituitary Cells Has the Pattern of a Primary Response Gene
Authors: Holt Elizabeth H. , Lu Charles , Dreyer Barbara E. , Dannies Priscilla S. , Broadus Arthur E.
Published: July 2010

The parathyroid hormone‐related peptide (PTHrP) gene has been reported to be subject to a wide variety of physiological and pharmacological controls. Two distinct patterns of PTHrP mRNA response have been recognized, one characterized by a prolonged or plateau response lasting many hours to days and the second characterized by rapid induction‐deinduction kinetics and lasting 1 to several hours. The kinetics of the second pattern are similar to those displayed by primary response genes like nuclear protooncogenes, cytokines, and growth factors. In GH4C1, rat pituitary cells, 17β‐estradiol induced a rapid and transient increase in PTHrP mRNA expression, with a peak response at 1–2 h. This response appeared to be due to a rapid and transient burst in gene transcription, which by runoff analysis was maximal at 20–40 min and declined thereafter. PTHrP mRNA half‐life was 30 min in these cells and was unaltered by estradiol. Cy‐cloheximide did not block the 17β‐estradiol‐induced response but rather prolonged it, and runoff analysis revealed that this effect was due to a prolongation or persistence of PTHrP gene transcription. These findings suggest that the transient nature of the native response reflects the effects of an estrogen‐inducible represser. All of these features are characteristic of a prototypical primary response gene.
A Rat Brain cDNA Encodes Enzymatically Active GABA Transaminase and Provides a Molecular Probe for GABA‐Catabolizing Cells
Authors: Medina‐Kauwe Lali K. , Tillakaratne Niranjala J. K. , Wu Jang‐Yen , Tobin Allan J.
Published: July 2010

Abstract: cDNAs encoding γ‐aminobutyric acid aminotransferase (GABA‐T) were isolated from a λZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA‐T antibody and plaque hybridization with a GABA‐T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA‐T from a rat brain cDNA containing the full‐length GABA‐T coding region. Our rat brain GABA‐T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA‐T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA‐T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD85 and GAD87.
Overexpressed Monomeric Human Acetylcholinesterase Induces Subtle Ultrastructural Modifications in Developing Neuromuscular Junctions of Xenopus laevis Embryos
Authors: Seidman Shlomo , Aziz‐Aloya Revital Ben , Timberg Rina , Loewenstein Yael , Velan Baruch , Shafferman Avigdor , Liao Jian , Norgaard‐Pedersen Bent , Brodbeck Urs , Soreq Hermona
Published: July 2010

Abstract: Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle‐specific proteins, among them the acetylcholine‐hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over‐expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell‐associated rHAChE accumulated in myotomes of 2‐and 3‐day‐old embryos within the same sub‐cellular compartments as native Xenopus AChE. NMJs from 3‐day‐old DNA‐injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.
Sequences Distal to the AP1/E Box Motif Are Involved in the Cell Type‐Specific Expression of the Rat Tyrosine Hydroxylase Gene
Authors: Wong Shou C. , Moffat Mark A. , O'Malley Karen L.
Published: July 2010

Abstract: In order to define cell type‐specific elements associated with the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), transient transfections of promoter deletion constructs were used to test relative reporter‐gene activities in TH‐expressing and‐nonexpressing cell lines. Such assays demonstrated that a region between‐503 and‐578 contributed to rat TH promoter activity in the pheochromocytoma cell line PC12. Deletion of these sequences resulted in a 66% loss in cell type‐specific activity. Mutations within the E box/dyad symmetry element (CAGGTGCCTGTGACAGTG) did not affect the basal and cell type‐specific pattern of expression exhibited by the rat TH promoter. Promoter fusion constructs between the rat TH promoter (‐741 and‐197) and the human TH promoter (‐197 and +1) exhibited reporter‐gene activities equivalent to that of wild‐type‐741 rat TH constructs, further demonstrating that sequence elements upstream of the rat E box/dyad symmetry are important for cell type‐specific expression. Gel‐shift experiments indicated that a PC12 nuclear factor could bind to a 39‐bp sequence within this region in a cell type‐specific manner. The size of this factor was 52 kDa as determined by UV cross‐linking experiments.
β‐Lumicolchicine Interacts with the Benzodiazepine Binding Site to Potentiate GABAAReceptor‐Mediated Currents
Authors: Mihic S. John , Whatley Valerie J. , McQuilkin Susan J. , Harris R. Adron
Published: July 2010

Abstract:An analogue of colchicine,β‐lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. β‐Lumicolchicine was found to competitively inhibit [3H]flunitrazepam binding and to enhance muscimol‐stimulated36Cr‐uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses inXenopusoocytes expressing human α1β2γ2S, but not α1β2, GABAAreceptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of β‐Lumicolchicine on the GABAAreceptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, β‐Lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.
Resistance of Golden Hamster to l‐Methyl‐4‐Phenyl‐1,2,3,6 Tetrahydropyridine: Relationship with Low Levels of Regional Monoamine Oxidase B
Authors: Mitra N. , Mohanakumar K. P. , Ganguly D. K.
Published: July 2010

Abstract: Effects of acute and chronic administration of 1 ‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) were investigated for dopamine (DA) and its metabolites, 3,4‐dihydroxyphenylacetic acid and 4‐hydroxy‐3‐methoxyphenylacetic acid, in nucleus caudatus putamen (NCP), limbic system, and substantia nigra (SN) of golden hamster and BALB/c and C57/BL mice to obtain a clue for the variance of MPTP toxicity between the strains and species. Regional differences in the levels of monoamine oxidase (MAO) and the in vitro effects of MAO inhibitors were also determined and correlated with MPTP neurotoxicity. Concentrations of MPTP in the brains of mice and golden hamster at 10 min were comparable. Golden hamster was found to be resistant to the administration of MPTP as indicated by a lack of any alteration from the normal content of DA in NCP, limbic system, and SN. Both strains of mice exhibited >50% and >75% depletion of DA (C57/BL and BALB/c, respectively). The metabolites‐to‐DA ratios were decreased and increased in golden hamster and mouse strains, respectively, after acute or chronic treatment. Whereas the content of total MAO in golden hamster was one‐third to one‐sixth of any nuclei or mitochondria of both strains of mice, the ratio of MAO A to B was significantly higher in the former species. A possible involvement of discrete regional MAO activity in determining the extent of susceptibility of a species to MPTP toxicity is indicated from the study because (1) susceptibility as evidenced by DA depletion of a species coincided with high levels of MAO activity in SN and NCP, and (2) a highly positive correlation existed with total MAO and MAO B activity, there was a lack of correlation with MAO A activity, and a negative correlation existed with MAO A‐to‐B ratio and DA depletion. Hence, we propose that the resistance of a species to MPTP toxicity may depend on the content as well as the ratios of the two forms of MAO in NCP and SN. In other words, a higher MAO activity and a relative dominance of MAO B in these nuclei are critical in determining the susceptibility of a species to MPTP neurotoxicity.
Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate
Authors: Ereciska Maria , Nelson David , Nissim Itzhak , Daikhin Yevgeny , Yudkoff Marc
Published: July 2010

Abstract: Alanine transport and the role of alanine amino‐transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high‐and low‐affinity uptake systems. The high‐affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na+. Rapid accumulation of the Na+‐alanine complex by synaptosomes stimulated activity of the Na+/K+ pump and increased energy utilization; this, in turn, activated the ATP‐producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na+ also caused a small depolarization of the plasma membrane, a rise in [Ca2+]1, and a release of glutamate. Intra‐synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [15N]alanine formation from [15N]glutamine was seven to eight times higher than the rate of [15N]glutamate generation from [15N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.
Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m‐Calpain
Authors: Figueiredo‐Pereira Maria E. , Banik Naren , Wilk Sherwin
Published: July 2010

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m‐calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β‐casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC50 values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl‐peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m‐calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.
Assessing the Extent of RNA Editing in the TMII Regions of GluR5 and GluR6 Kainate Receptors During Rat Brain Development
Authors: Bernard Anne , Khrestchatisky Michel
Published: July 2010

Abstract: Kainate (KA) is a potent neuroexcitatory agent that induces seizure and brain damage syndromes with increasing efficiency during maturation. It has been suggested that the selective neuronal damage induced by KA may result not only from its depolarizing actions, but also from intracellular accumulation of Ca2+. The effects of KA are mediated by specific high‐affinity receptors, enriched in the hippocampus. Members of this class of receptors, GluR5 and GluR6, have been characterized by cDNA cloning. Ca2+ permeability of the GluR6 receptor is determined by editing in the corresponding RNA. We report here a rapid PCR‐based approach to assess in all experimental conditions the levels of GluR5 and GluR6 editing in the transmembrane TMII region. We show that editing in both GluRS and GluR6 RNA is developmentally regulated and that different regions of the adult rat hippocampus demonstrate distinct levels of GluR6 editing.
Regulation of Angiotensin II Type 1 Receptor mRNA in Neuronal Cultures of Normotensive and Spontaneously Hypertensive Rat Brains by Phorbol Esters and Forskolin
Authors: Lu Di , Sumners Colin , Raizada Mohan K.
Published: July 2010

Abstract: Neuronal cells in primary culture from the brains of normotensive, Wistar‐Kyoto (WKY) rats and spontaneously hypertensive (SH) rats express angiotensin II type 1 (AT1) receptors. Treatment of WKY rat brain cultures with a phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), causes a time‐and dose‐dependent increase in the levels of an ˜2.3‐kb AT1 receptor mRNA transcript. A maximal stimulation of 4.5‐fold in the AT1 receptor mRNA transcript level is observed with 200 nM PMA in 4 h and is blocked by 1 μM staurosporine. Forskolin also increases the AT1 receptor mRNA levels in WKY rat brain neurons in a time‐and dose‐dependent manner, and a 4.5‐fold stimulation is achieved with 50μM forskolin in 4 h. The stimulatory effects of both PMA and forskolin are completely abolished by coincubation of neuronal cultures with 1 μM actinomycin D. In addition, nuclear run‐on assay indicated an increase in the transcription of AT1 receptor mRNA in WKY rat brain neurons treated with either PMA or forskolin. Both PMA and forskolin also stimulate levels of AT1 receptor mRNA in neuronal cultures from brain of the SH rat. The degree of stimulation in these cultures is comparable to that in WKY rat brain neurons. These observations show that although the basal AT1 receptor gene expression is significantly higher in SH rat brain neurons compared with WKY rat brain neurons, the protein kinase C‐and protein kinase A‐responsive stimulation is not altered. These data suggest a possible involvement of protein kinase C and protein kinase A response elements in AT1 receptor gene expression.
Measurement of Blood‐Brain Barrier GLUT1 Glucose Transporter and Actin mRNA by a Quantitative Polymerase Chain Reaction Assay
Authors: Boado Ruben J. , Pardridge William M.
Published: July 2010

Abstract: The expression of the blood‐brain barrier GLUT1 glucose transporter is down‐regulated in brain capillary endothelial cells in tissue culture. Consequently, the study of the regulation of this low‐abundance transcript requires the isolation of poly(A)+ mRNA from relatively large numbers of brain endothelial cells in culture (˜107). Therefore, in order to facilitate studies with smaller amounts of cells, we describe here a quantitative polymerase chain reaction (PCR) assay to measure the mRNA of GLUT1 and the mRNA of the housekeeping gene, actin, which is used as standard control. Bovine brain endothelial cells were grown as either a primary culture (EP cells) or as a brain endothelial cell line (ECL cells) in 25‐mm 6‐well cluster dishes, and total or poly(A)+ RNA was isolated. Following synthesis of cDNA with AMV reverse transcriptase and oligo(dT)18 primer, PCR was performed with sense and antisense primers for bovine GLUT1 and 7‐actin, respectively. Reactions were performed in the presence of 2.5 μCi of [α‐32P]dCTP, and products were resolved in agarose gels and quantified by scanning densitometry of autoradiograms. A direct relationship between RNA‐cDNA and PCR products was observed for GLUT1 after 30 cycles, and for actin after 15 PCR cycles. The method was reproducible within specified ranges of starting RNA‐derived cDNA, and the intraassay coefficient of variation averaged 7.2 β 1.8%. The GLUT1/actin mRNA ratio was as follows: brain capillaries β EP > ECL. In addition, it is demonstrated that tumor necrosis factor‐α induced a three‐to fourfold increase in the GLUT1/actin mRNA ratio in ECL cells. This method provides a 100‐200‐fold increase in the sensitivity of detection of blood‐brain barrier GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture compared with the conventional northern blotting technique using poly(A)+ mRNA.
Cloning, Characterization, and Distribution of a μ‐Opioid Receptor in Rat Brain
Authors: Zastawny Roman L. , George Susan R. , Nguyen Tuan , Cheng Regina , Tsatsos Jim , Briones‐Urbina Rosario , O'Dowd Brian F.
Published: July 2010

Abstract:We report the isolation and characterization of a rat cDNA clone encoding a μ‐opioid receptor. This receptor, a 398 amino acid protein, shares 59% overall identity with the mouse Δ‐andK‐opioid receptors. Transient expression of the receptor in COS cells revealed high‐affinity binding of μ‐selective opioid antagonists and agonists, with aKDfor naloxone ∼1.5 nM, and for [D‐Ala2,N‐Me‐Phe4, Gly5‐ol]‐enkephalin (DAMGO) and morphine at the high‐affinity site of 2–4 nM, confirming a μ‐opioid pharmacological profile. Northern blotting and in situ hybridization histoohemistry revealed that the μ‐opioid receptor mRNA was expressed in many brain regions, including cerebral cortex, caudate putamen, nucleus accumbens, olfactory tubercle, septal nuclei, thalamus, hippocampus, and medial habenular nucleus, in keeping with the known distribution of the μ‐opioid receptor.
Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C
Authors: Doherty Patrick , Furness Josie , Williams Emma J. , Walsh Frank S.
Published: July 2010

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N‐and L‐type calcium channels.
Expression of Neuronal Proteins in Cells from Normal Adult Rat Brain Propagated in Serial Culture
Authors: Black Ronald S. , DeGiorgio Lorraine A. , Sheu Kwan‐Fu Rex , Darzynkiewicz Zbigniew , Duffy Janet T. , Blass John P.
Published: July 2010

Abstract: Cells have been cultured from the brains of 60‐day‐old rats and propagated through 12 passages. The cells contain the high and middle, but not low, molecular weight neurofilament subunits and neuron‐specific enolase, demonstrated by immunoblotting and immunocyto‐chemistry with redundant antibodies. The cells did not have the morphology of neurons when cultured in medium containing fetal calf serum and growth factors. In low serum medium containing the same growth factors with the addition of dibutyryl cyclic AMP, the cells became smaller and developed long processes. Three clonal lines derived from these cultures had the same properties. These observations are in agreement with recent observations using mouse and human brain tissue and demonstrate that proteins normally associated with neurons can be found in dividing cells cultured from the brains of young adult rats.
Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain
Authors: Katoh‐Semba Ritsuko , Semba Reiji , Kato Hiroyuki , Ueno Masataka , Arakawa Yoshihiro , Kato Kanefusa
Published: July 2010

Abstract: Our previous studies showed that the concentration of the β subunit of nerve growth factor (β‐NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β‐NGF protein and its mRNAs were measured in male, female, and castrated male CD‐1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β‐NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β‐NGF levels disappeared. Average levels of β‐NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12‐fold higher), hypothalamus (sixfold higher), and hypophysis (72‐fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β‐NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β‐NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β‐NGF protein in testicular feminization mice were similar to those in female CD‐1 mice in all regions. The concentrations of mRNA for β‐NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain‐derived neurotrophic factor and neurotrophin‐3 between the two sexes. Thus, in some regions of the brain, the production of β‐NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β‐NGF in these region.
Evolution of Acetylcholinesterase Transcripts and Molecular Forms During Development in the Central Nervous System of the Quail
Authors: Anselmet Alain , Fauquet Mireille , Chatel Jean‐Marc , Maulet Yves , Massoulié Jean , Vallette Francois‐Marie
Published: July 2010

Abstract: We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes, and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiplemRNA species (4.5 kb, 5.3 kb, and 6 kb); (b) translation and/or stability of the AChE protein; (c) production of active and inactive AChE molecules; (d) production of amphiphilic and nonamphiphilic AChE forms; and (e) proportions of tetrameric G4, dimeric G2, and monomeric G1 forms. The large transcripts present distinct temporal patterns and disappear in the adult, which possesses only the 4.5‐kb mRNA; these changes are unlikely to be related to those observed for the AChE protein, because all transcripts seem to encode the same catalytic subunit (type T). In addition, the levels of mRNA and AChE are not correlated in the three regions, especially at the adult stage. The proportion of inactive AChE was found to be markedly higher at the hatching period (E16) than at earlier stages (E10 and E13) or in the adult. The G4 form is pre‐dominant already at E10, and in the adult its proportion reaches 80% of the activity in the cerebellum and optic lobes, and 65–70% in the neuroretina. This form is largely nonamhiphilic in embryonic tissues, but it becomes progressively more amphiphilic with development. Thus, the different processing and maturation steps appear to be regulated in an independent manner and potentially correspond to physiologically adaptative mechanisms.
NMR Spectroscopy of Cultured Astrocytes: Effects of Glutamine and the Gliotoxin Fluorocitrate
Authors: Hassel Bjørnar , Sonnewald Ursula , Unsgård Geirmund , Fonnum Frode
Published: July 2010

Abstract:Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with13C and31P NMR spectroscopy. f2–13C]Acetate and [1–13C]glucose were used as labeled precursors. In some experiments glutamine (2.5 mM) was added to the culture medium. Fluorocitrate (20 μM)inhibited the tricarboxylic acid (TCA) cycle without affecting the level of ATP. The net export of glutamine was reduced significantly, and that of citrate increased similarly, consistent with an inhibition of aconitase. Fluorocitrate (100 μM)inhibited TCA cycle activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mMglutamine, 100 μMfluorocitrate did not affect ATP levels, although glutamine synthesis was nearly fully blocked. The consumption of the added glutamine increased with increasing concentrations of fluorocitrate, whereas the consumption of glucose decreased. This shows that glutamine fed into the TCA cycle, substituting for glucose as an energy substrate. These findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, viz., not by depleting glial cells of ATP, but by causing a rerouting of 2‐oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase. Analysis;of the13C labeling of the C‐2 versus the C‐4 positions in glutamine obtained with [2–13C]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycle. Glutamine and citrate were the main TCA cycle intermediates to be released, but a large amount of lactate formed from TCA cycle intermediates was also released, showing that recycling of pyruvate takes place in astrocytes.
Functional Studies with Substance P Analogues: Effects of N‐Terminal, C‐Terminal, and C‐Terminus‐Extended Analogues of Substance P on Nicotine‐Induced Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells
Authors: Cheung Nam Sang , Karlsson Per , Wang Jian‐Xin , Bienert Michael , Oehme Peter , Livett Bruce G.
Published: July 2010

Abstract:Substance P (SP) and SP analogues, including C‐terminal, N‐terminal, and C‐terminus‐extended analogues, have been investigated for their ability to modulate nicotine‐induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on‐line luciferin‐luciferase bioluminescence technique. SP is known to have the following two effects on nicotine‐induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10‐5Mnicotine was inhibited 70‐80% by SP, SP‐methyl ester, and the C‐terminus‐extended analogue SP‐Tyr12‐NH2, 65% by (Ala3)SP‐NH2, 45% by the C‐terminal analogue SP(4‐11), and 20 and 5% by the N‐terminal analogues SP(1‐7) and SP(1‐5), respectively, when these peptides were present at 3 ×; 10‐5Mconcentrations. The order of potency was SP = SP‐methyl ester = SP‐Tyr12‐NH2> (Ala3)SP‐NH2> SP(4‐11) > SP(1‐7) > SP(1‐5). SP, SP‐methyl ester, and (Ala3)SP‐NH2protected against nicotinic desensitization by 40‐55%, and SP(4‐11) protected by 20% (all at 3 ×; 10‐5M). In contrast, the N‐terminal analogues SP(1‐7) and SP(1‐5) and the C‐terminus‐extended analogue SP‐Tyr12‐NH2at 3 × 10‐5Mdid not protect against nicotinic desensitization. Cyclo‐SP(3‐9), Ac‐SP(3‐9)‐NH2, SP(3‐9), and SP(3‐6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine‐evoked catecholamine secretion. The present work indicates that for inhibition of nicotine‐evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine‐induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine‐evoked secretion and protection against nicotinic desensitization, are regulated independently.
Serotonergic Facilitation of Acetylcholine Release In Vivo from Rat Dorsal Hippocampus via Serotonin 5‐HT3Receptors
Authors: Console Silvana , Bertorelli Rosalia , Russi Giovanni , Zambelli Matilde , Ladinsky Herbert
Published: July 2010

Abstract:The serotonin (5‐HT) releaserd‐fenfluramine and its active metabolited‐norfenfluramine, or the 5‐HT‐uptake inhibitor citalopram, by increasing synaptic 5‐HT availability, facilitated in vivo release of acetylcholine (ACh) from dorsal hippocampi of freely moving rats as determined by the microdialysis technique. The effects ofd‐norfenfluramine (7.5 mg/kg i.p.) and citalopram (10 μM, applied by reverse dialysis) were prevented by a 14‐day chemical lesion of the raphe nuclei, suggesting mediation by the 5‐HT system in the cholinergic action of the drugs. The increase in extracellular ACh content induced byd‐norfenfluramine (5 mg/kg i.p.) was antagonized by the 5‐HT3receptor antagonists tropisetron (0.5 mg/kg i.p.) and DAU 6215 (60 μg/kg i.p.), but not by the mixed 5‐HT1and 5‐HT2receptor antagonist metergoline (2 mg/kg s.c.). In accordance with an involvement of the 5‐HT3receptor in the ACh facilitation induced by d‐norfenfluramine is the finding that the selective 5‐HT3receptor agonist 2‐methyl‐serotonin (250 μg i.c.v., or 10 μMapplied by reverse dialysis) raised ACh release. The effect of the intracerebroventricular drug was prevented by the 5‐HT3antagonists DAU 6215 (60 μg/kg i.p.) and ondansetron (60 μg/kg s.c.). These antagonists by themselves did not modify the basal ACh release, indicating that 5‐HT does not tonically activate the 5‐HT3receptors involved. In conclusion, the overall regulatory control exerted by 5‐HT in vivo is to facilitate hippocampal ACh release. This is mediated by 5‐HT3receptors probably located in the dorsal hippocampi.
Stimulation of Postsynaptic and Inhibition of Presynaptic Adenylyl Cyclase Activity by Metabotropic Glutamate: Receptor Activation
Authors: Musgrave Melinda A. , Madigan Maureen A. , Bennett Brian M. , Goh Joanne W.
Published: July 2010

Abstract:To determine the subcellular distribution of cyclic AMP‐coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre‐and postsynaptic elements. In SY, a water‐soluble analogue of forskolin (7β‐forskolin) increased enzyme activity ˜ 10‐fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) inhibited enzyme activity as did glutamate and quisqualate.l‐Amino‐4‐phosphobutanoate (l‐AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonistl‐2‐amino‐3‐phosphopropionic acid (l‐AP3) was not effective in the SY in antagonizing the agonist‐induced decreases in adenylyl cyclase activity by glutamate or 1S,3R‐ACPD. It was, however, effective at antagonizing quisqualate‐induced decreases in enzyme activity. In SN, at the highest concentration tested, 7β‐forskolin produced a 60‐fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3R‐ACPD, quisqualate, andl‐AP4 increased adenylyl cyclase activity. In the SN,l‐AP3 was ineffective in antagonizing any agonist‐induced increases (1S,3R‐ACPD,l‐AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.
Role of Ca2+in α‐Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid‐Mediated Polyphosphoinositide Turnover in Primary Neuronal Cultures
Authors: Lee Yi‐Hsuan , Deupree David L. , Chen Shine‐Chi , Kao Lung‐Sen , Wu Jang‐Yen
Published: July 2010

Abstract:Excitatory amino acid (EAA) receptors and EAA‐mediated stimulation of polyphosphoinositide (poly‐PI) turnover were studied in cultured neurons at different days in vitro (DIV). Six main observations have emerged from these studies: (a) Neurons increased their sensitivity to EAAs as a function of time in culture, indicated by increasing EAA‐mediated poly‐PI turnover, (b) Extracellular Ca2+concentration played an important role in α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐stimulated poly‐PI turnover in cells at 4 DIV, whereas poly‐PI turnover mediated byl‐glutamate andtrans‐1‐amino‐cyclopentane‐1,3‐dicarboxylic acid was not Ca2+‐dependent. (c) A marked stimulation of poly‐PI turnover by AMPA was seen in the cultured neurons at 4 DIV, but not at 17 DIV, suggesting that a distinct EAA receptor sensitive to AMPA is transiently expressed, (d) The Ca2+ionophore A23187 increased poly‐PI turnover in cultured neurons, suggesting that Ca2+entry is involved in stimulating poly‐PI turnover, (e) Stimulation of poly‐PI turnover by carbachol was greater in neurons at 17 DIV as compared with −4 DIV, and appeared to be Ca2+‐dependent across DIV. (f) 6‐Cyano‐7‐nitroquinoxaline‐2,3‐dione, an antagonist for non‐N‐methyl‐d‐aspartate ionotropic EAA receptors, inhibited 100% and 35% of AMPA‐and quisqualate‐induced poly‐PI turnover, respectively, suggesting an involvement of ionotropic AMPA/quisqualate receptors in stimulating poly‐PI turnover.