bluuebunny/crossref_metadata_2025 huggingface dataset:
Galactose‐1‐Phosphatase in Rat Brain
Authors: Gulavita Srima J. , Zhang Li‐Ping , Dougherty John J. , Dain Joel A.
Published: October 2006

A prominent galactose‐1‐phosphatase was isolated from rat brain and partially purified by chromatography on diethylaminoethyl‐Sephacel, hydroxylapatite, and Sephacryl S‐300 columns. The galactose‐1‐phosphatase was separated from alkaline phosphatase, and from two forms of glucose‐1‐phosphatase. The three columns gave a 10‐fold increase in specific activity to 290 mol/min/mg of protein, with a yield of 15%. Of the eight sugar phosphates tested, galactose‐1‐phosphate was the best substrate for the purified enzyme, followed by glucose‐1‐phosphate, which was hydrolyzed 40% as rapidly as galactose‐1‐phosphate. Galactose‐1‐phosphatase had an optimum pH of 8.5 and a Km value of 2.5 mM for galactose‐1‐phosphate hydrolysis. Mg2+ was required for activity, and supported half‐maximal activity at a concentration of 1.25 mM. Phosphate was the only potent inhibitor found. ATP, arsenate, and vanadate caused moderate inhibition of 10 mM levels, whereas AMP, L‐homoarginine, and L‐phenylalanine stimulated enzyme activity. Galactose‐1‐phosphatase was determined to have a Stokes radius of 30 A and a sedimentation coefficient of 4.IS. These values were used to calculate a molecular weight of 50,200 and a factional ratio showing the enzyme to be a globular protein. It is hypothesized that a similar phosphatase may play a role in reducing brain galactose‐1 ‐phosphate concentrations in patients with galactosemia.
Tyrosine Hydroxylase mRNA Expression by Dopaminergic Neurons in Culture: Effect of 1 ‐Methyl‐4‐Phenylpyridinium Treatment
Authors: Beck Klaus D. , Knusel Beat , Pasinetti Giulio , Michel Patrick P. , Zawadzka Halina , Goldstein Menek , Hefti Franz
Published: October 2006

To enable us to study expression of tyrosine hydroxylase [TH; tyrosine 3‐monooxygenase; L‐tyrosine tetrahydropteridine: oxygen oxidoreductase (3‐hydroxylating); EC 1.14.16.2] as a measure of dopaminergic neuron function in future experiments, methods were developed to quantify TH mRNA levels in cultures of dopaminergic mesencephalic cells. The model of selective dopaminergic toxicity of 1‐methyl‐4‐phenylpyridinium (MPP+) was used to verify the specificity of our methods. Fetal (embryonic day 15) rat ventral mesencephalic cell cultures were treated with 15 μMMPP+ for 48 h, conditions previously shown to reduce the number of TH‐immunoreactive neurons, TH activity, and dopamine uptake to 5–10% of control values. This treatment decreased the number of neurons labeled by TH in situ hybridization to 9% of untreated controls and caused a strong reduction of the abundance of TH mRNA in Northern blots. Our findings establish TH mRNA expression as a parameter for future studies of toxic and trophic effects on cultured dopaminergic neurons, and they support the view that MPP+ destroys dopaminergic neurons.
Iminodipropionitrile‐Induced Dyskinesia in Mice: Striatal Calcium Channel Changes and Sensitivity to Calcium Channel Antagonists
Authors: Bangalore Ramesh , Hawthorn Mark , Triggle David J.
Published: October 2006

Administration of 3, 3′‐iminodipropionitrile (IDPN) (1g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN‐induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200–110 binding sites (Bmax) in whole brains of IDPN‐treated mice. Similarly, the K+‐depolarization‐dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 ± 7 fmol/mg to 237 ± 31 fmol/mg in control and treated groups, respectively) of PN 200–110 binding to the striatum without change of KD value (38 ± 4.7 pM versus 33 ± 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 ± 10.1 pM to 45 ± 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 ± 51 fmol/mg versus 485 ± 41 fmol/mg) of PN 200–110 binding to the cortex. IDPN did not alter ω‐conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment are inhibited by L‐type calcium channel antagonists and this may be associated with the observed increase in striatal L‐type calcium channels.
Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases
Authors: Mitsui Kazuhiko , Tsuji Shuichi , Yamazaki Mitsuo , Nagai Yoshitaka
Published: October 2006

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro‐2a (a murine neuroblastoma cell line) showed dose‐dependent (1 μM‐1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro‐2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple‐neurite‐bearing)‐type neuritogenesis. A similar type of neurite outgrowth was induce by 8‐bromo‐cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro‐2a cells were treated with retinoic acid, neurite formation was of the monopolar (single‐neurite‐bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar‐type cell shape change in Neuro‐2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.
Nerve Growth Factor Potentiates Bradykinin‐Induced Calcium Influx and Release in PC12 Cells
Authors: Bush Andrew B. , Borden Laurence A. , Greene Lloyd A. , Maxfield Frederick R.
Published: October 2006

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin‐induced calcium increases in PC12 cells. Short‐term (1h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10‐fold, whereas long‐term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100‐fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short‐term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long‐term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short‐term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1, 4, 5‐trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.
Presence of Calmodulin and Calmodulin‐Binding Proteins in the Nuclei of Brain Cells
Authors: Vendrell Montserrat , Aligué Rosa , Bachs Oriol , Serratosa Joan
Published: October 2006

The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 γg/ mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin‐binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of a‐spectrin and a 62‐kDa calmodulin‐binding protein (p62) in the nuclei of neurons and glial cells by irnmunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by irnmunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.
Stimulatory and Inhibitory Effects of N‐Methyl‐D‐Aspartate on 3H‐Inositol Polyphosphate Accumulation in Rat Cortical Slices
Authors: Baird John G. , Nahorski Stefan R.
Published: October 2006

The actions of the excitatory amino acid N‐methyl‐D‐aspartate (NMDA) on the accumulation of 3H‐inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose‐dependent accumulation of only [3HJinositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and (3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1, 3, 4, 5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol‐induced accumulation of 3H‐inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 γM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 γMNMDA on carbachol responses. These studies, therefore, reveal Ca2+‐dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.
Modulation of Proteolytic Activity During Neuritogenesis in the PC12 Nerve Cell: Differential Control of Plasminogen Activator and Plasminogen Activator Inhibitor Activities by Nerve Growth Factor and Dibutyryl‐Cyclic AMP
Authors: Leprince P. , Register B. , Delrée P. , Rigo J. M. , Andre B. , Moonen G.
Published: October 2006

Extracellular proteolysis is considered to be required during neuritic outgrowth to control the adhesiveness between the growing neurite membrane and extracellular matrix proteins. In this work, PC12 nerve cells were used to study the modulation of proteolytic activity during neuronal differentiation. PC12 cells were found to contain and release a 70–75‐kDa tissue‐type plasminogen activator (tPA) and a much less abundant 48‐kDa urokinase‐type plasminogen activator. A plasminogen activator inhibitor (PAI) activity with molecular sizes of 54 and 58 kDa was also detected in PC12 cell conditioned medium and formed high‐molecular‐mass complexes with released tPA. Release of PAI activity was dependent on treatment with nerve growth factor (NGF), whereas tPA synthesis and release were under control of a cyclic AMP‐ dependent mechanism and increased on treatment with dibutyryl‐cyclic AMP [(But)2cAMP] or cholera toxin. Simultaneous treatment with NGF and (But)2cAMP resulted in increases of both tPA and PAI release and enhancement of tPA‐PAI complex formation. The resulting plasminogen activator activity in conditioned medium was high in (But)2cAMP‐treated cultures with short neuritic outgrowth but remained low in NGF‐ or NGF plus (But)2 cAMP‐treated cultures, where neurite extension was, respectively, large and very large. These results suggest that excess proteolytic activity may be detrimental to neuritic outgrowth and that not only PAI release but also tPA‐PAI complex formation is associated with production of large and stable neuritic outgrowth. This can be understood as an involvement of PAI in the protection against neurite‐destabilizing proteolytic activity.
Guanine Nucleotides Regulate 2‐[125I]Iodomelatonin Binding Sites in Chick Retinal Pigment Epithelium but Not in Neuronal Retina
Authors: Chong Nelson W. S. , Sugden David
Published: October 2006

The characteristics of the binding sites labeled by the radioligand 2‐[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2‐[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 ± 55 pMand a total number of binding sites (8max) of 25.4 ± 2.2 fmol/ mg of protein for neuronal retina. For RPE the KD was 34.1 ±2.2 pM and the Bmax 59.5 ± 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2‐iodomelatonin < 2‐chloromelatonin > melatonin > 6‐chloromelatonin > 6‐hydroxymelatonin > N‐acetylserotonin > 6‐methoxyharmalan > 5‐hydroxytryptamine. Linear regression of log Kivalues from neuronal retina and RPE gave a highly significant dorrelation (r =0.994, n = 8; p <0.001). GTP inhibited specific binding to RPE membranes in a concentration‐dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide‐binding regulatory protein (G protein), but that in neuronal retina is not.
Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain
Authors: Siemens I. R. , Swanson G. N. , Fluharty S. J. , Harding J. W.
Published: October 2006

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30–40% using the synthetic detergent 3[(3‐cholamidopropyl)dimethylammonio)]‐1 ‐propane‐sulfonate. Kinetic analysis employing the high‐affinity antagonist I25I‐Sar1, lle8‐Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r −0.99) between the respective pIC50values of a series of agonist and antagonists competing for 125I‐Sarl,IIe8‐Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I‐ Ang II to solubilized receptors with the homo‐bifunctional cross‐linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross‐linking of I25l‐Ang II to solubilized brain membranes followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I‐Sar1.IIe8‐Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isodectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.
Angiotensin II Increases Catecholamine Release from Bovine Adrenal Medulla but Does Not Enhance That Evoked by K+ Depolarization or by Carbachol
Authors: Powis D. A. , O'Brien K. J.
Published: October 2006

: The effect of angiotensin II on catecholamine release from bovine adrenal medulla has been investigated. In retrogradely perfused, isolated bovine adrenal glands, angiotensin II increased basal efflux of catecholamines, but the presence of angiotensin II did not increase the release of catecholamines evoked either by bolus injections of the secretogogue carbachol or by depolarization with a perfusing solution containing a raised concentration of K+. In chromaffin cells maintained in primary tissue culture, angiotensin II increased 3H release from cells preloaded with [3H]‐noradrenaline but did not enhance the release evoked by carbachol or by depolarization with K+. The increase in 3H release evoked by angiotensin II from chromaffin cells in tissue culture was inhibited by its analogue antagonist Sar1,Ala8‐angiotensin II (saralasin) and was entirely dependent on the presence of Ca2+ in the experimental medium. These findings suggest that, in the chromaffin cells of the bovine adrenal medulla, angiotensin II acts on specific receptors to cause a calcium‐dependent catecholamine release but triggers no additional response that acts synergistically with depolarizing or nicotinic stimuli to augment catecholamine release.
Age‐Dependent Changes in the Subcellular Distribution of Rat Brain μ‐Opioid Receptors and GTP Binding Regulatory Proteins
Authors: Bem Wojciech T. , Yeung S. Jim , Belcheva Mariana , Barg Jacob , Coscia Carmine J.
Published: October 2006

: The relative subcellular distributions of μ‐opioid receptors and guanine nucleotide binding regulatory proteins (G proteins) in 1‐day‐old (P1) and adult rat forebrain were compared. Light membranes (LMs) were resolved from heavy membranes (HM) by sucrose density gradient centrifugation. Marker enzyme analyses indicated that LMs contained most of the endoplasmic reticulum and Golgi complexes, whereas HMs were enriched in plasma membranes. Binding distribution and properties of μ‐opioid sites were assessed using [3H][d‐Ala2,Me‐Phe4,Gly‐ol5]enkephalin. P1 LMs possessed 43% of die total μ‐opioid binding detected compared to 16% in the adult. Although NaCl inhibited μ binding in LMs to a greater extent than in HMs, age‐dependent differences were not observed. P1 LM μ binding possessed greater sensitivity to 5′‐guanylylimidodiphosphate than their adult counterpart. Moreover, P1 LMs contained more Gocr. protein than P1 HMs or adult LMs, as demonstrated by immunoblotting with antisera against Gocr after one‐or two‐dimensional gel electrophoresis. These results suggest that P1 LMs contain a greater proportion of newly synthesized intracellular μ sites than adult LMs.
In Vitro Release of Endogenous Dopamine from the Striatum of the Weaver Mutant Mouse
Authors: Simon J. R. , Yu H. , Richter J. A. , Vasko M. R. , Ghetti B.
Published: October 2006

: The weaver mutant mouse has a genetically determined defect in the nigrostriatal dopaminergic system. The present study was undertaken to test the hypothesis that in the weaver mutant mouse, striatal nerve terminals undergo compensatory changes in response to this deficiency. To test this hypothesis, we studied the basal and stimulated release of dopamine from striatal slices of weaver mutant mice and matched controls. By using a superfusion system and concentrating the superfusate by passage over alumina, resting dopamine release could be determined in the weaver mutant despite the fact that striatal tissue content of dopamine in these mice is reduced by >75% compared with control mice. Fractional resting release of dopamine in weaver striatal slices was significantly elevated compared with that in controls, suggesting that the release mechanisms in the weaver may be adapting to overcome the dopamine deficit. Potassium‐evoked release (24 and 48 mM potassium) was not significantly different between the two genotypes. In contrast, amphetamine‐evoked release (1 μM) was significantly greater in the weaver mice than in controls. In both genotypes, release evoked by amphetamine was completely inhibited by cocaine, implicating the dopamine uptake carrier in this release process. These findings suggest that fundamental differences in dopamine release mechanisms exist between weaver and control mice and support the hypothesis that compensatory mechanisms may develop in neurons in response to dopamine deficits.
Metabolism of Extracellular Adenine Nucleotides by Cultured Rat Brain Astrocytes
Authors: Lai Kam‐Ming , Wong Patrick C. L.
Published: October 2006

: Intact astrocytes cultured from newborn rat cerebral cortex rapidly converted extracellular ATP to ADP. The ATPase responsible was apparently not saturated, even at 750 μM ATP. In contrast, the conversion of ADP to AMP was slow, and the reaction was limiting for the subsequent dephosphorylation process. Adenosine formation was the only fate for AMP. The reaction was catalyzed by S'‐nucleotidase with an apparent Km of 55 μM for AMP and appeared to be inhibited by high concentrations of ATP and ADP.

Astrocytes were able to take up adenosine with an apparent Km value of 45 μM. Uptake was inhibited by dipyridamole but not by anti‐5′‐nucleotidase IgG. The results support the proposal that astrocytes play a role in modulating synaptic events involving ATP and adenosine.
Effect of Loading Doses of l‐Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain
Authors: Smith C. Beebe , Sun Y. , Deibler G. E. , Sokoloff L.
Published: October 2006

: “Flooding” amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and “flooded” (10‐28 mM) by comparing the steady‐state specific activity of the tRNA‐bound valine with that of the plasma valine. Under normal and “flooding” conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; “flooding” with a plasma level of 28 mM raised the brain acid‐soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than “flooding” with a plasma level of 17 mM valine, which raised the brain acid‐soluble level only to 2.3 mM. The results of these studies show that “flooding” amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.
Proteolysis of Microtubule‐Associated Protein 2 and Tubulin by Cathepsin D
Authors: Johnson Gail V. W. , Litersky Joel M. , Whitaker John N.
Published: October 2006

: The in vitro degradation of microtubule‐associated protein 2 (MAP‐2) and tubulin by the lysosomal aspartyl endopeptidase cathepsin D was studied. MAP‐2 was very sensitive to cathepsin D‐induced hydrolysis in a relatively broad, acidic pH range (3.0–5.0). However, at a pH value of 5.5, cathepsin D‐mediated hydrolysis of MAP‐2 was significantly reduced and at pH 6.0 only a small amount of MAP‐2 was degraded at 60 min. Interestingly, the two electro‐phoretic forms of MAP‐2 showed different sensitivities to cathepsin D‐induced degradation, with MAP‐2b being significantly more resistant to hydrolysis than MAP‐2a. To our knowledge, this is the first clear demonstration that MAP‐2 is a substrate in vitro for cathepsin D. In contrast to MAP‐2, tubulin was relatively resistant to cathepsin D‐induced hydrolysis. At pH 3.5 and an enzyme‐to‐substrate ratio of 1: 20, only 35% of the tubulin was degraded by cathepsin D at 60 min. The cathepsin D‐mediated hydrolysis of tubulin was optimal only at pH 4.5. These results demonstrate that MAP‐2 and tubulin are unequally susceptible to degradation by cathepsin D. These data also imply a potential for rapid degradation of MAP‐2 in vivo by cathepsin D either in lysosomes or perhaps autophagic vacuoles of the neuron.
Ultrastructural Changes Induced by 12‐O‐Tetradecanoylphorbol 13‐Acetate in Pure Cholinergic Synaptosomes of Torpedo Electric Organ
Authors: Egea Gustavo , Guitart Xavier , Marsal Jordi
Published: October 2006

: We have studied the morphological changes induced by the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze‐fracture techniques We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acctylcholme release, did not change the density of mtramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcrioline release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.
Genomic Organization of Drosophila Choline Acetyltransferase
Authors: Sugihara Hidemitsu , Andrisani Veronica , Salvaterra Paul M.
Published: October 2006

: Choline acetyltransferase (ChAT; acetyl‐CoA:cho‐line‐O‐acetyltransferase; EC 2.3.1.6) is the enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is thus the genetic determinant of neurons with a cholinergic phenotype. We have screened a Drosophila genomic library using a cRNA probe, transcribed from Drosophila ChAT cDNA, and isolated three independent clones representing all the exons of this gene. The gene spans more than 26 kb of DNA and is organized into eight exons containing 594, 80,192, 759, 408, 147, 201, and 1,612 nucleotides. All inserts that hybridized with a cRNA probe have been subcloned and the sequence of intron/exon boundaries determined. The only part of the ChAT gene not represented in our clones is a part of the first intron. A minimum size for this uncloned DNA has been deduced from Southern analysis of Drosophila genomic DNA. We also have probed the transcripts of the ChAT gene by northern analysis of total Drosophila RNA using two different exon‐specific antisense RNA probes. An exon I probe detected two bands of RNA whereas an exon VHI probe hybridized with only the smaller band, previously identified as ChAT mRNA. These results indicate a complex transcription pattern for the ChAT locus in Drosophila.
Specific Brain Protein Changes Correlated with Behaviourally Effective Brain Transplants
Authors: Wets Kathleen M. , Sinden John , Hodges Helen , Allen Yvonne , Marchbanks R. M.
Published: October 2006

: The objective of this study was to identify cellular proteins that are associated with foetal brain transplants effective in reinstating memory function in adult rats with brain lesions. Quantitative memory deficits can be created in rats by lesioning the cholinergic projection system, using ibotenic acid. Previous work suggested that injection of cell suspensions prepared from presumptive cholinergic cells of foetal basal forebrain into adult brain, after such lesions, are most effective in restoring cognitive function. It was not clear, however, whether it was the cholinergic nature of the transplants that was critical for their success or whether other factors were involved. In this study, the proteins present in transplanted tissues and control brains were analysed by two‐dimensional polyacrylamide gel electrophoresis to identify markers for the cells that were specifically correlated with restoration of cognitive function. On each gel, the relative optical densities of the same 33 selected proteins were measured on an interactive computerised image analyser. The amount of each protein was compared between treatment groups and correlated with four behavioural measurements. Seven of the proteins analysed had levels of expression that were either related to transplantation or correlated with behavioural performance. The proteins of interest were divided into the following three groups: (1) transplant‐related proteins, (2) cholinergic transplant‐specific proteins, and (3) behaviour‐related proteins. Notable among the proteins of interest was one of the cholinergic transplant‐specific proteins that was positively correlated with three of the four behavioural measurements and was also the only protein among those analysed that was significantly correlated with choline acetyltransferase (ChAT) levels. This has been identified, by immunoblotting, as glial fibrillary acidic protein, an astrocytic cell marker. These results suggest, therefore, that at least two cell types, astrocytes and ChAT+‐staining cells, play an important role in the successful recovery of cognitive function. This study also identified possible protein markers for cognitive performance. The level of expression of two of the proteins analysed was not affected by lesioning or transplantation, but was significantly correlated with behaviour. One of these proteins, whose amounts correlated negatively with behavioural measurements, has been identified as neurone‐specific enolase, a brain‐specific neuronal cell marker.
Conservation of Docosahexaenoic Acid in Rod Outer Segments of Rat Retina During n‐3 and n‐6 Fatty Acid Deficiency
Authors: Wiegand Rex D. , Koutz Cynthia A. , Stinson Ann M. , Anderson Robert E.
Published: October 2006

: We investigated the mechanism by which rat retina conserves docosahexaenoic acid during essential fatty acid deficiency. Weanling female albino rats were fed diets containing either 10% by weight hydrogenated coconut oil, safflower oil, or linseed oil for 15 weeks. Plasma and rod outer segment (ROS) membranes were prepared for fatty acid and phospholipid molecular species analysis. In addition, retinas were removed for morphometric analysis. We found the following: (1) Plasma phospholipids and cholesterol esters from coconut oil, safflower oil, and linseed oil diet groups were enriched in 20:3(n‐9), 20:4(n‐6), and 20:5(n‐3), respectively. The levels of these 20‐carbon fatty acids in the ROS, however, were only slightly affected by diet. (2) The fatty acids and molecular species of ROS phospholipids from the safflower oil and coconut oil groups showed a selective replacement of 22:6(n‐3) with 22:5(n‐6), as evidenced by a reduction of the 22:6(n‐3)‐22:6(n‐3) molecular species and an increase in the 22:5(n‐6)‐22:6(n‐3) species. (3) The renewal rate of ROS integral proteins, determined by autoradiography, was 10% per day for each diet group. (4) Morphometric analysis of retinas showed no differences in the outer nuclear layer area or in ROS length between the three groups. We conclude that the conservation of 22:6(n‐3) in ROS is not accomplished through reductions in the rate of membrane turnover, the total amount of ROS membranes, or in the number of rod cells. The retina may conserve 22:6(n‐3) through recycling within the retina or between the retina and the pigment epithelium, or through the selective uptake of 22‐carbon poly‐unsaturated fatty acids from the circulation.
Interactions of Glutamate Receptor Agonists Coupled to Changes in Intracellular Ca2+ in Rat Cerebellar Granule Cells in Primary Culture
Authors: Holopainen Irma , Louve Michael , Åkerman Karl E. O.
Published: October 2006

: Changes in cytosolic free Ca2+ concentrations in response to glutamate receptor agonists and their interactions were studied in rat cerebellar granule cells grown on cover‐slips. The intracellular Ca2+ as measured with fura‐2 increased by applying kainate (KA), quisqualate (QU), and N‐methyl‐d‐aspartate (NMDA). The effect of KA could not be blocked by the NMDA receptor blocker 2‐amino‐5‐phosphonovalenc acid (APS). The KA‐and QU‐induced increase in intracellular free Ca2+ was also observed in a Na+‐free medium, indicating that this response is not secondarily due to the depolarization. The effect of 10 μM QU on the KA‐induced changes in cytosolic free Ca2+ was additive only at low KA concentrations, but QU at 0.1 mM totally blocked the response to KA. In the presence of 10 μM KA, the dose‐response curve of QU became biphasic, whereas with 50 μM KA, a reduction of the response was seen around 1–100 μM QU. The effect of NMDA on the QU‐induced response was additive only at low QU concentrations. It is proposed that rat cerebellar granule cells in primary culture express separate receptor‐channel complexes for NMDA, QU, and KA, but interactions between agonists for these receptor sites exist. Thus, QU when present at intermediate concentrations seems to interact with the KA type of receptor, causing its desensitization. At high QU concentrations, an interaction of QU with the NMDA receptor site is apparent.
The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain
Authors: Hallak Marta E. , Bongiovanni Guillermina , Barra Héctor S.
Published: October 2006

: The posttranslational incorporation of arginine into proteins catalyzed by arginyl‐tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.
Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor
Authors: Brown Alice B. , Carpenter Graham
Published: October 2006

: PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I‐epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I‐epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I‐epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I‐epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high‐and low‐affinity 125I‐epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.
Effects of Chronic and Subchronic Nicotine on Tyrosine Hydroxylase Activity in Noradrenergic and Dopaminergic Neurones in the Rat Brain
Authors: Smith Katherine M. , Mitchell Stephen N. , Joseph Michael H.
Published: October 2006

: Chronic nicotine (0.8 mg/kg by daily subcutaneous injection) over a 7 to 28‐day period was found to increase the activity of tyrosine hydroxylase in predominantly noradrenergically innervated regions but not in dopaminergic projection areas. Increases in tyrosine hydroxylase activity were observed in dopaminergic cell body regions only after nicotine treatment for 3 to 5 days. The increase in tyrosine hydroxylase activity in noradrenergic neurones was evident first in the cell bodies in the locus coeruleus from 3 to 7 days, reaching 223% of control activities, and was followed by increases of up to 205% in the terminals up to 3 weeks later. It was then established that nicotine for 7 days was sufficient to increase the activity of the enzyme to the same extent in the terminals at 21 days even without further nicotine administration. This is consistent with axonal transport preceded by induction of the enzyme in noradrenergic cell bodies, whereas “delayed activation” might account for the transient effect seen in dopaminergic cell body regions. The response in the locus coeruleus to nicotine for 7 days was completely blocked by daily preinjection with mecamylamine but not with hexamethonium, which is consistent with the effect of nicotine on tyrosine hydroxylase being mediated by central nicotinic receptors.
Regulation of Five Tubulin Isotypes by Thyroid Hormone During Brain Development
Authors: Aniello Francesco , Couchie Dominique , Gripois Daniel , Nunez Jacques
Published: October 2006

: Nucleic acid probes derived from the 3’noncoding region of five tubulin cDNAs were used to study the effects of thyroid hormone deficiency on the expression of the mRNAs encoding two α (α1 and α2)‐and three β (β2, β4, and β5)‐tubulin isotypes in the developing cerebral hemispheres and cerebellum. The content of α1, which markedly declines during development in both brain regions, is maintained at high levels in the hypothyroid cerebellum, whereas it is decreased in the cerebral hemispheres. The α2 level also declines during development and is decreased in both regions by thyroid hormone deficiency, but only during the two first postnatal weeks. Thyroid hormone deficiency slightly increases at all stages the β2 level in the cerebellum, whereas a decrease is observed at early stages in the cerebral hemispheres. The ft level seems to be independent of thyroid hormone in the cerebral hemispheres, whereas it decreases at early stages in the hypothyroid cerebellum. Finally, the expression of the brain‐specific β4 isotype is markedly depressed by thyroid hormone deficiency, particularly in the cerebellum. These data suggest that the genes encoding the tubulin isotypes are, directly or not, differently regulated by thyroid hormone during brain development This might contribute to abnormal neurite outgrowth seen in the hypothyroid brain and therefore to impairment in brain functions produced by thyroid hormone deficiency.
Autoradiographic Localization of Dopaminergic and Noradrenergic Receptors in the Bovine Pineal Gland
Authors: Simonneaux V. , Happe H. K. , Ebadi M. , Murrin L. C.
Published: October 2006

: Dopamine and norepinephrine are involved in regulation of melatonin synthesis in the pineal gland. In bovine pineal gland, D1‐and D2‐dopaminergic and α1‐adrenergic receptors have been characterized pharmacologically in several laboratories, while β1‐adrenergic receptors have been studied using physiological techniques. The current study presents a quantitative autoradiographic analysis of these four dopaminergic and noradrenergic receptors in bovine pineal gland. The density order of the receptors is D1 > α1 > D2β1. The Bmax of dopamine D1 receptors is about 5 to 6 times higher than the Bmax for α1adrcnergic receptors and about 20 times higher than the Bmax values for β1adrenergic and D2‐dopaminergic receptors. Dopamine D1 receptors are significantly denser in the pineal cortex than in the medulla. Both dopamine receptors are more concentrated in the distal area than in the proximal area (close to the habenula), whereas both noradrenergic receptors are homogeneously distributed along the longitudinal axis. Only D1‐dopaminergic receptors display a heterogeneous distribution between the superior and the inferior areas, being denser in the inferior area. The observation of a much higher concentration of D1‐dopaminergic receptors relative to the other receptors suggests an important role for dopamine in the regulation of bovine pineal physiology.
Phorbol Ester Pretreatment Desensitizes the Inhibition of Ca2+ Channels Induced by k‐Opiate, α2‐Adrenergic, and Muscarinic Receptor Agonists
Authors: Attali Bernard , Nah Seung‐Yeol , Vogel Zvi
Published: October 2006

: Acute treatment of rat spinal cord‐dorsal root ganglion cocultured neurons with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), a known activator of protein kinase C, inhibited the dihydropyridinc‐sensitive voltage‐dependent 45Ca2+ influx measured in these cells (IC50 of⋍100 nM, 66% inhibition at 1 νM TPA). However, prolonged preincubation (24 h) of the cells with 100 nM TPA followed by extensive washing completely abolished, i.e., desensitized, the capacity of a second application of TPA to inhibit the activity of the voltage‐dependent Ca2+ channels. Moreover, this treatment also abolished the inhibition of Ca2+ influx produced by k‐opiate as well as by α2‐adrenergic and muscarinic receptor agonists. Substantial desensitization was already observed following a 1‐h pretreatment with 100 nMTPA. In contrast to TPA, an inactive phorbol ester (4β‐phorbol 13‐acetate) did not affect the inhibition of the voltage‐dependent Ca2+ influx by these receptor agonists. These results suggest that protein kinase C may have a role in the modulation of Ca2+channels by k‐opiate, α2‐adrenetgic, and muscarinic receptor agonists.
Age‐Dependent Increase in Neuropeptide Y Gene Expression in Rat Adrenal Gland and Specific Brain Areas
Authors: Higuchi Hiroshi , Yokokawa Kiyoshi , Iwasa Atsushi , Yoshida Hiroshi , Miki Naomasa
Published: October 2006

Age‐dependent changes in the expression of neuropeptide Y (NPY) peptides and prepro‐NPY mRNA (NPY mRNA) were studied in rat adrenal gland and brain areas by means of radioimmunoassay, immunohistochemistry, and northern blot analysis. In the adrenal gland, NPY immunoreactívity (NPY‐I) increased by 80‐fold, mainly in the chromaffin cells, during aging (from 7 to 33 weeks old). The increase in NPY‐I was accompanied by a concomitant increase in the content of NPY mRNA (800 bases in size, by 16‐fold) and putative NPY pre‐mRNA, a result suggesting that this increase results from that in NPY gene expression, probably at the level of transcription. In contrast, in some brain areas, such as striatum and medulla oblongata plus pons, NPY‐I decreased in an age‐dependent manner, whereas NPY mRNA abundances in these areas increased by twofold with age (from 7 to 33 weeks old). The opposite changes between NPY and NPY mRNA content in specific brain areas suggested the accelerated turnover/degradation of NPY peptide in the brain areas. Furthermore, β‐actin mRNA abundance did not change in rat adrenal gland and brain areas during aging. Thus, the characteristic age‐related increase in NPY gene expression in rat adrenal gland and some brain areas seems to be important for physiological regulation of some neuronal functions, such as blood pressure, in aged animals.
Synaptic Regulation of Immediate Early Gene Expression in Primary Cultures of Cortical Neurons
Authors: Murphy T. H. , Worley P. F. , Nakabeppu Y. , Christy B. , Gastel J. , Baraban J. M.
Published: October 2006

Abstract: Neuronal stimulation can rapidly activate several immediate early genes that code for transcription factors. We have used primary cortical cultures to study the regulation of four of these genes, c‐fos, c‐jun, jun‐B, and zif268. Im‐munocytochemical studies with antibodies to Jun‐B, c‐Jun, and c‐Fos demonstrate intense staining in the nuclei of a subset of cortical neurons in mature cultures (21–25 days in vitro) but not young cultures (3–7 days in vitro). To assess whether this immunoreactivity may be induced by spontaneous synaptic activity that develops with a similar profile, we examined the effects of agents that reduce this synaptic activity. Tetrodotoxin or N‐methyl‐d‐aspartate receptor antagonists suppress basal immunoreactivity to Jun‐B and c‐Fos, but not c‐Jun, indicating that the basal level of c‐Jun expression is not dependent on electrical activity. Pierotoxin, an agent that increases synaptic excitation indirectly by blocking inhibitory synaptic currents mediated by γ‐aminobutyric acidA receptors, markedly increases the percentage of neurons displaying immunoreactivity to c‐Fos, c‐Jun, Jun‐B, and Zif268. Northern analysis suggests that the increases in immunostaining induced by picrotoxin are secondary to a rapid increase in mRNA for these proteins. These findings provide evidence for rapid transcriptional regulation of immediate early genes in cortical neurons by synaptic activity.
A Fast Axonally Transported Protein of the Frog Sciatic Sensory Axons Undergoes Similar Qualitative Changes During Regeneration In Vitro and In Vivo
Authors: Remgård Pär , Ekström Per A. R. , Edström Anders
Published: October 2006

The adult frog sciatic sensory neurons have been shown to regenerate in vitro. If a crush injury is made at the beginning of culture, regeneration starts after 3.4 days and proceeds at a rate of ∼0.8 mm/day for several days. Two‐dimensional gel electrophoresis was used to study the patterns of radiolabeled, fast axonally transported proteins during the first 7 days of regeneration. Interest was focused on one protein, referred to as rrp31 (regeneration‐related protein 31), which changed in apparent pl from 4.9 to 5.3 when the outgrowth of new fibers started. The change was noticeable 3 days after injury and became prominent during day 5 of culturing. By day 7 the pl changed again, this time toward the original value. The in vitro results were supported by experiments in vivo. In this case the change occurred earlier, with a peak only 3 days after injury, after which the pl decreased. If adenosine at 1 mM was included in the culturing medium, the outgrowth of sensory axons was inhibited in a nontoxic way, and the pl changes of rrp31 were prevented. The temporal nature of the pl changes suggests a role for rrp31 in the initiation of the regeneration process.
Solubilization, Characterization, and Partial Purification of α‐Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid‐, Quisqualate‐, Kainate‐Sensitive l‐Glutamate Binding Sites from Porcine Brain Synaptic Junctions
Authors: Chang Yen‐Chung , Lin Yih‐Hong , Lee Yun‐Huey , Leng Chih‐Hsiang
Published: October 2006

l‐[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X‐114 in the presence of KC1. The solubilized binding sites bound l‐[3H]glutamate reversibly with Kd and Bmax values of 1.48 ± 0.18 μM and 178.2 ± 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3‐fold purification of these binding sites was achieved by Triton X‐114 solubilization, anion‐exchange chromatography, and affinity chromatography using wheat germ agglutinin‐Sepharose. The apparent molecular mass of the partially purified binding sites was 620 ± 50 kDa. l‐[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non‐N‐methyl‐d‐as‐partate (NMDA) l‐glutamate receptors but not by NMDA or α‐amino‐4‐phosphonobutyrate. The rank order for the competitive ligands in displacing l‐[3H]glutamate was: quisqualate > α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid > l‐glutamate > kainate.