bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolving Concepts About the Role of Acidosis in Ischemic Neuropathology
Authors: Tombaugh Geoffrey C. , Sapolsky Robert M.
Published: October 2006

Abstract: Cerebral ischemia is one of the most common neurological insults. Many pathological events are undoubtedly triggered by ischemia, but only recently has it become accepted that ischemic cell injury arises from a complex interaction between multiple biochemical cascades. Tissue acidosis is a well established feature of ischemic brain tissue, but its role in ischemic neuropathology is still not fully understood. Within the last few years, new evidence has challenged the historically negative view of acidosis and suggests that it may play more of a beneficial role than previously thought. This review reintroduces the concept of acidosis to ischemic brain injury and presents some new perspectives on its neuroprotective potential.
Time‐Related Loss of Glutamine from Hippocampal Slices and Concomitant Changes in Neurotransmitter Amino Acids
Authors: Kapetanovic Izet M. , Yonekawa Wayne D. , Kupferberg Harvey J.
Published: October 2006

Abstract: A dramatic, time‐dependent loss of l‐glutamine was observed in mouse and rat hippocampal slices equilibrated in normal artificial CSF under static (no‐flow) and super‐fused (constant‐flow) conditions. Concomitant with the decline in l‐glutamine, there was a significant, but less pronounced, decrease in levels of the neurotransmitter amino acids, γ‐aminobutyric acid, l‐aspartate, and l‐glutamate. The disappearance of l‐glutamine was a result of diffusion from the tissue to the artificial CSF rather than chemical or biochemical transformation. The loss of amino acids from the hippocampal slices was prevented to different degrees by the addition of 0.5 mM exogenous l‐glutamine to the artificial CSF. The levels of newly synthesized amino acids were also determined, because they may be more indicative of the neuronal activity than the total tissue levels of amino acids. The effects of perturbations in glutamine (length of the equilibration time and addition of exogenous. glutamine) on newly synthesized glutamate were more pronounced under 4‐aminopyridine‐stimulated than control (unstimulated) conditions. Therefore, a loss of l‐glutamine from the hippocampal slices may have neurophysiological effects and warrants further investigation.
Opposite Age‐Dependent Changes of α2A‐Adrenoceptors and Nonadrenoceptor [3H]Idazoxan Binding Sites (I2‐Imidazoline Sites) in the Human Brain: Strong Correlation of I2 with Monoamine Oxidase‐B Sites
Authors: Sastre Magdalena , Garcia‐Sevilla Jesus A.
Published: October 2006

Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [3H]RX 821002 (2‐methoxyidazoxan) to α2A‐adrenoceptors and that of [3H]idazoxan to l2‐imidazo‐line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α2A‐adrenoceptors and age were negatively correlated (r=‐0.71; p < 0.001). In contrast, the density of l2‐imidazo‐line sites was positively correlated with aging (r= 0.59; p < 0.005). The ratio of receptor densities (α2A/l2) also showed a marked negative correlation with age (r=‐0.76; p < 0.001). In an age‐selected group (range, 10–89 years), the density of monoamine oxidase (MAO)‐B sites labeled by [3H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p < 0.005). In these subjects, the density of l2‐imidazoline sites correlated well with the density of MAO‐B sites (r= 0.70; p < 0.005). The ratio of the density of these sites (MAO‐B/l2) did not correlate with the age of the subject at death (r=‐0.15). In the human frontal cortex, idazoxan displayed very low affinity (Ki= 89 μM) against the binding of [3H]Ro 19–6327 to MAO‐B, which discounted a direct interaction of [3H]idazoxan with the active center of the enzyme and indicated that the l2‐imidazoline site cannot be identified with MAO‐B. However, l2‐imidazoline sites and MAO‐B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l2‐imidazoline site has a specific location on glial (astrocyte) cells.
Nonenzymatic Conversion of 3,4‐Dihydroxyphenylalanine to 2,4,5‐Trihydroxyphenylalanine and 2,4,5‐Trihydroxyphenylalanine Quinone in Physiological Solutions
Authors: Newcomer Timothy A. , Palmer Alan M. , Rosenberg Paul A. , Aizenman Elias
Published: October 2006

Abstract: 2,4,5‐Trihydroxyphenylalanine (TOPA) oxidizes in solution to form, a quinone derivative that is a non‐N‐methyl‐d‐aspartate agonist and neurotoxin. Although pathways have been postulated for the formation of both TOPA and TOPA quinone from closely related catechol‐amines, the generation of these compounds has not been conclusively demonstrated by analytical techniques. Reverse‐phase HPLC with a dual electrode coulometric detector was used to analyze TOPA containing solutions in an effort to rigorously characterize the behavior of this substance under physiological conditions. Electrode potential, buffer system, and methanol concentration were varied to obtain optimal conditions to selectively detect and quantify TOPA and TOPA quinone from closely related catecholamines. TOPA was shown to rapidly autoxidize to TOPA quinone by a process dependent on pH. TOPA was the dominant species at acidic pHs (below 5–6), whereas TOPA quinone was dominant at physiological pHs. This conversion was reversible upon acidification. In addition, we found that 3,4‐dihydroxyphenylalanine can autoxidize to form both TOPA and TOPA quinone under physiological conditions. This partial conversion (0.5%) is time dependent and can be substantially decreased (0.2%) in acidic conditions (pH ≤ 3). These results suggest that some of the excitatory and excitotoxic properties that some investigators have attributed to DOPA may actually be due to its conversion to TOPA and TOPA quinone.
Microdialysis of Melatonin in the Rat Pineal Gland: Methodology and Pharmacological Applications
Authors: Drijfhout W. J. , Grol C. J. , Westerink B. H. C.
Published: October 2006

Abstract: The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on‐line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluori‐metric detection, was set up. With this system it is possible to monitor the melatonin levels on‐line in the pineal gland during day‐and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the β‐agonist isoprenaline (ISO). Also, local infusion of ISO or 8‐bromoadenosine 3′,5′‐cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of β‐adrenergic receptors, coupled to a cyclic AMP‐based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO‐induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of α1‐receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.
Heterogeneity in the Phosphorylation of Micro tubule‐Associated Protein MAP 1B During Rat Brair Development
Authors: Ulloa Luis , Avila Jesús , Díaz‐Nido Javier
Published: October 2006

Abstract: The patterns of isoforms and of immunoreactivity of the microtubule‐associated protein MAP1 B toward a panel of antibodies to phosphorylation‐sensitive epitopes are different in distinct rat brain regions and change during development. This suggests the occurrence of a considerable degree of heterogeneity in the phosphorylation state of rat brain MAP1 B. It appears that MAP1 B can be phosphorylated at multiple sites that may be conventionally classified into at least two modes of phosphorylation. Mode I of phosphorylation induces significant upward shifts in the electrophoretic mobility of the protein, giving rise to “high” MAP1B isoforms, whereas the mode II of MAP1B phosphorylation does not greatly affect the electrophoretic mobility of the protein. These MAP1B phosphorylation modes are differentially regulated throughout development and show some regional specificity. Cytosolic MAP1 B is highly phosphorylated both at mode I and mode II sites in the developing rat brain, as well as in the adult olfactory bulb, where axonal growth takes place. In most adult rat brain regions, cytosolic MAP1B is highly phosphorylated at mode II sites but largely dephosphorylated at certain mode I sites. However, MAP1 B present in the particulate fraction of most rat brain region homogenates may be partially dephosphorylated at certain mode II sites, although it contains some phosphorylated mode I sites. These data are compatible with the view that different protein kinases, possibly including casein kinase II and proline‐directed protein kinases, might regulate the state of phosphorylation of MAP1B in distinct localizations along the development of different neuronal populations in the brain.
Tetrahydrobiopterin Cofactor Biosynthesis: GTP Cyclohydrolase I mRNA Expression in Rat Brain and Superior Cervical Ganglia
Authors: Hirayama Kei , Lentz Stephen I. , Kapatos Gregory
Published: October 2006

Abstract: GTP cyclohydrolase I (GTPCH) is the rate‐limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription‐polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridisation. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1,2‐kb form predominating in the liver and the 3.8‐kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA‐containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA‐depleting drug reserpine; both the 1.2‐and 3.8‐kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.
Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum
Authors: Jenkinson Stephen , Patel Neela , Nahorski Stefan R. , Challiss R. A. John
Published: October 2006

Abstract: The effects of lithium on muscarinic cholinoceptor‐stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [3H]inositol or [3H]cytidine prelabelling and inositol 1,4,5‐trisphosphate [lns(1,4,5)P3] and inositol 1,3,4,5‐tetrakisphosphate [lns(1,3,4,5)P4] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P3 and lns(1,3,4,5)P4 mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30‐min incubation period. Carbachol‐stimulated lns(1,4,5)P3 and lns(1,3,4,5)P4 accumulations were inhibited by lithium in all brain regions studied in a time‐and concentration‐dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC50 values for inhibition of agonist‐stimulated lns(1,4,5)P3 and lns(1,3,4,5)P4 accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [3H]CMP‐phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo‐inositol to reverse these effects, as well as lithium‐suppressed lns(1,4,5)P3 accumulation, implicates myo‐inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [3H]inositol monophosphate and [3H]CMP‐phosphatidate levels and inhibitions of lns(1,4,5)P3 and lns(1,3,4,5)P4 accumulations. However, the inability of striatal slices to sustain carbachol‐stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo‐inositol may indicate differences in phosphoinositide signalling in this brain region.
Basic Fibroblast Growth Factor‐Induced Increase in zif/268 and c‐fos mRNA Levels Is Ca2+ Dependent in Primary Cultures of Hippocampal Neurons
Authors: Ferhat Lotfi , Khrestchatisky Michel , Roisin Marie‐Paule , Barbin Gilles
Published: October 2006

Abstract: Basic fibroblast growth factor (bFGF) is present in the developing rat brain and has been shown to provide critical trophic support for hippocampal neurons in culture. The influence of bFGF on the expression of mRNAs encoding the transcription factors zif/268 and c‐fos was studied in primary cultures of hippocampal neurons (derived from rat embryos) using reverse transcription‐coupled PCR. In these cultures grown for 3 days in the absence of serum, bFGF causes a dramatic and transient increase in the levels of zif/268 and c‐fos, within 15 and 30 min, respectively. A similar induction of these two early genes occurs following activation of protein kinase C (PKC). The bFGF‐induced activation persists after PKC desensitization but is inhibited by chelation of intracellular Ca2+. These results suggest that in primary cultures of hippocampal neurons, bFGF induces the expression of immediate early genes through a pathway that requires Ca2+ mobilization.
Subunit Association and Glycosylation of Acetylcholinesterase from Monkey Brain
Authors: Liao Jian , Nørgaard‐Pedersen Bent , Brodbeck Urs
Published: October 2006

Abstract: Cercopithecus monkey brain acetylcholinesterase (AChE; EC 3.1.1.7) consists of about 15% hydrophilic, salt‐soluble enzyme and 83% amphiphilic, detergent‐soluble enzyme. Sucrose density gradient centrifugation showed that hydrophilic, salt‐soluble AChE was composed of about 85% tetramer (10.3S) and 15% monomer (3.3S). In amphiphilic, detergent‐soluble AChE, 85% tetramer (9.7S), 10% dimer (5.7S), and 5% monomer (3.2S) were seen. The enzyme is N‐glycosylated, and no O‐linked carbohydrate could be detected. Use of two monoclonal antibodies, one directed against the catalytic subunit and the other against the hydrophobic anchor, gave new insights into the subunit assembly of brain AChE. It is shown that in tetrameric AChE, not all of the subunits are disulfide‐bonded and that two populations of tetramers exist, one carrying one and the other carrying two hydrophobic anchors.
Altered Transition Between Agonist‐and Antagonist‐Favoring States of μ‐Opioid Receptor in Brain Membranes with Modified Microviscosity
Authors: Lazar Dan F. , Medzihradsky Fedor
Published: October 2006

Abstract: In unmodified synaptosomal brain membranes the presence of NaCl inhibited the binding to μ receptors of the tritiated opioid agonists etorphine, Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol, and sufentanil by 53, 43, and 37%, respectively, and increased that of the antagonist [3H]naltrexone by 54%. On the other hand, in membranes whose microviscosity was increased by incorporation of cholesteryl hemi‐succinate (CHS) the effects of sodium on opioid agonist and antagonist binding were abolished and strongly reduced, respectively. Furthermore, in the modified membranes the ability of sodium to protect the opioid receptor from inactivation by the sulfhydryl‐reactive agent N‐ethyl‐maleimide (NEM) was diminished. In CHS‐treated membranes whose elevated microviscosity was reduced by the incorporation of oleic acid, the effectiveness of sodium in modulating opioid binding and attenuating receptor inactivation by NEM was restored. The results implicate membrane microviscosity in the mechanism by which sodium modulates the conversion between agonist‐and antagonist‐favoring states of μ opioid receptor.
Haloperidol but Not Clozapine Increases Neurotensin Receptor mRNA Levels in Rat Substantia Nigra
Authors: Bolden‐Watson Carolyn , Watson Michael A. , Murray Karl D. , Isackson Paul J. , Richelson Elliott
Published: October 2006

Abstract: We examined the effects of chronic (2 weeks) treatment with a typical neuroleptic, haloperidol (1 mg/kg, s.c.), and an atypical neuroleptic, clozapine (20 mg/kg, s.c.), on neurotensin receptor (NTR) mRNA levels by in situ hybridization histochemistry. Quantitative OD analysis showed haloperidol‐induced NTR mRNA levels in the substantia nigra/ventral tegmental area (SN/ VTA) 110% over control levels (significant difference from the control, p < 0.05). In contrast, the same analysis applied to the sections from clozapine‐treated animals showed no significant change in NTR mRNA levels compared with matched control sections (p > 0.05). Thus, chronic treatment with haloperidol but not clozapine resulted in elevated levels of NTR mRNA within the SN/VTA. These results suggest that the high incidence of extrapyramidal side effects of typical neuroleptics could result from changes in NTR expression in the SN/VTA.
Neurofilament Proteins Are Synthesized in Nerve Endings from Squid Brain
Authors: Crispino Marianna , Capano Carla Perrone , Kaplan Barry B. , Giuditta Antonio
Published: October 2006

Abstract: It is generally believed that the proteins of the nerve endings are synthesized on perikaryal polysomes and are eventually delivered to the presynaptic domain by axoplasmic flow. At variance with this view, we have reported previously that a synaptosomal fraction from squid brain actively synthesizes proteins whose electrophoretic profile differs substantially from that of the proteins made in nerve cell bodies, axons, or glial cells, i.e., by the possible contaminants of the synaptosomal fraction. Using western analyses and immunoabsorption methods, we report now that (a) the translation products of the squid synaptosomal fraction include neurofilament (NF) proteins and (b) the electrophoretic pattern of the synaptosomal newly synthesized NF proteins is drastically different from that of the IMF proteins synthesized by nerve cell bodies. The latter results exclude the possibility that NF proteins synthesized by the synaptosomal fraction originate in fragments of nerve cell bodies possibly contaminating the synaptosomal fraction. They rather indicate that in squid brain, nerve terminals synthesize NF proteins.
Transglutaminase Catalyzes the Formation of Sodium Dodecyl Sulfate‐Insoluble, Alz‐50‐Reactive Polymers of τ
Authors: Dudek Serena M. , Johnson Gail V. W.
Published: October 2006

Abstract: Paired helical filaments, a constituent of neurofibrillary tangles in Alzheimer's disease, consist primarily of the microtu‐bule‐associated protein τ. However, the process by which the detergent‐insoluble filaments of the neurofibrillary tangles are formed from soluble τ remains unknown. Here, we present a potential mechanism for the abnormal aggregation of τ in Alzheimer's disease: the covalent cross‐linking of τ by the enzyme transglutaminase. Macromolecular complexes of τ, formed in the presence of transglutaminase, were found to be insoluble in ionic detergent, β‐mercaptoethanol, guanidine‐HCI, and urea and, furthermore, demonstrated an increased immunoreactivity with the monoclonal antibody Alz‐50. Electron microscopic studies revealed that τ cross‐linked by transglutaminase has a defined filamentous structure. These results indicate that transglutaminase, the activity of which has been shown to increase during programmed cell death, may play a role in the formation of pathology associated with Alzheimer's disease.
Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells
Authors: Williams Robert J. , Kelly Eamonn
Published: October 2006

Abstract: Recent work has shown that chronic ethanol treatment inhibits receptor‐stimulated phosphoinositide hydrolysis in NG108–15 cells and that ethanol exerts this effect primarily at the level of the guanine‐nucleotide binding protein (G protein). Here we investigated the effects of ethanol exposure on the expression of Gqα/11α, two highly homologous G protein α‐subunits that have been implicated as regulators of phosphoinositidase C. Addition of ethanol (10–200 mM) to the culture medium for 48 h caused a concentration‐dependent decrease in the immunologically detectable levels of Gqα/11α. A small (∼15%) reduction in Gqα/11α, was observed after only 6 h of exposure to 200 mM ethanol, but membrane levels were reduced by 31% at 48 h. The ethanol‐induced loss of Gqα/11α was apparently independent of factors present in the foetal calf serum component of the culture medium. These results suggest that the decrease in receptor‐mediated phosphoinositide hydrolysis following chronic ethanol treatment of NG108–15 cells may be mediated in part by a reduction in the membrane levels of Gqα/11α.
Down‐Regulation of Angiotensin II Receptor Subtypes and Desensitization of Cyclic GMP Production in Neuroblastoma N1E‐115 Cells
Authors: Reagan L. P. , Ye X. , Maretzski C. H. , Fluharty S. J.
Published: October 2006

Abstract: Murine neuroblastoma N1E‐115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E‐115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I‐AngII or the high‐affinity antagonist 125I‐[Sarc1,Ile8]‐AngII. This apparent down‐regulation was dose related with an ED50 of 1 nM, and maximal decreases of ∼90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist‐exposed cells with non‐hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long‐term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down‐regulation of receptors, and the high‐affinity antagonist [Sarc1, Thr8]‐AngII blocked agonist‐induced down‐regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E‐115 cells. These results suggest that differentiated N1E‐115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII receptor subtypes in the nervous system.
Transport of Phosphatidylserine from Microsomes to the Inner Mitochondrial Membrane in Brain Tissue
Authors: Corazzi Lanfranco , Pistolesi Roberto , Carlini Enrico , Arienti Giuseppe
Published: October 2006

Abstract: Phosphatidylserine was labeled by incubating rat brain homogenates with [3‐14C]serine in the presence of Ca2+ (base‐exchange conditions). Some labeled phosphati‐dylethanolamine also forms, in spite of the inhibition of Ca2+ on phosphatidylserine decarboxylase. Phosphatidylserine labeling and decarboxylation also occur on incubating a mixture of purified mitochondria and microsomes, suggesting that no soluble factors are necessary for the synthesis and the decarboxylation of phosphatidylserine. Ca2+ favors the transfer of phosphatidylserine from microsomes (where it forms) to mitochondria (where it is decarboxylated). The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmentalized in microsomes and that radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospholipid.
Expression of Gangliosides GD3 and 3′‐isoLM1 in Autopsy Brains from Patients with Malignant Tumors
Authors: Fredman P. , von Holst H. , Collins V. P. , Dellheden B. , Svennerholm L.
Published: October 2006

Abstract: Three autopsy brains from patients who succumbed to malignant gliomas have been analyzed in various regions with regard to their ganglioside content. The study focused on the gangliosides GD3 and 3′‐isoLM1, which in a previous study of biopsies were found to be associated with these tumors. In particular, 3′‐isoLM1, was suggested to be a marker for malignant gliomas. The highest concentrations (200–1,000 nmol of sialic acid/g wet weight) of GD3 was found in specimens of macroscopically pure tumor, where the proportion of GD3 was, at the most, 78% (range, 11–78%) of the total ganglioside sialic acid compared with <10% in normal brain tissue. The proportion of the total ganglioside sialic acid made up by GD3 was also elevated in the periphery of the tumor and in the same region in the opposite hemisphere, where no tumor cells were detected. In four of eight brain metastases of various carcinomas, GD3 was >10% of the total ganglioside sialic acid (range, 3–37%). The ganglioside 3′‐isoLM1, as determined by TLC‐enzyme‐linked immunosorbent assay using a specific monoclonal antibody (SL‐50), was not present at detectable levels in any of the macroscopically homogenous tumor areas. It was, however, found in the periphery of the tumor, in the corpus callosum, and at highest concentrations in the region of the opposite hemisphere corresponding to the tumor. The concentration varied between 0.1 and 6.0 nmol/g wet weight of tissue. The 3′‐isoLM1 ganglioside was not detected in normal gray or white matter or in the normal corpus callosum, but in one of three breast cancer metastasis, one of two low differentiated cancer metastases, and one stomach cancer. The concentration was 1–4 nmol/g wet weight. These results indicate a unique distribution of the gangliosides GD3 and 3′‐isoLM1 and suggest that they play distinct roles in interaction between tumor cells and brain.
Rate of 59Fe Uptake into Brain and Cerebrospinal Fluid and the Influence Thereon of Antibodies Against the Transferrin Receptor
Authors: Ueda F. , Raja K. B. , Simpson R. J. , Trowbridge I. S. , Bradbury M. W. B.
Published: October 2006

Abstract: Uptake of 59Fe from blood into brains of anaesthetized rats and mice has been studied by intravenous infusion of [59Fe]ferrous ascorbate or of 59Fe‐transferrin, the results not being significantly different. Uptakes in the rat were linear with time, but increased at longer times in the mouse. Transfer constants, Kin (in ml/g/h × 103), for cerebral hemispheres were 5.2 in the adult rat and 5.6 in the mouse. These Kin values corresponded to 59Fe influxes of 145 and 322 pmol/g/h, respectively. 59Fe uptake into the mouse brain occurred in the following order: cerebellum > brainstem > frontal cerebral cortex > parietal cortex > occipital cortex > hippocampus > caudate nucleus. In genetically hypotransferrinaemic mice, 59Fe uptake into brain was 80–95 times greater than in To strain mice. Pretreatment of young rats and mice with monoclonal antibodies to transferrin receptors, i.e., the anti‐rat immunoglobulin G OX 26 and the anti‐mouse immunoglobulin M RI7 208, inhibited 59Fe uptake into spleen by 94% and 98%, respectively, indicating saturation of receptors. The antibodies reduced 59Fe uptake into rat brain by 35–60% and that into mouse brain by 65–85%. Although a major portion of iron transport across the blood‐brain barrier is normally transferrin‐mediated, non‐transferrin‐bound iron readily crosses it at low serum transferrin levels.
The Cholinergic Stimulating Effects of Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor Are Mediated by Protein Kinase C
Authors: Kalberg Christopher , Yung Shau Yu , Kessler John A.
Published: October 2006

Abstract: The intracellular mechanisms through which two trophic factors, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), regulate cholinergic development were examined in sympathetic neuron cultures. Treatment with CNTF or LIF increased levels of choline acetyltransferase (ChAT) activity by 375 and 350%, respectively. However, in neuronal cultures depleted of protein kinase C (PKC) activity by chronic phorbol ester treatment, neither CNTF nor LIF elevated ChAT activity. Further, the stimulation of ChAT due to increased cell density was not observed in PKC‐depleted sympathetic neurons. The inhibition of CNTF‐stimulated ChAT by phorbol ester occurred in a dose‐dependent manner and chronic phorbol ester treatments did not alter the levels of the catecholamine biosynthetic enzyme tyrosine hydroxylase. Moreover, increased levels of diacylglycerol, an endogenous activator of PKC, were observed in sympathetic neurons treated with CNTF. However, neither CNTF nor LIF stimulated the hydrolysis of phosphatidylinositol 4,5‐bisphosphate. These observations suggest that a common PKC‐dependent pathway, which is independent of phosphatidylinositol 4,5‐bisphosphate hydrolysis, mediates the cholinergic stimulating effects of CNTF, LIF, and cell‐cell contact in cultured sympathetic neurons.
Characterization of Basic Proteins from Goldfish Myelin
Authors: Mastronardi Fabrizio G. , Boulias Chris , Roots Betty I. , Moscarello Mario A.
Published: October 2006

Abstract: Myelin basic protein (MBP) from common goldfish (Carassius auratus) myelin was extracted with dilute mineral acid. Immunological cross‐reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with 125I and chromatographed it on a carboxymethyl cellulose‐52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C‐1, C‐2, C‐3, C‐4, and C‐8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet‐erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed‐phase HPLC. Amino acid compositions were determined for both the 17‐ and 14‐kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17‐ and 14‐kDa proteins was higher than that of the human C‐1, the mouse C‐1 protein, and the shark proteins. The HPLC‐purified 14‐kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.
Subtypes of Sodium‐Dependent High‐Affinity L‐[3H]Glutamate Transport Activity: Pharmacologic Specificity and Regulation by Sodium and Potassium
Authors: Robinson Michael B. , Sinor Jeroo D. , Dowd Lisa A. , Kerwin James F.
Published: October 2006

Abstract: Some data suggest that the sodium‐dependent, high‐affinity L‐glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium‐dependent transport of L‐[3H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by <65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were <10‐fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L‐Glu, L‐aspartate, and L‐cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10‐fold selective as inhibitors. These included β‐N‐oxalyl‐L‐α,β‐diaminopropionate, α‐methyl‐DL‐glutamate, (2S, 1′S,2′S)‐2‐(carboxycyclopropyl)glycine, and (2S, 1′S,2′S,3′S)‐2‐(2‐carboxy‐3‐methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S, 1′R,2′R)‐2‐(Carboxycyclopropyl)glycine (L‐CCG‐II) was identified as the most potent (IC50= 5.5 μM) and selective (60–100‐fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L‐homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L‐CCG‐II and L‐homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.
Production of Adenosine from Extracellular ATP at the Striatal Cholinergic Synapse
Authors: James Stephen , Richardson Peter J.
Published: October 2006

Abstract: The components of the ectonucleotidase pathway at the immunoaffinity‐purified striatal cholinergic synapse have been studied. The ecto‐ATPase (EC 3.6.1.15) had a Km of 131 γM, whereas the ecto‐ADPase (EC 3.6.1.6) had a Km of 58 γM, was Ca2+‐dependent, and was inhibited by the ATP analogue 5′‐adenylylimidodiphosphate (AMPPNP). The ecto‐5′‐nucleotidase (EC 3.1.3.5) had a Km of 21 γM, was inhibited by AMPPNP and α,β‐methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5′‐nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto‐adenylate kinase activity was detected on these purified synapses. The intraterminal 5′‐nucleotidase enzyme, which amounted to 40% of the total 5′‐nucleotidase activity, was inhibited by AMPPNP, α,β‐methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5′‐nucleotidase, and was inversely proportional to the ecto‐5′‐nucleotidase activity. The function and characteristics of this pathway and the central role of 5′‐nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.
Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells
Authors: Bessho Yasumasa , Nawa Hiroyuki , Nakanishi Shigetada
Published: October 2006

Abstract: Metabotropic glutamate receptor (type 1; mGluR1) is expressed predominantly in the hippocampus and the cerebellum. Using cultured cerebellar granule cells, we investigated the regulation of the mGluR1 mRNA expression. Levels of mGluR1 mRNA were decreased to less than half by high potassium stimulation and by glutamate and quisqualate. Although these glutamate receptor agonists tested are also known to cause neuronal cell death in culture, the effect of cell death cannot explain the observed reduction in mGluR1 mRNA because of the following reasons: (a) antagonists of N‐methyl‐D‐aspartate and non‐N‐methyl‐D‐aspartate receptors inhibited cell death, but not the reduction of the level of mGluR1 mRNA; (b) mGluR1 mRNA returned to its initial level 48 h after the agonist application; and (c) the mRNA level of one of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate/kainate receptors (GluR1) was not altered by these conditions. Therefore, we conclude that the glutamate or quisqualate stimulation can specifically inhibit the expression of mGluR1 mRNA. The dose response of quisqualate for the reduction in mGluR1 mRNA is consistent with that for inositol phosphate formation stimulated through the cloned mGluR1. The mRNA reduction did not require extracellular calcium. Desensitization of mGluR1 with phorbol ester abolished the mRNA reduction. These results suggest that the reduction in mGluR1 mRNA is mediated by the activation of the metabotropic receptor itself.
Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth
Authors: Hotta Taeko , Asai Kiyofumi , Takeda Naohito , Tatematsu Akira , Nakanishi Keiko , Eksioglu Yaman Z. , Isobe Ichiro , Kato Taiji
Published: October 2006

Abstract: Human glioma cell extracts were found to elicit a marked growth‐promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography‐mass spectrometry. The authentic uridine showed a strong growth‐promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses.
Age‐Dependent Vulnerability of the Striatum to the Mitochondrial Toxin 3‐Nitropropionic Acid
Authors: Brouillet Emmanuel , Jenkins Bruce G. , Hyman Bradley T. , Ferrante Robert J. , Kowall Neil W. , Srivastava Rachana , Roy Deborah Samanta , Rosen Bruce R. , Beal M. Flint
Published: October 2006

Abstract: The mechanisms of delayed onset and cell death in Huntington's disease (HD) are unknown. One possibility is that a genetic defect in energy metabolism may result in slow excitotoxic neuronal death. Therefore, we examined the effects of age on striatal lesions produced by local administration of the mitochondrial toxin 3‐nitropropionic acid in rats. In vivo chemical shift magnetic resonance imaging showed marked increases in striatal lactate concentrations that significantly correlated with increasing age. Histologic and neurochemical studies showed a striking age dependence of the lesions, with 4‐ and 12‐month‐old animals being much more susceptible than 1‐month‐old animals. Continuous systemic administration of low doses of 3‐nitropropionic acid for 1 month resulted in striatal lesions showing growth‐related changes in dendrites of striatal spiny neurons using the Golgi technique. These results show that a known mitochondrial toxin can produce selective axon‐sparing striatal lesions showing both the age dependence and striatal spiny neuron dendritic changes that characterize HD.
Analysis of the Human Dopamine β‐Hydroxylase Promoter: Transcriptional Induction by Cyclic AMP
Authors: Lamouroux Annie , Houhou Leila , Biguet Nicole Faucon , Serck‐Hanssen Guldborg , Guibert Bernard , Icard‐Liepkalns Christine , Mallet Jacques
Published: October 2006

Abstract: We have analyzed some functional aspects of the promoter of the human dopamine β‐hydroxylase (DBH) gene. A fragment of 1,247 bp directly 5′ to the transcriptional start was progressively shortened, placed in front of a reporter gene, and tested in a human neuroblastoma cell line expressing DBH (SK‐N‐SH‐TFM) and in a monkey kidney cell line (CV‐1). A remarkably short region (267 bp), directly upstream from the transcription start, was sufficient to confer activity and tissue‐specific expression. Furthermore, the expression of the DBH gene was shown to be inducible by cyclic AMP in SK‐N‐SH‐TFM cells. This effect was demonstrated to occur at the transcriptional level, as shown by run‐on assays, and was due to the presence of a near‐consensus cyclic AMP‐responsive element located in the untranscribed 5′ regulatory region of the gene.
Differences in the Ligand Binding Properties of the Short and Long Versions of the D2 Dopamine Receptor
Authors: Castro Sandra W. , Strange Philip G.
Published: October 2006

Abstract: The short and long forms of the D2 dopamine receptor have been expressed at similar levels in fibroblast Ltk− cells, and the long form of the receptor has also been expressed in Chinese hamster ovary cells. The ligand binding properties of the two forms of receptor expressed in different systems have been compared in saturation analyses with [3H]spiperone and in competition studies with a range of antagonists. The long form of the receptor expressed in two different cell hosts exhibits essentially identical properties. However, whereas the long and short forms of the receptor show very similar affinities for several compounds representative of different chemical classes, the short form shows a two‐ to fivefold higher affinity for several substituted benzamide drugs. The conformation of the receptor binding site may therefore be different in the two receptor isoforms.
A Single Amino Acid Difference Accounts for the Pharmacological Distinctions Between the Rat and Human 5‐Hydroxytryptamine1B Receptors
Authors: Parker Eric M. , Grisel Darcy A. , Iben Lawrence G. , Shapiro Robert A.
Published: October 2006

Abstract: Molecular cloning of the rat and human 5‐hydroxytryptamine1B (5‐HT1B) receptors has revealed that the primary amino acid sequence of these two receptors is >90% identical. Despite this high degree of primary sequence homology, these two receptors have significantly different pharmacological properties. A mutant human 5‐HT1B receptor was constructed in which Thr355 was replaced by Asn, the corresponding residue at this position in the rat 5‐HT1B receptor. The pharmacology of the mutant human 5‐HT1B receptor was very similar to that of the rat 5‐HT1B receptor. Specifically, the mutant receptor had much higher affinity for pindolol, [125I]‐iodocyanopindolol, propranolol, and CP‐93,129 than the wild‐type receptor. In contrast, the mutant had significantly lower affinity for sumatriptan, N,N‐dipropyl‐5‐carboxamidotryptamine, 5‐methoxy‐N,N‐dimethyltryptamine, methysergide, metergoline, and rauwolscine. These data suggest that a single amino acid difference at position 355 is responsible for the pharmacological differences between the rat and human 5‐HT1B receptors.
Evidence for D2 Receptor Regulation of Dopamine Release in the Goldfish Retina
Authors: Rashid Kashif , Baldridge William H. , Ball Alexander K.
Published: October 2006

The possible existence of a dopamine D2 receptor‐mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [3H]dopamine and superfused with D2 dopamine receptor agonists or antagonists to determine if there was an effect on [3H]dopamine release. The D2 receptor antagonist sulpiride increased both baseline [3H]‐ dopamine release and [3H]dopamine release induced by an increase in extracellular potassium concentration. The D2 receptor agonists LY‐171555 and RU‐24213 did not reduce baseline [3H]dopamine release but completely inhibited [3H]dopamine release induced by an increase in [K±]o. This action of the D2 agonists was blocked by sulpiride. These studies demonstrate the existence of D2 receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.
Developmental Changes in τ Phosphorylation: Fetal τ Is Transiently Phosphorylated in a Manner Similar to Paired Helical Filament‐τ Characteristic of Alzheimer's Disease
Authors: Brion Jean‐Pierre , Smith Carthage , Couck Anne‐Marie , Gallo Jean‐Marc , Anderton Brian H.
Published: October 2006

Rat and human fetal brain τ were probed with a panel of monoclonal antibodies (tau‐1, AT8, 8D8, RT97, SMI31, SMI34) that distinguish between paired helical filament (PHF)‐τ of Alzheimer's disease and normal adult brain τ. These antibodies discriminate between normal and PHF‐τ because their epitopes are phosphorylated in PHF‐τ. Although only one molecular isoform of τ was shown to be expressed in fetal brain, two fetal τ species could be distinguished on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and the slower migrating species was recognized by all of the PHF‐τ‐specific antibodies. Moreover, this immunoreactivity was shown to be phosphorylation dependent. Our observations suggest that the abnormal phosphorylation of τ in Alzheimer's disease may be the result of reactivation of pathways governing the phosphorylation of τ in the developing brain.
Differential Processing of the Ferritin Heavy Chain mRNA in Human Liver and Adult Human Brain
Authors: Dhar M. S. , Joshi J. G.
Published: October 2006

Northern blot analyses of the poly(A)± RNAs from human brain and liver, using a human brain ferritin heavy chain (FTH) cDNA as the probe, shows the presence of two transcripts of 1.4 and 1.1 kb. The larger, 1.4‐kb RNA, is expressed predominantly in the brain, whereas the smaller, 1.1 kb, is expressed abundantly in the liver. Screening of two normal human brain cDNA libraries yielded two types of human brain FTH cDNAs. One type corresponds to the previously characterized 1.1‐kb RNA from liver and lymphocytes. The other is also identical to the previously characterized FTH cDNA except that it contains an additional 279‐bp sequence at the 3′ untranslated region. This additional sequence shows 94.1%, 62.5%, and 58.9% identity to the 3′ flanking sequence of the human liver and mouse and rat FTH genomic clones, respectively. A fragment of a genomic clone containing the 279‐bp sequence was also isolated and sequenced. These data suggest that differential processing of the primary transcript for the FTH mRNA in human brain and liver could generate two mature mRNAs of 1.4 and 1.1 kb. This could be due to the use of alternative polyadenylation sites in the pre‐mRNA.