bluuebunny/crossref_metadata_2025 huggingface dataset:
The Iron Chelator Desferrioxamine (Desferal) Retards 6‐Hydroxydopamine‐Induced Degeneration of Nigrostriatal Dopamine Neurons
Authors: Ben‐Shachar D. , Eshel G. , Finberg J. P. M. , Youdim M. B. H.
Published: October 2006

Abstract: A selective increase in content of iron in the pars compacta of the substantia nigra has been implicated in the biochemical pathology of Parkinson's disease. Iron is thought to induce oxidative stress by liberation of oxygen free radicals from H2O2. Because 6‐hydroxydopamine (6‐OHDA) is thought to induce nigrostriatal dopaminergic neuronal lesions via metal‐catalyzed free radical formation, the effect of the iron chelator desferrioxamine was investigated on 6‐OHDA‐induced dopaminergic neuron degeneration in the rat. Intracerebroventricular injection of 6‐OHDA (250 μg) caused a 88, 79, and 70% reduction in striatal tissue content of dopamine (DA), 3,4‐dihydroxyphenylacetic acid, and homovanillic acid (HVA), respectively, and a 2.5‐fold increase in DA release as indicated by the HVA/DA ratio. Prior injection of desferrioxamine (130 ng i.c.v.) resulted in a significant protection (<60%) against the 6‐OHDA‐induced reduction in striatal DA content and a normalization of DA release. Dopaminergic‐reiated behavioral responses, such as spontaneous movements in a novel environment and rearing, were significantly impaired in the 6‐OHDA‐treated group. By contrast, the desferrioxamine‐pretreated rats exhibited almost normal behavioral responses. The ability of iron chelators to retard dopaminergic neurodegeneration in the substantia nigra may indicate a new therapeutic strategy in the treatment of Parkinson's disease.
Characterization of the Carbohydrate Chain on the Substance P Receptor in the Rat Brain Cortex: Effect of Lectins on [3H]Substance P Binding
Authors: Takamatsu Hiroyuki , Tani Yoshie , Akiyama Mitoshi , Nakata Yoshihiro , Segawa Tomio
Published: October 2006

Abstract: The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose‐dependent and diminished by the addition of α‐methyl‐D‐mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex‐type or a high mannose‐type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system.
α‐[3H]Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid Binding to Human Cerebral Cortical Membranes: Minimal Changes in Postmortem Brains of Chronic Schizophrenics
Authors: Kurumaji Akeo , Ishimaru Masahiko , Toru Michio
Published: October 2006

Abstract: The binding of α‐[3H]amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid ([3H]AMPA), a selective ligand for the ion channel‐linked quisqualate receptor, was evaluated in Triton X‐100‐treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p‐chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]‐AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD= 220 nM, Bmax= 235 fmol/mg of protein), curvilinear Scatchard plots of, specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KDI= 5.82 nM, Bmaxx= 247 fmol/mg of protein; KD2= 214 nM, Bmax2= 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included β‐N‐oxalylamino‐l‐alanine (l‐BOAA), cysteine‐S‐sulfate, l‐glutamate, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and 6,7‐dinitroquinoxaline‐2,3‐dione. Kainate, l‐homocysteine sulfinic acid, and l‐homocysteic acid were active with an IC50 value of a micromolar concentration, whereas l‐cysteic acid and l‐cysteine sulfinic acid were weakly active. N‐Methyl‐d‐aspartate, l‐aspartate, N‐acetylaspartylglutamate, quinolinate, and 1‐naphthylacetyl spermine, an analogue of Joro spider toxin, were inactive at 1 mM. These results suggest that l‐glutamate has an important effect on AMPA receptors in the human cerebral cortex and that l‐BOAA and cysteine‐S‐sulfate exhibit their neurotoxicity through AMPA receptors. Specific [3H]AMPA binding, using the ligand at 6 nM, in the presence of 100 mM KSCN exhibited a heterogeneous distribution pattern in the human cerebral cortex: [3H]AMPA binding values were high in the frontal and occipital cortex, whereas they were low in the parietotemporal cortex. However, no statistically significant changes in [3H]AMPA binding were observed in 22 brain areas of the cerebral cortex of chronic schizophrenics, compared with controls, suggesting that AMPA receptors in the cerebral cortex are minimally involved in an abnormality of excitatory amino acidergic neurons hypothesized in schizophrenic brain.
Characterization of Soluble Neural Cell Adhesion Molecule in Rat Brain, CSF, and Plasma
Authors: Krog Lisbeth , Olsen Marianne , Dalseg Anne‐Marie , Roth Jürgen , Bock Elisabeth
Published: October 2006

Abstract: The polypeptide composition and glycosylation of soluble isoforms of neural cell adhesion molecule (NCAM) in developing rat brain, CSF, and plasma were characterized. Soluble NCAM in rat brain consisted of several glycosylated isoforms. The degree of glycosylation was developmentally regulated. After desialylation, four polypeptides of Mr values of ∼ 190,000 (sl), 135,000 (s2), 115,000 (s3), and 110,000 (s4) were observed. Polypeptides si, s2, and s3 were also present in CSF, whereas only s3 and s4 were observed in plasma. Treatment of soluble brain NCAM with N‐glycosidase F, which removes N‐linked carbohydrates, produced polypeptides of Mr values of ∼ 190,000, 125,000, and 108,000–97,000. The monoclonal antibody OB11, which recognizes an epitope on the cytoplasmic part of transmembrane forms of NCAM, did not react with any of the soluble isoforms. Purified soluble NCAM, consisting mainly of s3, contained an N‐terminal sequence identical to that of membrane‐associated NCAM. Gel nitration of s3 indicated that it was present as a dimer under the chosen conditions. NCAM‐expressing glioma cells adhered specifically to immobilized soluble NCAM. This implies that functionally significant soluble forms of NCAM are present in the extracellular fluid.
Depolarization‐Dependent Tyrosine Phosphorylation in Rat Brain Synaptosomes
Authors: Woodrow Sarah , Bissoon Nankie , Gurd James W.
Published: October 2006

Abstract: Synaptosomes from rat forebrain were analyzed for the presence of phosphotyrosine‐containing proteins by immunoblotting with antiphosphotyrosine antibodies. Using this technique, 10–11 phosphotyrosine‐containing proteins were detected. Depolarization of synaptosomes by transfer to a high (41 mM) K+ medium resulted in increases in the phosphotyrosine content of several synaptosomal proteins, the most pronounced increase being associated with a membrane protein of Mr 117,000 (ptp 117). Additional proteins exhibiting depolarization‐dependent increases in phosphotyrosine content had molecular weights of 39,000, 104,000, 135,000, and 160,000. The depolarization‐dependent increase in the phosphotyrosine content of ptp117 was apparent within 30 s of the onset of depolarization, reached a maximum between 3 and 5 min, and then decreased to near control values by 30 min. The increase in tyrosine phosphorylation of ptp117 was dependent on the concentration of K+ in the depolarizing medium and was maximal with [K+] in excess of 50 mM. It was also calcium dependent and did not occur in the absence of extracellular calcium. The addition of veratridine to the incubation medium also resulted in an increase in the tyrosine phosphorylation of ptpl 17. The results suggest that the phosphorylation of synaptic proteins on tyrosine residues may be involved in the regulation or modulation of synaptic activity.
Determination of Regional Rates of Cerebral Protein Synthesis Adjusted for Regional Differences in Recycling of Leucine Derived from Protein Degradation into the Precursor Pool in Conscious Adult Rats
Authors: Sun Y. , Deibler G. E. , Sokoloff L. , Smith C. Beebe
Published: October 2006

Abstract: The quantitative autoradiographic L‐[1–14C]leucine method for the determination of regional rates of cerebral protein synthesis in vivo takes into account recycling of unlabeled leucine derived from protein degradation into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the apparent steady‐state leucine specific activity in the precursor amino acid pool (tRNA‐bound leucine) to that in the arterial plasma. In the whole brain of the conscious rat this ratio (λWB) equals 0.58. The equivalent ratio for leucine in the acid‐soluble pool in whole brain (ΨWB) is 0.49. A first‐degree polynomial equation for λWB as a function of ΨWB was fitted from paired determinations. To determine the degree of recycling in local regions of the brain, we have measured in individual brain regions (i) Ψi and calculated λi assuming that the fitted equation also applies to these localized regions. Our results indicate that the degree of recycling into the precursor pool does vary regionally; λi in the individual regions varies from 0.62 in the hypoglossal nucleus to 0.50 in the globus pallidus. Local rates of protein synthesis were then determined by the autoradiographic technique with regional corrections for recycling of unlabeled leucine. Rates of leucine incorporation into protein averaged 6.1 nmol/g of tissue/min in the brain as a whole, with the rates in gray matter about twice those in white matter.
Effect of Nitroprusside (Nitric Oxide) on Endogenous Dopamine Release from Rat Striatal Slices
Authors: Zhu Xing‐Zu , Luo Lu‐Guang
Published: October 2006

Abstract: It is becoming apparent that the synthesis of nitric oxide (NO) from l‐arginine not only explains endothelium‐dependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3–100 μM, induced a dose‐dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 μM) produced an inhibitory effect on the spontaneous release of DA. l‐Arginine (10 and 100 μM), a substrate in the NO‐forming enzyme system, also produced an elevation of DA release. l‐Arginine‐induced DA release was attenuated by NG‐monomethyl‐l‐arginine, an inhibitor of NO synthase. NADPH (1 μM), a cofactor of NO synthase, enhanced l‐arginine‐induced DA release. These results suggest a possible involvement of NO in the DA release process in rat striatum.
Modulatory Role of Glutathione on μ‐Opioid, Substance P/Neurokinin‐1, and Kainic Acid Receptor Binding Sites
Authors: Liu Y. F. , Quirion R.
Published: October 2006

Abstract: Reduced glutathione (l‐γ‐glutamyl‐l‐cysteinyl‐glycine; GSH) is an endogenous tripeptide involved in the formation and maintenance of protein thiol groups as well as in various detoxification reactions. Because multiple receptor types contain thiol groups or disulfide bridges, effects of GSH treatments on μ‐opioid, neurokinin‐1/substance P, and kainic acid receptor binding sites were investigated and compared with those produced by dithiothreitol (DTT), a potent synthetic reducing agent. GSH inhibited binding more potently than did DTT at all three receptor types in porcine striatal membrane homogenates as well as in CHAPS‐solubilized preparations of the μ and neurokinin‐1 sites. GSH‐induced inhibitory effects were associated with decreases in maximal binding capacity (Bmax) without significant alteration in apparent affinity (KD). Cysteine, the functional moiety of GSH, mimicked GSH effects albeit with lower potencies, whereas oxidized glutathione had no effects at similar concentrations. In CHAPS‐solubilized preparations, the combination of low concentrations of GSH and guanylylimidodiphosphate markedly decreased the Bmax values of the binding of [3H][D‐Ala2, Gly‐ol5]enkephalin and [3H]substance P. This GSH‐mediated mechanism may be important to prevent cell overstimulation by accelerating receptor uncoupling, desensitization, and/or intemalization. This is in keeping with purported roles of GSH related to the maintenance of cellular integrity.
Extracellular Intermediates of Glucose Metabolism: Fluxes of Endogenous Lactate and Alanine Through Extracellular Pools in Embryonic Sympathetic Ganglia
Authors: Larrabee Martin G.
Published: October 2006

Abstract: The flux rates of lactate and alanine in and out of the cells of an intact tissue, which cannot be measured directly because some of the released materials are reabsorbed, were determined by computer analysis of uptakes and outputs by the whole tissue in the presence of various concentrations of these substances. The outputs of labeled lactate and alanine from [U‐14C]glucose and the uptakes of [U‐14C]lactate and [U‐14C]alanine were measured on intact sympathetic ganglia excised from 15‐day‐old chicken embryos. The volume and time constant of the extracellular space were measured using labeled lactate, alanine, and sucrose. Models, which mathematically described the cellular uptakes and outputs as functions of the extracellular concentrations, were used to predict the exchanges that would be observed on the whole tissue, and their parameters were adjusted for best fit to the actual observations. The fitted models were then used to calculate the fluxes in and out of the cells and the concentrations in the extracellular space. The following results were obtained: (1) Cellular uptakes of lactate and alanine were both well described by familiar Michaelis‐Menten kinetics. (2) The cellular output of [14C]‐lactate from [14C]glucose declined with increase in the extracellular lactate concentration, whereas the cellular output of [14C]alanine from [14C]glucose rose with the extracellular alanine concentration. (3) Half‐saturation values for cellular uptake, determined from the fitted equations, were 0.45 mM for lactate and 1.17 mM for alanine, both several‐fold lower than less relevant estimates for the whole tissue made directly from the uptake observations. (4) As much as 45% of the carbon in the glucose consumed was released into the extracellular space as lactate and alanine, but much of this was reabsorbed. Implications for brain metabolism are discussed.
Unsaturated Fatty Acids Esterified in 2‐Acyl‐1‐Lysophosphatidylcholine Bound to Albumin Are More Efficiently Taken up by the Young Rat Brain than the Unesterified Form
Authors: Thiès F. , Delachambre M. C. , Bentejac M. , Lagarde M. , Lecerf J‐
Published: October 2006

Abstract: The aim of the present study was to investigate whether unsaturated 2‐acyl‐lysophosphatidylcholine bound to plasma albumin is a relevant delivery form of unsaturated fatty acids to the developing brain. Twenty‐day‐old rats were perfused for 30 s with labeled palmitic, oleic, linoleic, and arachidonic acids in either their unesterified form or esterified in 2‐acyl‐lysophosphatidylcholine labeled on the choline and fatty acid moieties. Both forms were bound to albumin. Incorporation in brain lipid classes was followed within 1 h. The brain uptake of the unesterified fatty acids reached a plateau at 5–155 mim and was maximal for arachidonic acid (0.45% of the perfused dose). The brain uptake of palmitoyl‐lysophosphatidylcholine was similar to that of palmitic acid, whereas that of other lysophosphatidylcholines increased with the degree of unsaturation (rate and maximal uptake) and was six‐ to 10‐fold higher than that of the corresponding unesterified fatty acid. 2‐Acyl‐lysophosphatidylcholines were taken up without prior hydrolysis and reacylated into doubly labeled phosphatidylcholine, which was the most labeled lipid class, whereas lipid distribution of the unesterified fatty acid was more diversi fied. Partial hydrolysis of 2‐acyl‐lysophosphatidylcholine occurred in the brain tissue, and redistribution of the fatty acyl moiety into other phospholipid classes was also observed and was the highest for arachidonic acid. In this case, the percentage of esterification of this fatty acid in phosphatidylinositol (expressed as a percentage of the total lipid fraction) was relatively lower than that observed when the unesterified form was used. 1‐Acyl‐lysophosphatidylcholine (palmitoyl) was taken at the same extent that 2‐acyllysophosphatidylcholine but was more hydrolyzed and reesterified in other lipid classes than 2‐acyl isomer. All these results suggest that 2‐acyl‐lysophosphatidylcholine bound to albumin could be an efficient delivery form of unsaturated fatty acids to the developing rat brain and that the fatty acid delivery form could modulate their fate in the tissue.
Combined Microdialysis and Fos Immunohistochemistry for the Estimation of Dopamine Neurotransmission in the Rat Caudate‐Putamen
Authors: Morelli M. , Carboni E. , Cozzolino A. , Tanda G. L. , Pinna A. , Di Chiara G.
Published: October 2006

Abstract: Extracellular dopamine (DA) concentrations estimated by transcerebral dialysis and D1‐dependent c‐fos expression, as demonstrated by Fos immunohistochemistry, were studied after blockade of DA reuptake by GBR‐12909. Rats implanted with dialysis probes in the dorsal caudate‐putamen did not show any Fos‐positive neuronal labeling in the implanted area or in the rest of the caudate‐putamen. Administration of GBR‐12909 dose‐dependently increased DA output in dialysates and resulted in the appearance in the caudate‐putamen of Fos‐positive neurons whose density was related to the dose of GBR‐12909 and to the increase in extracellular concentrations of DA. The D1 antagonist SCH‐23390 blocked GBR‐12909‐induced activation of Fos while potentiating the stimulation of DA output. The results show that following blockade of DA reuptake by GBR‐12909, the induction of Fos is related to stimulation of D1 receptors by extracellular DA. Combination of brain dialysis with Fos immunohistochemistry might provide a method for estimating the functional significance of extracellular DA as measured by brain microdialysis.
Iron‐Melanin Complex in Substantia Nigra of Parkinsonian Brains: An X‐Ray Microanalysis
Authors: Jellinger K. , Kienzl Elisabeth , Rumpelmair G. , Riederer P. , Stachelberger H. , Ben‐Shachar Dorit , Youdim M. B. H.
Published: October 2006

Abstract: Using energy‐dispersive x‐ray analysis on an electron microscope working in the scanning transmission electron microscopy mode equipped with a microanalysis system, we studied the subcellular distribution of trace elements in neuromelanin‐containing neurons of the substantia nigra zona compacta (SNZC) of three cases of idiopathic Parkinson's disease (PD) [one with Alzheimer's disease (AD)] and of three controls, in Lewy bodies of SNZC, and in synthetic dopamine‐melanin chemically charged or uncharged with Fe. Weak but significant Fe peaks similar to those of a synthetic melanin‐Fe3+ complex were seen only in intraneuronal highly electron‐dense neuromelanin granules of SNZC cells of PD brains, with the highest levels in a case of PD plus AD. whereas a synthetic melanin‐Fe2+ complex showed much lower iron peaks, indicating that neuromelanin has higher affinity for Fe3+ than for Fe2+. No detectable Fe was seen in nonmelanized cytoplasm of SNZC neurons and in the adjacent neuropil in both PD and controls, in Lewy bodies in SNZC neurons in PD, and in synthetic dopamine‐melanin uncharged with iron. These findings, demonstrating for the first time a neuromelanin‐iron complex in dopaminergic SNZC neurons in PD, support the assumption that an iron‐melanin interaction contributes significantly to dopaminergic neurodegeneration in PD and PD plus AD.
Gene Expression of Aromatic l‐Amino Acid Decarboxylase in Cultured Rat Glial Cells
Authors: Li Xin‐Min , Juorio Augusto V. , Paterson I. Alick , Walz Wolfgang , Zhu Meng‐Yang , Boulton Alan A.
Published: October 2006

Abstract: Northern blot hybridization was performed to detect aromatic L‐amino acid decarboxylase (AADC) mRNA in primary cultures of astrocytes and C6 glioma cells. The cDNA probe for rat AADC was generated by reverse transcription from rat adrenal gland total RNA and was amplified by the polymerase chain reaction method. AADC mRNA from cultured astrocytes and C6 glioma cells was present as a single band, 2.2 kbp in size, that comigrated with the RNA from rat kidney. Western immunoblot showed a single protein band at 52 kDa for AADC enzyme protein. These findings demonstrate that AADC is expressed in rat glial cells.
Interleukin 1 and Tumor Necrosis Factor‐α Stimulate the Production of Colony‐Stimulating Factor 1 by Murine Astrocytes
Authors: Théry Clotilde , Stanley E. Richard , Mallat Michel
Published: October 2006

Abstract: Astrocytes have the ability to secrete colony‐stimulating factor 1 (CSF‐1), a growth factor known to stimulate the proliferation of brain macrophages. We have studied the effect of cytokines such as interleukin 1 (IL‐1), tumor necrosis factor‐α (TNFα), and interleukin 6 (IL‐6) on the production of CSF‐1 by cultured primary astrocytes and an astrocytic cell line derived from embryonic mouse brain. We observed that both TNFα and IL‐1 increased CSF‐1 mRNA and protein levels in the astrocytic cultures. In contrast, IL‐6 was ineffective. The CSF‐1 mRNA levels were strongly reduced by incubating immortalized astrocytic cells with staurosporine, a protein kinase C inhibitor, both in the absence and in the presence of cytokines. Conversely, 12‐O‐tetradecanoylphorbol 13‐acetate, a protein kinase C activator, increased CSF‐1 mRNA levels. These results suggest a mechanism whereby mononuclear phagocytes could favor their own recruitment in the CNS by producing cytokines.
Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium‐ and Calmodulin‐Dependent Kinase II
Authors: Churn Severn B. , Taft William C. , Billingsley Melvin S. , Sankaran Banumathi , DeLorenzo Robert J.
Published: October 2006

Abstract: The activity of multifunctional calcium/calmodulin‐dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia‐induced inhibition of the enzyme, CaM kinase II was purified to > 1,000‐fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the β subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent‐binding ATP analog 8‐azidoadenosine‐5′‐[α‐32P]triphosphate was used. Covalent binding of 25 μM azido‐ATP was decreased 40.4 ± 12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p < 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.
Ethanol Reduces Norepinephrine‐Stimulated Melatonin Synthesis in Rat Pinealocytes
Authors: Chik Constance L. , Ho Anthony K.
Published: October 2006

Abstract: In this study, the in vitro effects of ethanol on norepinephrine‐stimulated cyclic AMP (cAMP), N‐acetyltransferase (NAT), and melatonin (MT) production were examined in dispersed rat pinealocytes. Cellular cAMP content was determined 15 min after treatment; whereas NAT activity and MT release in the medium were determined 4.5 h after treatment. It was found that ethanol ≤200 mM had no effect on norepinephrine‐stimulated cAMP response, whereas 25 mM ethanol resulted in a significant inhibition of norepinephrine‐stimulated NAT and MT levels. Furthermore, ethanol was equally effective in inhibiting the dibutyryl cAMP‐stimulated NAT and MT levels. The inhibitory action of ethanol was not due to a direct effect or a delay in the onset of NAT activity. When alcohols with different chain lengths were used, it was found that their inhibitory potencies were related to their chain lengths with butanol > propanol > ethanol > methanol. Taken together, these findings indicate that (1) ethanol has an inhibitory action on norepinephrine‐stimulated MT synthesis, (2) one site of ethanol action is distal to cAMP elevation, and (3) the inhibitory effect of ethanol on pineal MT synthesis appears to be secondary to its hydrophobic action.
Characterization of the 5‐Hydroxytryptamine Receptor Modulating the Release of 5‐[3H]Hydroxytryptamine in Slices of the Human Neocortex
Authors: Galzin A. M. , Poirier M. F. , Lista A. , Chodkiewicz J. P. , Blier P. , Ramdine R. , Loô H. , Roux F. X. , Redondo A. , Langer S. Z.
Published: October 2006

Abstract: In the rat brain, the presynaptic 5‐hydroxytrypt‐amine (5‐HT) autoreceptors located on 5‐HT terminals correspond to the 5‐HT1B subtype. The presence of a 5‐HT receptor probably located on 5‐HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5‐HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5‐HT by various 5‐HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5‐HT1A/1B/1D receptor agonist 5‐carboxamidotryptamine produced a potent inhibition (70% at 0.03 μM) of the electrically evoked release of [3H]5‐HT which was blocked by 5‐HT receptor antagonists with the following relative order of potency: methiothepin > metergoline = methysergide > propranolol. The selective 5‐HT1A receptor agonist 8‐hydroxy‐2–(di‐n‐propylamino)tetralin at 0.1 μM did not modify the electrically evoked release of [3H]5‐HT. The 5‐MT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5‐HT overflow in the rat frontal cortex than in the human neocortex. The potent 5‐HT1B receptor antagonist cyanopindolol did not modify the 5‐carboxamidotryptamine‐induced inhibition of the electrically evoked release of [3H]5‐HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5‐HT overflow. The α2‐adrenoceptor antagonist yohimbine, which possesses affinity for the 5‐HT1D receptor subtype, decreased the release of [3H]5‐HT, but only in the presence of the selective α2‐adrenoceptor antagonist idazoxan, which by itself increased significantly [3H]5‐HT overflow. Taken together, these results support the view that the 5‐HT receptor modulating the electrically evoked release of [3H]5‐HT in slices of the human neocortex could be of the 5‐HT1D subtype. Moreover, preliminary results obtained with idazoxan confirm the existence of a presynaptic α2‐adrenoceptor modulating the release of [3H]5‐HT in the human neocortex. These α2‐heteroreceptors could exert a tonic inhibitory modulation on 5‐HT neurotransmission in the human neocortex.
Involvement of γ‐Aminobutyric Acid and N‐Methyl‐D‐Aspartate Receptors in the Inhibitory Effect of Ethanol on Pentylenetetrazole‐Induced c‐fos Expression in Rat Brain
Authors: Le Fei , Wilce Peter A. , Hume David A. , Shanley Brian C.
Published: October 2006

Abstract: The expression of c‐fos mRNA in rat brain was induced by intraperitoneal administration of pentylenetetrazole (PTZ) and picrotoxin, which act on the picrotoxin binding site of the γ‐aminobutyric acid‐benzodiazepine (GABA‐BZ) receptor complex, by N‐methyl‐D‐aspartate (NMDA) and kainic acid, agonists of different classes of glutamate receptors and by caffeine, an antagonist of adenosine receptors. The actions of PTZ and picrotoxin but not that of NMDA were blocked by ethanol and the NMDA‐receptor antagonist, MK‐801. Ro 15–4513 partially reversed the inhibitory effect of ethanol on PTZ‐induced c‐fos mRNA synthesis. Acute ethanol administration blocked the actions of PTZ and NMDA without affecting the response to kainic acid or caffeine. Taken together, these data suggest that ethanol blocks c‐fos gene activation by noncompetitive antagonists of the GABA‐BZ receptor via actions on both the NMDA and GABA receptors.
β‐Amyloid Precursor Protein Isoforms in Various Rat Brain Regions and During Brain Development
Authors: Löffler Jürgen , Huber Gerda
Published: October 2006

Abstract: To address the question of the possible functions of different Alzheimer's disease β‐amyloid precursor protein (β‐APP) isoforms in the brain, we studied their expression at different times during postnatal rat brain development and in various regions of the adult rat brain. Polyclonal antibodies directed to two peptide antigens were used. The majority of all β‐APP forms was found to be soluble as revealed by western blot analysis. The highest level of most β‐APP forms was reached in the second postnatal week, which is the time of brain maturation and completion of synaptic connections. Strikingly high concentrations of the Kunitz protease inhibitor‐containing β‐APP were present in the adult olfactory bulb, where continuous synaptogenesis occurs in the adult animal. These findings support the idea of an involvement of β‐APPs in the processes of cell differentiation and, probably, in the establishment of synaptic contacts.
Evidence for a New Member of the Myosin I Family from Mammalian Brain
Authors: Li Deqin , Chantler Peter D.
Published: October 2006

Abstract: Myosin I is an actin‐based motor responsible for powering a wide variety of motile activities in amebae and slime molds and has been found previously in vertebrates as the lateral bridges within intestinal epithelial cell microvilli. Although neurons exhibit extensive cellular and intracellular motility, including the production of ameboid‐like growth cones during development, the proteins responsible for the motor in these processes are unknown. Here, we report the isolation of a partially purified protein fraction from bovine brain that is enriched for a 150‐kDa protein; immunochemical and biochemical analyses suggest that this protein possesses a number of functional properties that have been ascribed to myosin I from various sources. These properties include an elevated K+‐EDTA ATPase, a modestactin‐activated Mg2+‐ATPase, the ability to bind calmodulin, and a ready association with phospholipid vesicles made from phosphatidylserine, but not from phosphatidylcholine. The combination of these properties, together with a molecular mass of 150 kDa (most myosin I molecules found to date have molecular masses in the range 110–130 kDa) yet recognition by an anti‐myosin I antibody, suggests the presence of a new member of the myosin I family within mammalian brain.
Preparation of Antibodies to β Subunits of γ‐Aminobutyric AcidA Receptors
Authors: Endo Shuichi , Olsen Richard W.
Published: October 2006

Abstract: Antisera were produced in rabbits against synthetic peptides based on two regions of the cDNA sequence of the β1 subunit of bovine γ‐aminobutyric acidA (GABAA) receptors. The deduced amino acid sequences were similar in other β subunits of bovine, rat, and chick receptors, predicting cross‐reactability with all β subunits. One antiserum (anti‐βe) was raised against an extracellular moiety near the invariant disulfide loop thought to be located near the neuro‐transmitter binding domain; the other (anti‐(βc) was raised against an intracellular moiety containing a consensus sequence for cyclic AMP‐dependent protein kinase phos‐phorylation of a serine residue. Predicted secondary structures suggested high potential immunogenicity for the chosen antigen peptides. Both antisera at high dilutions recognized the same polypeptide bands on western blots of GABAA receptors purified from three regions of bovine brain (four bands at 57, 54, 53, and 52 kDa in cerebral cortex) but fewer bands (57, 54, and 52 kDa) in hippocampus and cerebellum (one major band at 54 kDa, traces at 57 and 53 kDa). This is consistent with the presence of multiple β subunits whose expression varies with brain region, as shown by molecular cloning. The anti‐βc antibody was able to immunoprecipitate purified GABAA receptor [3H]‐muscimol binding, 87% in bovine cortex and 75% in total rat brain; the anti‐J.3e was unable to immunoprecipitate any antigen. These antibodies indicate a region‐dependent heterogeneity of p subunits and should be useful for analyzing structure, function, and localization of GABAA receptor subtypes in brain.
Human Brain βA4 Amyloid Protein Precursor of Alzheimer's Disease: Purification and Partial Characterization
Authors: Moir Robert D. , Martins Ralph N. , Bush Ashley I. , Small David H. , Milward Elizabeth A. , Rumble Baden A. , Multhaup Gerd , Beyreuther Konrad , Masters Colin L.
Published: October 2006

Abstract: The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4‐kDa βA4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100–110 kDa and 120–130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus‐directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane‐associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.
Amino Acid Neurotransmitter Receptor Changes in Cerebral Cortex in Alcoholism: Effect of Cirrhosis of the Liver
Authors: Dodd Peter R. , Thomas Gregory J. , Harper Clive G. , Kril Jillian J.
Published: October 2006

Abstract: γ‐Aminobutyric acidA/benzodiazepine receptor binding sites and the N‐methyl‐D‐aspartate subclass of glutamate receptor sites were assessed in synaptic plasma membrane homogenates of cerebral cortex tissue obtained at autopsy from cirrhotic and noncirrhotic alcoholic patients and matched control subjects. The alcoholic patients consumed an average of >80 g of ethanol/day, the control subjects <20 g/day. Postmortem delays up to ∼ 100 h caused no significant loss of any of the binding sites; the patient and subject groups were closely matched for age. The affinities (KD) of the receptor sites did not differ between the patient and subject groups, nor between cortical regions. Using three different radioligands ([3H]muscimol, [3H]flunitrazepam, and [3H]diazepam), the γ‐aminobutyric acidA/benzodiazepine receptor complex was found to have greater density (Bmax) in superior frontal gyrus in alcoholic patients (which selectively shows morphological change in alcoholic patients), but was unchanged in motor cortex. Alcoholic patients with cirrhosis had much less pronounced changes. The density of the N‐methyl‐D‐aspartate subclass of glutamate receptors, assessed with [3H]MK‐801. did not vary across patient and subject groups.
Effects of Sulfate Ions on Alzheimer β/A4 Peptide Assemblies: Implications for Amyloid Fibril‐Proteoglycan Interactions
Authors: Fraser Paul E. , Nguyen Jack T. , Chin David T. , Kirschner Daniel A.
Published: October 2006

Abstract: To model the possible involvement of sulfated proteoglycans in amyloidogenesis, we examined the influence of sulfate ions, heparan, and Congo red on the conformation and morphology of peptides derived from the Alzheimer β/A4 amyloid protein. The peptides included residues 11–28, 13–28, 15–28, and 11–25 of β/A4. Negative‐stain electron microscopy revealed a sulfate‐specific tendency of the preformed peptide fibrillar assemblies of β(11–28), β(13–28), and β(11–25), but not β(15–28), to undergo extensive lateral aggregation and axial growth into “macrofibers” that were ∼0.1–0.2 μm wide by ∼20–30 μm long. Such effects were observed at low sulfate concentrations (e.g., 5–50 mM) and could not be reproduced under comparable conditions with Na2HPO4, Na2SeO4, or NaCl. Macrofibers in NaCl were only observed at 1,000 mM. At physiological ionic strength of NaCl, fibril aggregation was observed only with addition of sulfate ions at 5–50 mM. Selenate ions, by contrast with sulfate ions, induced only axial and not substantial lateral aggregation of fibrils. X‐ray diffraction indicated that the original cross‐β peptide conformation remained unchanged; however, sulfate binding did produce an intense ∼65 Å meridional reflection not recorded with control peptides. This new reflection probably arises from the periodic deposition of the electron‐dense sulfate along the (long) axis of the fibril. The sulfate binding could provide sites for the binding of additional fibrils that generate the observed lateral and axial aggregation. The binding of heparan to β(11–28) also produced extensive aggregation, suggesting that in vivo sulfated compounds can promote macrofibers. The amyloid‐specific, sulfonated dye Congo red, even in the presence of sulfate ions, produced limited aggregation and reduced axial growth of the fibrils. Therefore, electrostatic interactions are important in the binding of exogenous compounds to amyloid fibrils. Our findings suggest that the sulfate moieties of certain molecules, such as glycosaminoglycans, may affect the aggregation and deposition of amyloid fibrils that are observed as extensive deposits in senile plaques and cerebrovascular amyloid.
Intraventricular Infusions of Anti‐Neural Cell Adhesion Molecules in a Discrete Posttraining Period Impair Consolidation of a Passive Avoidance Response in the Rat
Authors: Doyle E. , Nolan P. M. , Bell R. , Regan C. M.
Published: October 2006

Abstract: Intraventricular infusions of anti‐neural cell adhesion molecule (anti‐NCAM) are demonstrated to inhibit consolidation of a passive avoidance response when administered in the 6‐8‐h posttraining period. Anti‐NCAM was ineffective when administered during training or at any other time up to 10 h thereafter, and no amnesic effects were observed with absorbed anti‐NCAM or anti‐neurofilament protein. Amnesia was observed only at the 48‐h recall time, and this could not be attributed to poor antibody penetration or a prolonged residence time, as studies with 125I‐labelled anti‐NCAM in trained animals demonstrated a rapid accumulation into all brain regions, and this was marked in the olfactory bulb and hippocampus, areas showing an inherent and paradigm‐specific increase in NCAM sialylation state, respectively. The lack of an amnesic action at the 24‐h recall time is attributed to anti‐NCAM‐impaired synapse structuring becoming apparent following the paradigm‐specific increases in NCAM sialylation state.
Ischemia‐Induced Changes in Extracellular Lévels of Striatal Cyclic AMP: Role of Dopamine Neurotransmission
Authors: Prado Ricardo , Busto Raul , Globus Mordecai Y.‐T.
Published: October 2006

Abstract: Dopamine has been demonstrated to be involved in the development of ischemic neuronal damage in the striatum. This detrimental effect of dopamine may involve activation of second messenger systems, such as the cyclic AMP (cAMP) cascade, which may enhance the susceptibility of striatal neurons to ischemia. In the present study, we have evaluated the relationship between ischemia‐induced changes in cAMP and dopamine neurotransmission. Microdialysis probes were implanted in both striata, and a D1 antagonist (SCH‐23390, 100 μM) was administered through one probe and modified Ringer's solution through the other. After a stabilization period, rats (n = 6) were subjected to 20 min of ischemia by two‐vessel occlusion plus hypotension. Extracellular samples were collected from both striata, before, during, and after ischemia, and analyzed for cAMP by radioimmunoassay. Ischemia induced a significant increase in extracellular cAMP (means ± SE, fmol/μl; baseline: 4.35 ± 1.1, ischemia: 12.2 ± 1.98), which was also observed at 4 h of recirculation (mean level of 8.45 ±1.14). Treatment with the D1 antagonist significantly inhibited the rise in extracellular cAMP during ischemia and recirculation. These results indicate that an ischemia‐induced surge in dopamine and activation of D1 receptors are involved in the generation of cAMP during ischemia and recirculation. Because activation of the adenylate cyclase cascade may modulate the effects of glutamate, generation of cAMP through this pathway may play a role in facilitating the injurious effects of dopamine during ischemia.
Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites
Authors: Krieger Timothy J. , Mende‐Mueller Liane , Hook Vivian Y. H.
Published: October 2006

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys‐Arg, Arg‐Arg, and Lys‐Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin‐containing peptides, peptide E and BAM‐22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM‐22P at the Lys‐Arg site between the two basic residues. The Arg‐Arg site of both peptide E and BAM‐22P was cleaved at the NH2‐terminal side of the paired basic residues to generate [Met]‐enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH2‐terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys‐Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH2‐terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.
Uptake and Distribution of Iron and Transferrin in the Adult Rat Brain
Authors: Morris C. M. , Keith A. B. , Edwardson J. A. , Pullen R. G. L.
Published: October 2006

Brain uptake of iron‐59 and iodine‐125‐labelled transferrin from blood in the adult rat has been investigated using graphical analysis to determine the blood‐brain barrier permeability to these tracers in experiments that lasted between 5 min and 8 days. The blood‐brain barrier permeability (Kin) to 59Fe was 89 ± 10−5 ml/min/g compared to the value of 7 ± 10−5 ml/min/g for 125I‐transferrin, which is similar to that of albumin, a plasma marker. The autoradiographic distribution of these tracers in brain was also studied to determine any regional variation in brain uptake after the tracers had been administered either system‐ically or applied in vitro. No regional uptake was seen for 125I‐transferrin even after 24 h of circulation. In contrast, 59Fe showed selective regional uptake by the choroid plexus and extra‐blood‐brain barrier structures 4 h after administration. After 24 h of circulation, 59Fe distribution in brain was similar to the transferrin receptor distribution, as determined in vitro, but was unlike the distribution of non‐haem iron determined histochemically. The data suggest that brain iron uptake does not involve any significant transcytotic pathway of transferrin‐bound iron into brain. It is proposed that the uptake of iron into brain involves the entry of iron‐loaded transferrin to the cerebral capillaries, deposition of iron within the endothelial cells, followed by recycling of apotransferrin to the circulation. The deposited iron is then delivered to brain‐derived transferrin for extracellular transport within the brain, and subsequently taken up via transferrin receptors on neurones and glia for usage or storage.
A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma
Authors: Asai Kiyofumi , Hirano Takayoshi , Kaneko Shuji , Moriyama Akihiko , Nakanishi Keiko , Isobe Ichiro , Eksioglu Yaman Z. , Kato Taiji
Published: October 2006

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion‐exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross‐reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemi‐cally visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent Mr of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent Mr of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50‐kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor‐stimulated astroblasts, but not neuronal cells.