bluuebunny/crossref_metadata_2025 huggingface dataset:
The Distribution of Cathepsin D Activity in Adult and Aging Human Brain Regions
Authors: Banay‐Schwartz M. , DeGuzman T. , Kenessey A. , Palkovits M. , Lajtha A.
Published: October 2006

Abstract: We measured the activity of cathepsin D, the major cerebral protease, in 50 separate areas of the central nervous system of adult and aged humans, using hemoglobin as the substrate. The activity showed significant regional heterogeneity, with average differences of 50–100% between the lower and higher level areas, and a more than threefold difference between the lowest and highest levels. The forebrain, midbrain, and hindbrain each had areas of high and low activity; cerebellum and cord areas were among those with low activity. Cathepsin levels tended to increase with age in about half of the areas analyzed, and the increases were significant in 14. Statistically significant decreases with aging were observed in two areas. The increases varied between 30 and 60%, and the decreases were 20%. Enzyme activity in thalamus, hypothalamus, pons, medulla, and cerebellum increased with age. In the ventrolateral medulla, which contains the major portion of the cerebral noradrenergic cells, the cathepsin D levels increased with age; in the dorsal raphe area, which contains the major portion of the cerebral serotonergic cells, the enzyme levels decreased. The change with age in human brain seems to be less than what we observed in rat brain, where activity more than doubled in most areas. The changes in enzyme levels need to be tested at more ages to establish a pattern of changes in activity throughout life.
Butyrylcholinesterase from Chicken Brain Is Smaller than That from Serum: Its Purification, Glycosylation, and Membrane Association
Authors: Treskatis Sven , Ebert Christoph , Layer Paul G.
Published: October 2006

Abstract: Applying a new four‐step isolation procedure, we have purified butyrylcholinesterase (BChE) from chicken serum to homogeneity with more than 250 U/mg specific activity. The serum enzyme was used for producing monoclonal antibodies. These BChE‐specific antibodies also recognize BChE from brain, and thus enabled us to isolate the enzymes from embryonic and adult brain that occur only in minute amounts. More than 50% of the brain BChE is membrane‐bound. The catalytic and inhibition properties of brain BChE are similar to those of serum BChE. However on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the serum enzyme is represented by a double‐band of 79/82 kDa, whereas the brain enzyme has a size of 74 kDa. Limited digestion of the serum and brain preparations by V8‐protease leads to similar peptide patterns. Enzymatic deglycosylation shows that their core proteins consist of 59‐kDa subunits and that the different molecular weights are due to different glycosylation patterns. The differently sized glycosylation parts of brain and serum BChE may indicate that they subserve different functions. Furthermore, the membrane‐bound brain BChE can be solubilized by Pronase or protease K, but not by phosphatidylinositol‐specific phospholipase C.
Selective Expression of DM‐20, an Alternatively Spliced Myelin Proteolipid Protein Gene Product, in Developing Nervous System and in Nonglial Cells
Authors: Ikenaka Kazuhiro , Kagawa Tetsushi , Mikoshiba Katsuhiko
Published: October 2006

Abstract: Mutations within the gene for myelin proteolipid protein (PLP), a major myelin structural protein, result in abnormal glial differentiation, suggesting that the PLP gene products play some other functional roles. Transcripts from the PLP gene were analyzed in the developing mouse brain by a sensitive method using polymerase chain reaction. The mRNA for DM‐20, an alternatively spliced transcript from the PLP gene, was detected in the embryonic mouse brain as early as embryonic day 11, long before the appearance of oligodendrocytes, which were considered to be responsible for PLP production. PLP gene expression was analyzed in various cell lines to determine whether synthesis of the DM‐20 mRNA is restricted to those of glial cell lineage. All of the nervous system cell lines examined, including nonglial cell lines, produced DM‐20 mRNA but no or very little PLP mRNA. Peripheral sciatic nerve from adult Wistar rats also produced mainly DM‐20 mRNA. These results indicate that DM‐20 is not only a myelin structural protein, but it also plays other roles in the nervous system that seem to relate, at least in part, to glial differentiation.
Effects of Feeding and Drinking on Acetylcholine Release in the Nucleus Accumbens, Striatum, and Hippocampus of Freely Behaving Rats
Authors: Mark Gregory P. , Rada Pedro , Pothos Emmanuel , Hoebel Bartley G.
Published: October 2006

Abstract: Extracellular levels of acetylcholine (ACh) were measured in the nucleus accumbens (NAC), striatum (STR), and hippocampus (HIPP) using microdialysis in 30‐min intervals before, during, and after free‐feeding in 20‐h food‐deprived rats. The effects on ACh in the NAC and STR were also observed in response to water intake in 20‐h water‐deprived animals. Neostigmine was used in the perfusate to improve ACh recovery. Basal ACh was sensitive to tetrodotoxin and low calcium, and therefore largely neuronal in origin. Feeding caused a 38% increase in extracellular ACh in the NAC and no change in the STR or HIPP. Dopamine was also increased in the NAC (48%) and to a lesser extent in the STR (21%) following feeding. Drinking caused 18–20% increases in ACh release in both the NAC and STR. In a separate experiment, ACh release in the NAC was monitored in 10‐min intervals during free‐feeding; ACh increased in the interval immediately following maximal food intake. These results suggest a site‐specific increase in ACh release following feeding that cannot be solely attributed to the activation associated with this behavior.
Effects of Handling or Immobilization on Plasma Levels of 3,4‐Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats
Authors: Kvetňansk Richard , Goldstein David S. , Weise Virginia K. , Holmes Courtney , Szemeredi Katalin , Bagdy Gyorgy , Kopin Irwin J.
Published: October 2006

Abstract: In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4‐dihydroxyphenylalanine (DOPA). NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4‐dihydroxyphenylglycol (DHPG) and 3,4‐dihydroxyphenylacetic acid (DOPAC), and their O‐methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1,3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE. EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.
Alternative Splicing of the Cytoplasmic Domain of Neural Cell Adhesion Molecule Alters Its Ability to Act as a Substrate for Neurite Outgrowth
Authors: Doherty Patrick , Rimon Galia , Mann Derek A. , Walsh Frank S.
Published: October 2006

Abstract: A full‐length cDNA encoding 180‐kDa neural ceil adhesion molecule (NCAM 180) has been transfected into mouse NIH‐3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.
Cloning and Characterization of the cDNA Encoding a Novel Brain‐Specific 14‐kDa Protein
Authors: Tobe Takashi , Nakajo Shigeo , Tanaka Atsutaka , Mitoya Akira , Omata Kumiko , Nakaya Kazuyasu , Tomita Motowo , Nakamura Yasuharu
Published: October 2006

Abstract: A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencg. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full‐length Sequence of the 14‐kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14‐kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14‐kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14‐kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60–80) flanked by highly hydrophilic stretches on both sides. Whereas the N‐terminal region of the 14‐kDa protein contained four repeating motifs, EKTKEGV, the C‐terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14‐kDa protein indicated no homology to any other protein reported so far.
Inhibition of Cyclic AMP Accumulation in Intact NCB‐20 Cells as a Direct Result of Elevation of Cytosolic Ca2+
Authors: Garritsen Anja , Zhang Yingxin , Firestone Jordan A. , Browning Michael D. , Cooper Dermot M. F.
Published: October 2006

Abstract: Earlier studies established that adenylyl cyclase in NCB‐20 cell plasma membranes is inhibited by concentrations of Ca2+ that are achieved in intact cells. The present studies were undertaken to prove that agents such as bradykinin and ATP, which elevate the cytosolic Ca2+ concentration ([Ca2+]i) from internal stores in NCB‐20 cells, could inhibit cyclic AMP (cAMP) accumulation as a result of their mobilization of [Ca2+]i and not by other mechanisms. Both bradykinin and ATP transiently inhibited [3H]cAMP accumulation in parallel with their transient mobilization of [Ca2+]i. The [Ca2+]i rise stimulated by bradykinin could be blocked by treatment with thapsigargin; this thapsigargin treatment precluded the inhibition of cAMP accumulation mediated by bradykinin (and ATP). A rapid rise in [Ca2+]i, as elicited by bradykinin, rather than the slow rise evoked by thapsigargin was required for inhibition of [3H]cAMP accumulation. Desensitization of protein kinase C did not modify the inhibitory action of bradykinin on [3H]cAMP. Effects of Ca2+ on phosphodiesterase were also excluded in the present studies. The accumulated data are consistent with the hypothesis that hormonal mobilization of [Ca2+]i leads directly to the inhibition of cAMP accumulation in these cells and presumably in other cells that express the Ca2+‐inhibitable form of adenylyl cyclase.
Biochemical Characterization of Recombinant Human Nerve Growth Factor
Authors: Schmelzer Charles H. , Burton Louis E. , Chan Wai‐Pan , Martin Evelyn , Gorman Cori , Canova‐Davis Eleanor , Ling Victor T. , Sliwkowski Mary B. , McCray Glynis , Briggs Jonathan A. , Nguyen Tue H. , Polastri Gian
Published: October 2006

Abstract: Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion‐exchange and reversed‐phase (RP) chromatography. The isolated product was shown to be consistent with a 120‐amino‐acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), RP‐HPLC, and mass spectrometry and with an N‐terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size‐exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120‐residue monomer produced additional species of 118 (trypsin, removal of the C‐terminal Arg119‐Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C‐terminal Arg118‐Arg119‐Ala120 sequence) residues. Each of these species was isolated by high‐performance ion‐exchange chromatography and characterized by amino acid and N‐terminal sequence analyses, SDS‐PAGE, RP‐HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.
Amygdala Kindling Alters Protein Kinase C Activity in Dentate Gyrus
Authors: Chen Shu‐Jen , Desai Manisha A. , Klann Eric , Winder Danny G. , Sweatt J. David , Conn P. Jeffrey
Published: October 2006

Abstract: Kindling is a use‐dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca2+‐independent) and Ca2+‐stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling‐associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.
Differences in the In Vitro and In Vivo 5‐Hydroxytryptamine Extraction Performance Among Three Common Microdialysis Membranes
Authors: Tao Rui , Hjorth Stephan
Published: October 2006

Abstract: The present study summarizes the results of an in vitro and in vivo comparison of the apparent 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindoleacetic acid, and 3,4‐dihydroxyphenylacetic acid dialysis performance of three types of membrane frequently used in intracerebral microdialysis experiments. The dialysis fiber types examined were a regenerated cellulose Cuprophan (GF), a proprietary polycarbonate ether (CMA), and a polyacrylonitrile/sodium methallylsulfonate copolymer (HOSPAL). The experiments unexpectedly revealed that the HOSPAL membrane‐equipped probes displayed clearly aberrant 5‐HT diffusion dynamics compared with GF and CMA probes, demonstrable not only in vitro, but also in in vivo experiments. In vitro, the GF and CMA membrane‐equipped probes exhibited maximum relative recovery for 5‐HT already in the first 20‐min sample, whereas the 5‐HT recovery of HOSPAL probes increased in a very slow and protracted manner over a period of a little less than 2 h. The GF and CMA probes further displayed an immediate washout of 5‐HT when the probes were subsequently transferred to artificial CSF only‐containing medium (no 5‐HT), whereas sim 2 h was required to yield near‐total extinction of dialysate 5‐HT with the standard HOSPAL probes. In vivo, the rat ventral hippocampal dialysate 5‐HT output responses to K+ (100 mM) infusion, to Ca2+ omission, and to systemic 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin injection were all markedly retarded and blunted when HOSPAL instead of GF membrane‐equipped probes were used. However, the 5‐hydroxyindoleacetic acid and 3,4‐dihydroxyphenylacetic acid extraction in vitro and in vivo were comparable using either of the membrane types. The findings are discussed in terms of an interaction between cationic 5‐HT and negative ionic charges in the HOSPAL membrane. The anomalous 5‐HT dialysis dynamics of the HOSPAL membrane are also shown to be dependent on the exposed fiber surface area (and thus the probe design), and may be influenced by changing the perfusion flow rate or the ionic strength of the media in‐ and outside the probe. Our findings seriously caution against uncritically using any dialysis membrane without controlling for its properties within the context of the application intended. In particular, the usefulness of the HOSPAL membrane for microdialysis of 5‐HT at physiological pH may be limited.
Pertussis Toxin‐Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells
Authors: Kanoh Hiroyuki , Kanaho Yasunori , Nozawa Yoshinori
Published: October 2006

Abstract: In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid‐labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]PEt, which reached a plateau within 1 min, in a concentration‐dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12‐myristate 13‐acetate (PMA), also caused an increase in [3H]PEt content, which reached a plateau at 30–60 min after exposure, but an inactive phorbol ester, 4a‐phorbol 12,13‐didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 μM), blocked PMA‐induced [3H]PEt formation by 77%, it had no effect on the CCh‐induced formation. These results suggest that mAChR‐induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP‐binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5′‐O‐(3‐thiotriphosphate) (OTPGmS), also stimulated [3H]PEt formation in intact and digitonin‐permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5′‐O‐(2‐thiodiphosphate) significantly inhibited CCh‐ and GTPΓS‐ induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 μM) had no effect on [3H]PEt formation by GTPγS. Pretreatment of cells with pertussis toxin (10–200 ng/ml) for 15 h failed to suppress CCh‐induced [3H]PEt formation, although the pertussis toxin‐sensitive G protein(s) in membranes was completely ADP‐ribosylated under the same conditions. From these results, we conclude that the mechanisms of PMA‐ and CCh‐stimulated PLD activation are different from each other and that CCh‐induced PLD activation is independent of PKC and mediated, at least in part, via a pertussis toxin‐insensitive G protein.
Tolerance of Low Intracellular pH During Hypercapnia by Rat Cortical Brain Slices: A 31P/1H NMR Study
Authors: Espanol Maryceline T. , Litt Lawrence , Yang Guo‐yuan , Chang Lee‐Hong , Chan Pak H. , James Thomas L. , Weinstein Philip R.
Published: October 2006

Abstract: Metabolic tolerance of low intracellular pH (pHi) was studied in well‐oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 mum thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (sim 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high‐energy phosphate metabolites, from which pHi and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pHi, ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation. During hypercapnia, pHi decreased from 7.18 pmn 0.22 to 6.79 pmn 0.13, 6.60 pmn 0.11, 6.33 pmn 0.40, and 6.37 pmn 0.14 when CO2 concentrations were increased to 10, 30, 50, and 70%, respectively. Lactate/N‐acetylaspartate ratios were unchanged until the administration of 70% CO2, which caused a relative increase from 0.83 pmn 0.15 to 1.35 pmn 0.32 (n = 5, p = 0.0024). The administration of 70% CO2 also caused complete depletion of PCr and a 46 pmn 22% (n = 5, p = 0.0024) decrease in ATP content. Metabolite and pH values returned to control values after restoration of normocapnia. The lowest tissue pHi attainable in our protocol using 30% O2/70% CO2 in our brain slice preparation was sim 6.3.
Activation of Dopamine D2 Receptors Linked to Voltage‐Sensitive Potassium Channels Reduces Forskolin‐Induced Cyclic AMP Formation in Rat Pituitary Cells
Authors: Memo Maurizio , Pizzi Marina , Belloni Marco , Benarese Marina , Spano PierFranco
Published: October 2006

Abstract: 3,4‐Dihydroxyphenylethylamine (dopamine) D2 receptor agonists, including BHT 920 and bromocriptine, and the potassium channel opener minoxidil share the property of hyperpolarizing the plasma membrane by activating voltage‐dependent potassium channels. These drugs were tested for their ability to inhibit the cyclic AMP formation induced by forskolin either in intact or in broken pituitary cells. In contrast to bromocriptine, which was active in both experimental systems, BHT 920 and minoxidil inhibited the forskolin‐induced cyclic AMP formation in intact‐cell but not in broken‐cell preparations. The effects of BHT 920 were (a) concentration dependent, with a calculated IC50 of 0.7 muM, (b) dopaminergic in nature, being specifically antagonized by sulpiride, (c) not additive with those induced by minoxidil, and (d) less effective in the presence of potassium channel blockers, such as 4‐aminopyridine and tetraethylammonium. These data indicate that the inhibition of forskolin‐induced cyclic AMP formation by BHT 920 in intact pituitary cells is not a primary consequence of receptor occupation, but a late event, possibly related to the opening of voltage‐dependent potassium channels elicited by this drug through the activation of a subtype of dopamine D2 receptors uncoupled to adenylyl cyclase.
Sodium Dependence of [3H]]Paroxetine Binding and 5‐ [3H]]Hydroxytryptamine Uptake in Rat Diencephalon
Authors: Mann Catherine D. , Hrdina Pavel D.
Published: October 2006

Abstract: The sodium dependence of binding of [3H]]‐paroxetine, a selective serotonin uptake inhibitor, to the serotonin transporter in rat diencephalon was studied in both brain membranes and tissue sections and compared with that of 5‐ [3H]]hydroxytryptamine ([3H]]5‐HT) uptake by synaptosomes from the same region. Binding of [3H]]‐paroxetine in both the membranes and sections displayed clear sodium dependence until a plateau occurring at 60 mMNaCl, the EC50 for sodium being 8 and 25 mM, respectively. The affinity (1/KD) of [3H]]paroxetine binding was a simple hyperbolic function of sodium concentration. In contrast, the density of [3H]]paroxetine sites was not affected by external Na+ concentration. The uptake of [3H]]5‐HT showed a similar pattern of sodium dependence with an EC50 for Na+ of 25 mM. Both the affinity (1/Km) and the rate (Vmax) of [3H]]5‐HT uptake were dependent on external [Na+] with sodium‐dependence curves fitting a rectangular hyperbola. The kinetic analysis of results indicates that one sodium ion is required for the binding of [3H]]paroxetine as well as for the binding and translocation of each [3H]]5‐HT molecule. The results concur with a single‐site model of the sodium‐dependent serotonin transporter with common or overlapping domains for 5‐HT and 5‐HT uptake inhibitors.
Characterization of Gene Organization and Promoter Region of the Rat Dopamine D1 Receptor Gene
Authors: Zhou Qun‐Yong , Li Chuanyu , Civelli ROlivier
Published: October 2006

Abstract: Genomic and cDNA clones encoding the rat Dl receptor were isolated and sequenced. Comparison of the Dl receptor cDNA and genomic sequences revealed that the rat Dl receptor gene is organized into two exons separated by a small intron in the 5’untranslated region of its mRNA. The transcription start site is located 864 bp upstream from the translational initiation site. The 5′‐flanking sequences of the Dl receptor gene do not contain TATA and CAAT canonical sequences, but have a high G + C content, potential cyclic AMP and glucocorticoid response element sequences, and binding sites for transcription factors such as Sp1, Ap1, and Ap2. Transfection studies using the Dl 5′‐flanking sequence and CAT gene fusion constructs have demonstrated that (1) the D1 promoter is active in Dl‐expressing neuroblastoma NS20Y cells, but inactive in D1‐deficient glioma C6 and kidney 293 cells, (2) the information contained within 735 bp of 5′‐flanking sequence of the Dl gene appears to be sufficient to confer its cell‐specific expression, and (3) the Dl gene promoter responds to cyclic AMP induction, suggesting the existence of an autoregulation mechanism by which the stimulation of Dl receptor exerts a positive feedback on its own gene expression.
Activity and Biospecificity of Proteolyzed Forms and Dimeric Combinations of Recombinant Human and Murine Nerve Growth Factor
Authors: Burton Louis E. , Schmelzer Charles H. , Szonyi Eva , Yedinak Cathleen , Gorrell Aldona
Published: October 2006

Abstract: Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland‐derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed‐phase HPLC (RP‐HPLC), and high‐performance ion‐exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120‐residue form of rhNGF produced a species of 109 residues (10–118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N‐terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N‐terminal sequence, PAGE, and RP‐HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma‐12 cell line neurite extension assay. The 118‐residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal‐modified molecules presented activity reduced by 50‐ to 100‐fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.
Use of the Neural Cell Adhesion Molecule VASE Exon by Neurons Is Associated with a Specific Down‐Regulation of Neural Cell Adhesion Molecule‐Dependent Neurite Outgrowth in the Developing Cerebellum and Hippocampus
Authors: Walsh Frank S. , Furness Josie , Moore Stephen E. , Ashton Sandra , Doherty Patrick
Published: October 2006

Abstract: The development of the CNS is associated with an increasing use of the 30‐bp variable alternative, spliced exon (VASE) in neural cell adhesion molecule (NCAM). We have assessed the relative usage of VASE by reverse transcriptase‐linked polymerase chain reaction in the developing cerebellum and hippocampus at times when neurons isolated from these tissues can respond to substrate‐associated NCAM by increased axonal growth and also at later developmental stages, when they are no longer responsive to substrate‐associated NCAM. Neurons isolated from the developing cerebellum at postnatal day 6 respond to NCAM with increased neurite growth. NCAM transcripts from these cells were found to have negligible levels of VASE usage. In contrast, neurons that are isolated at later stages of development (postnatal days 8, 10, and 11) and do not respond to NCAM were found to synthesise a much higher proportion of NCAM transcripts containing VASE. In the hippocampus, embryonic day 18 neurons, which are responsive to NCAM, express low levels of VASE, whereas postnatal days 4 and 5 neurons, which are not responsive to NCAM, have a greater proportion of transcripts containing VASE. Thus, the level of NCAM VASE exon usage by neurons appears to be a good indicator of the ability of these cells to respond to non‐VASE‐containing NCAM (expressed in a cellular substratum) by increased neurite outgrowth.
Distinct Mechanisms of Differentiation of SH‐SY5Y Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors
Authors: Leli Ubaldo , Cataldo Anne , Shea Thomas B. , Nixon Ralph A. , Hauser George
Published: October 2006

Abstract: Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH‐SY5Y differentiates when exposed to 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron‐specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF‐H). TPA, 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol, and staurosporine, but not DAG or 4‐O‐methyl‐TPA, caused neurite outgrowth. Neuron‐specific enolase was expressed in cells treated with TPA and 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol but not with DAG, staurosporine, or 4‐O‐methyl‐TPA. NF‐H increased in the perikarya of cells treated with DAG and 4‐O‐methyl‐TPA, in processes and to varying degrees in perikarya of TPA‐ and 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol‐treated cells, but much more in the processes than in the perikarya of staurosporine‐differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH‐SY5Y cells by different mechanisms, and that the five‐membered/seven‐membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.
Accumulation of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine in Cultured Cerebellar Astrocytes
Authors: Marini A. M. , Lipsky R. H. , Schwartz J. P. , Kopin I. J.
Published: October 2006

Abstract: Cultured cerebellar astrocytes rapidly accumulate 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) from the incubation medium, reaching a plateau within 10 min, whereas within that time negligible amounts of 1‐methyl‐4‐phenylpyridinium (MPP+) have entered the astrocytes. MPTP accumulation is essentially independent of temperature and is proportional to extracellular concentration at steady state: The steady‐state concentration achieved within these cells is about 50‐fold higher at relatively low extracellular concentrations. MPTP appears to accumulate intracellularly within lysosomes, because lysosomotropic agents such as ammonium chloride and chloroquine markedly diminish the accumulation. Moreover, a proton gradient is required, because MPTP accumulation is abolished by the hydrogen ion antiporter monensin. Over an interval of several days, MPTP is converted to MPP+ intracellularly, with a concomitant decrease in medium MPTP and increase in medium MPP+. A constant, small but significant amount of MPP+ is retained intracellularly over a 72‐h interval. Increasing the medium MPTP concentrations results in increased conversion of MPTP and enhanced intracellular retention of MPTP and MPP+. Neither MPTP nor MPP+ is neurotoxic to cultured cerebellar astrocytes as determined by cell counts and rate of conversion of MPTP to MPP+.
Stimulation of the P2Y Purinergic Receptor on Type 1 Astroglia Results in Inositol Phosphate Formation and Calcium Mobilization
Authors: Helen C. , Kastritsis C. , Salm A. K. , McCarthy Ken
Published: October 2006

Abstract: Cultured astroglia express purinergic receptors that initiate phosphoinositide metabolism and calcium mobilization. Experiments were conducted to characterize the purinergic receptor subtype on type 1 astroglia responsible for stimulating these second‐messenger systems. Inositol phosphate (IP) accumulation and calcium mobilization were measured after stimulation with ATP or purinergic receptor subtype‐selective ATP analogues. ATP (10–5M) increased IP accumulation severalfold. Dose–effect assays monitoring astroglial IP accumulation revealed the order of potency that defines the P2Y receptor: 2‐methylthioadenosine 5′‐triphosphate > ATP > αβ‐methyleneadenosine 5′‐triphosphate > βγ‐methyleneadenosine 5′‐triphosphate. The influence of ATP on intracellular calcium levels in individual type 1 astroglia was examined using the calcium indicator dye, fura‐2. Dose–effect experiments indicated that ATP was equally potent for generating inositol phosphates and increasing cellular calcium. The most prevalent response (87% of total responses) to ATP consisted of a rapid increase in calcium to a peak level that was approximately five times greater than the prestimulation level. This peak was followed by a decline to a plateau level that was significantly above baseline. This plateau phase of the calcium increase was maintained for at least 5 min in the presence of ATP and was dependent on external calcium. Many (23%) astroglia exhibited spontaneous calcium oscillations whose frequency and magnitude increased after the addition of 10–5M ATP. Immunocytochemical staining indicated that the responses occurred in glial fibrillary acidic protein positive cells. We conclude that type 1 astroglia express the P2Y purinergic receptor which regulates IP production and calcium mobilization.
First Characterization of 6‐Hydroxytryptamine in the Rat Midbrain by Using Specific Antibodies
Authors: Dabadie H. , Geffard M. , Charrier M. C. , Locuratolo D. , Berrier C. , Jacquesy J. C.
Published: October 2006

Abstract: The visualization of serotonin, 5‐methoxytryptamine, and tryptamine in the rat midbrain has been made possible by the development of antibodies raised against these conjugated molecules. It has been suggested that 6‐hydroxytryptamine (6‐HT) might also be a neurotransmitter in this region. To test this hypothesis, 6‐HT was synthesized and antibodies were raised in the rabbit. The high avidity (IC50= 5 × 10–9M) and specificity [cross‐reactivity ratio between 6‐HT–glutaraldehyde (G)–bovine serum albumin (BSA) and 5‐HT–G–BSA, the most immunoreactive compound, was 1,500] rendered these antibodies reliable tools for specific molecular detection of 6‐HT in the G‐fixed tissues. In the dopaminergic region, 6‐HT immunoreactivity was noted in the substantia nigra but was particularly intense in the red nuclei, where it seems to be localized in the magnocellular division in the form of large 6‐HT neurons. In contrast, there were few 6‐HT neurons in the raphe nuclei. Thus, 6‐HT may be a new putative neurotransmitter existing in the red nuclei, in addition to the other neurotransmitters already described in this region, in the nigrorubral pathway, and in the rubral projection from the dorsal raphe nuclei. 6‐HT is possibly implicated in motor control and might exert hallucinogenic properties as do other 6‐hydroxylated indoleamines.
Facilitated Transport of the Neurotoxin, β‐N‐Methylamino‐l‐Alanine, Across the Blood‐Brain Barrier
Authors: Smith Quentin R. , Nagura Hiroshi , Takada Yoshiaki , Duncan Mark W.
Published: October 2006

Abstract:β‐N‐Methylamino‐l‐alanine (BMAA) is a neuro‐toxic plant amino acid that has been implicated in the pathogenesis of the high incidence amyotrophic lateral sclerosis and related parkinsonism dementia of the western Pacific. Previous studies have demonstrated that BMAA is taken up into brain following intravenous or oral administration. To examine the kinetics and mechanism of brain transfer, BMAA influx across the blood–brain barrier was measured in rats using an in situ brain perfusion technique. BMAA influx was found to be saturable with a maximal transfer rate (Vmax) of 1.6 ± 0.3 × 10–3μmol/s/g and a half‐saturation constant (Km) of 2.9 ± 0.7 mMbased on total perfusate BMAA concentration. Uptake was sodium independent and inhibitable by excessl‐leucine, but not byl‐lysine,l‐glutamate, or methylaminoisobutyric acid, indicative of transfer by the cerebrovascular large neutral amino acid carrier.l‐BMAA competitively reduced brain influx ofl‐[14C]leucine, as expected for cross‐inhibition. The results demonstrate that BMAA is taken up into brain by the large neutral amino acid camer of the blood‐brain bamer and suggest that uptake may be sensitive to the same factors that affect neutral amino acid transport, such as diet, metabolism, disease, and age.
Nuclear Sialyl Cholesterol Causes Changes in the Structure of Chromatin and Its Transcription Level Followed by the Promotion of Neuritogenesis
Authors: Yamashita Tatsuro , Tsuji Shuichi
Published: October 2006

Abstract: Exogenously added sialyl cholesterol (SC) induced neuritogenesis and accumulated in the nuclear fraction of a mouse neuroblastoma cell line, Neuro2a, as reported elsewhere. To clarify the significance of nuclear SC, we examined the structure of chromatin and its transcriptional level. The chromatin of SC‐treated cells exhibits greater circular di‐chroism amplitude (260–310 nm) than that of nontreated cells. The transcriptional level of chromatin from SC‐treated cells was significantly higher than that of nontreated cells. The chromatin structural change has a good correlation to the transcriptional enhancement. On the addition of SC to the chromatin of nontreated cells, the circular dichroism spectra increased in amplitude and the transcriptional level also increased in a concentration‐dependent manner. α‐Amanitin‐treated cells did not show SC‐dependent neuritogenesis. These results strongly suggest that nuclear SC affects gene expression, which results in promotion of the neuritogenesis of cells.
Identification of Elastase as a Secretory Protease from Cultured Rat Microglia
Authors: Nakajima Kazuyuki , Shimojo Masato , Hamanoue Makoto , Ishiura Shoichi , Sugita Hideo , Kohsaka Shinichi
Published: October 2006

Abstract: In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7‐8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 μM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N‐methoxysuccinyl‐Ala‐Ala‐Pro‐Val‐methyl chloride, eluted at a position corresponding to a molecular mass of ∼ 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase‐like protease. This suggestion was confirmed by the finding that the 25‐kDa protein band was stained with anti‐elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopoly‐saccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.
D1 Dopamine Receptor Activation of Multiple Transcription Factor Genes in Rat Striatum
Authors: Cole Andrew J. , Bhat Ratan V. , Patt Cary , Worley Paul F. , Baraban Jay M.
Published: October 2006

Abstract: Recent studies have shown that dopamine receptor agonists induce expression of Fos‐like immunoreactivity in rat striatal neurons. The protooncogene c‐fos belongs to a family of immediate early genes that are rapidly induced in fibroblasts by growth factors. In light of previous findings that several immediate early gene mRNAs that encode proven or putative transcription factors are differentially regulated by neuronal stimulation in vivo, we have examined the effect of dopaminergic agents on mRNA levels of several such genes using in situ hybridization and northern blot analysis. d‐Amphetamine (2.5‐10 mg/kg i.p.) causes a rapid but transient dose‐dependent increase in zif268 and jun‐B mRNA levels in striatum that was abolished by striatal 6‐hydroxydopamine lesions or by pretreatment with the specific D1 receptor antagonist SCH‐23390 but not by specific D2 receptor antagonists. Apomorphine, a dopamine agonist that acts at both D1 and D2 receptors, and SKF‐38393, a specific D1 receptor agonist, produce similar mRNA changes in rats pretreated with either 6‐hydroxydopamine or reserpine, whereas LY‐171,555, a specific D2 receptor agonist, has no effect. Direct dopamine agonist effects on these immediate early gene mRNA levels are also blocked by D1 but not by D2 antagonists. We observed similar, although less robust, changes in c‐fos and fos‐B mRNA levels. These results demonstrate that striatal D1 dopamine receptors are coupled to activation of multiple transcription factor genes, including zif268 and jun‐B as well as members of the fos family.
Decrease of Extracellular Taurine in the Rat Dorsal Hippocampus After Central Nervous Administration of Vasopressin
Authors: Brust Peter , Christensen Thomas , Diemer Nils Henrik
Published: October 2006

Abstract: The extracellular amino acid concentrations in the left and right dorsal hippocampus of male rats were studied before and during application of vasopressin into the right hippocampus. The method of intracerebral microdialysis was used for both arginine vasopressin administration and monitoring of the composition of the extracellular fluid. The concentrations of 16 amino acids were measured by HPLC in the perfusate samples. The level of taurine declined 20% in the right hippocampus during perfusion with vasopressin, whereas o‐phosphoethanolamine decreased in both sides, the left 20% and the right 24%. These alterations may be related to cerebral osmoregulation. Also, the levels of tyrosine and phenylalanine increased 15% and 35%, respectively, during administration of vasopressin. No changes of other amino acids were observed.
Entry of Newly Synthesized Gangliosides into Myelin
Authors: Farrer Robert G. , Benjamins Joyce A.
Published: October 2006

Abstract: Ganglioside synthesis and transport to myelin was studied in brainstem slices prepared from 19–21‐day‐old rats. The slices were incubated for up to 2 h in the presence of [3H]glucosamine to label primarily the hexosamine portion of complex gangliosides. The amount of radioactivity incorporated into gangliosides during slice incubations was only 10–15% of the amount of the label incorporated during in vivo labeling of brainstem gangliosides using equivalent amounts of [3H]glucosamine. Among individual gangliosides this inhibition was greater for the more complex gangliosides. When labeled gangliosides were isolated from homogenate and myelin fractions prepared from brain slices, the complex total gangliosides of both fractions showed a lag in labeling kinetics but with a lower specific radioactivity for the myelin fraction, reflecting the larger pool size and slower turnover rate exhibited by myelin components. Chase experiments showed that more complex gangliosides in homogenate exhibited almost no effect of chase after 30 min. Addition of the Golgi‐disrupting agent monensin to slice incubations inhibited the labeling of all gangliosides except GM3, GM2, and GD3, and transport to myelin of all complex gangliosides except GM2. These results show that a monensin‐sensitive mode of transport is responsible for the translocation of most newly synthesized gangliosides into myelin.
Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures
Authors: Brosh Sara , Sperling Oded , Dantziger Esther , Sidi Yechezkel
Published: October 2006

Abstract: The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8‐Aminoguanosine (8‐AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP → IMP → XMP → GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of ∼8.5–13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8‐AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs. The incorporation of hypoxanthine into GuRNs and of guanine into IMP (and inosine) indicates that the two arms of the guanine nucleotide cycle are active, but the actual rate of activity of this cycle and its metabolic importance are yet uncertain.