bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Nerve Growth Factor on Poly amine Metabolism in PC12 Cells
Authors: Guroff Gordon , Dickens Geneva
Published: December 2006

Abstract: Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S‐adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S‐adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor‐induced neurite outgrowth in the cells.
Quantitation and Characterization of Thyrotropin‐Releasing Hormone in Vagal Nuclei and Other Regions of the Medulla Oblongata of the Rat
Authors: Kubek M. J. , Rea M. A. , Hodes Z. I. , Aprison M. H.
Published: December 2006

Abstract: Since evidence is now available to support a nonendocrine autonomic function for thyrotropin‐releasing hormone (TRH), quantitative measurements of TRH were made in nuclei of the vagal complex and other areas of the caudal medulla oblongata of the rat. Regions containing the dorsal motor nucleus of the vagus (DMN), nucleus tractus solitarius (NTS), hypoglossal nucleus, dorsal column nuclei, descending nucleus V (DNV), nucleus ambiguus (NA), raphe nuclei (MR) dorsomedial and ventromedial reticular formation, and inferior olivary nuclei were isolated from 300‐μm‐thick frozen sections of medulla by the micropunch technique. Each region was pooled bilaterally, homogenized in 0.1 M HCl, and vacuum‐dried. Extracts were assayed for TRH by specific radioimmunoassay (RIA). TRH levels varied 100‐fold among medulla nuclei. Highest content (ng/mg protein ± SEM) was found in DMN (14 ± 1.38) and NTS (4.7 ± 0.68), whereas lowest levels occurred in the DNV and MR (0.13, 0.06). Nearly 65% of the total medullary TRH was localized in nuclei associated with vagal complex (DMN, NTS, NA). Characterization of tissue immunoreactivity (TRHi) in these regions suggests the presence of TRH, since (1) medullary tissue extracts competed with 125I‐TRH for antibody binding sites with the same affinity as authentic TRH; (2) TRHi in tissue extracts co‐migrated with synthetic TRH when subjected to reverse‐phase high performance liquid chromatography and Sephadex G‐10 chromatography; and (3) rat serum TRH peptidases degraded TRHi and authentic TRH at similar rates. Another group of rats was subjected to unilateral (right side) vagotomy. At 33 weeks post‐vagotomy, the vagal preganglionic cell population in the ipsilateral DMN was depleted 50–75%, while the contralateral side was unaffected. Interestingly, the content of TRH in the ipsilateral (right) DMN remained unchanged, whereas TRH in the contralateral DMN increased by 50%. In contrast, TRH was significantly elevated in the NA on the ipsilateral side of the lesion. TRH in both ipsi‐ and contralateral NTS was unchanged when compared with sham‐operated controls. These results indicate that (1) TRH is present in several specific loci of the medulla; (2) very high levels are found in the vagal complex; and (3) vagotomy may alter TRH in the contralateral DMN and ipsilateral NA.
Examination of the Role of Central Cholinergic Mechanisms in the Therapeutic Effects of HI‐6 in Organophosphate Poisoning
Authors: Lundy P. M. , Shih T.‐M.
Published: December 2006

Abstract: In atropine‐pretreated rats, HI‐6 (125 mg/kg i.p.) raised the LD50 of Soman (subcutaneous) 5.7 times. Addition of HI‐6 (25 μg i.c. v.) failed to enhance this protection further. HI‐6 (intraperitoneal) also protected animals from intracerebroventricular Soman. HI‐6, administered intracerebroventricularly either alone or in combination with intraperitoneal HI‐6, failed to increase protection, nor did it reactivate Soman‐inhibited acetylcholinesterase (AChE) in several brain areas. HI‐6 (125 or 62.5 mg/kg i.p.) protected rats from Sarin lethality, but only the higher dose significantly altered the brain AChE activity. Furthermore, HI‐6 (intraperitoneal) failed to block the Soman‐induced increase in acetylcholine (ACh) or choline (Ch) levels in any of the brain areas examined. These data indicate that HI‐6 is a very beneficial therapy against Soman, but that no definitive central anticholinergic activity of the compound could be found to explain its protective effects. It is possible that HI‐6 acts by noncholinergic central mechanisms, or that it produces its beneficial effects outside the CNS. Furthermore, brain AChE activity does not appear to be indicative of protective effects of this oxime. ACh or Ch levels in this study were not good parameters to predict the outcome of Soman poisoning.
Lipid Peroxide Distribution in Brain and the Effect of Hyperbaric Oxygen
Authors: Noda Y. , McGeer P. L. , McGeer E. G.
Published: December 2006

Abstract: Regional differences in peroxide levels in rat brain are relatively small but the highest levels were found in the substantia nigra. Lipid peroxides of rat whole brain were significantly increased (22%) immediately after exposure to oxygen at high pressure (OHP). However, this increase was transient. No significant difference from control was observed 3 h after exposure to OHP. The greatest increases were in the cerebral cortex and spinal cord. The olfactory bulb, caudate‐putamen, globus pallidus, corpus callosum, septum, hypothalamus, substantia nigra, and inferior colliculus all showed moderate but significant increases, whereas the accumbens, thalamus, hippocampus, cerebellum, and amygdala showed no significant change. This regional difference in lipid peroxidation may indicate a regional susceptibility to the damaging effects of free radical reactions.
Effects of Intrastriatal Kainic Acid Injection on [3H]Dopamine Metabolism in Rat Striatal Slices: Evidence for Postsynaptic Glial Cell Metabolism by Both the Type A and B Forms of Monoamine Oxidase
Authors: Schoepp Darryle D. , Azzaro Albert J.
Published: December 2006

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ‐aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8‐day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [3H]DA (10−7 M) was examined in 8‐day KA‐lesioned rat striatal slices, an increase in [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) formation was observed. The KA‐induced elevation of [3H]DOPAC formation (but not [3H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [3H]DA (10−5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA‐lesioned striata. These results suggest that KA‐sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.
Calcium‐Stimulated Protein Phosphorylation in Synaptic Membranes
Authors: Sorensen Roger G. , Mahler Henry R.
Published: December 2006

Abstract: Synaptic membranes from rat brain contain several calcium‐requiring protein kinase (PK) activities with different substrate specificities: (a) an activity (CaH‐PK) effective at high concentrations of Ca2+ ion in the absence of Mg2+ (active on class F substrates); (b) a (Ca + Mg)‐PK activity that is mediated by Ca2+ ion in the presence of Mg2+ (active on class B substrates); (c) (Ca‐CaM)‐PK activities that exhibit simultaneous requirements for both Ca2+ ion and CaM (for class C and D substrates). Also described are three activities (d–f) that do not require Ca2+ ion: (d) a Mg‐PK activity in which the presence of Ca2+ causes the inhibition of phosphorylation (active on class A substrates); (e) an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg2+ ion alone (Mg‐PK activity) and is unaffected by the addition of Ca2+ ion and CaM, the substrates of which show different responses to several types of inhibitors; and, finally, (f) the previously well characterized cAMP‐dependent PK activities. Several of the substrates of these kinases have been identified in a fairly unambiguous manner: among them are P43 (class A), as the α subunit of pyruvate dehydrogenase; P54 (class B), as the presynaptic protein B50; and the doublet P75–P80, as proteins IA and IB of Ueda and Greengard. The most interesting activity is that requiring both Ca2+ and CaM. The half‐maximal stimulation (K0.5) for Ca2+ in the presence of CaM was found to be 1.0 μM Ca2+F in untreated membranes. There is little change in this value on prior EGTA extraction of the membranes, which removes the bulk of its Ca2+ and reduces its residual CaM by ≥ 50%. The apparent K0.5 for CaM in the presence of excess Ca2+ ion was found to equal 0.4 μg per reaction mixture (8 μg/ml) or 1.35 μg per reaction mixture (27 μg/ml), for the untreated and EGTA‐treated membranes, respectively.
Actin‐Stimulated Myosin Mg2+‐ATPase Inhibition by Brain Protein
Authors: Berl S. , Chou M. , Mytilineou C.
Published: December 2006

Abstract: A low‐molecular‐weight protein, isolated from bovine brain, inhibits the actin‐stimulated Mg‐ATPase activity of myosin from striated muscle. This inhibition is probably related to its ability to cause actin to revert from its polymerized to its depolymerized state and to prevent the polymerization of actin. Its effect on the polymeric state of the actin has been demonstrated by viscosity studies, DNase inhibition assay, and electron microscopy. Heavy meromyosin can overcome the effect of the brain protein and stimulate the polymerization of actin. The inhibition of ATPase activity is in part dependent upon the relative amounts of brain protein, actin, and myosin. The apparent molecular weight of the brain protein is approximately 20,000 daltons. It appears to be a heat‐labile glycoprotein containing 5–6% carbohydrate.
Characterization of Cholecystokinin Binding Sites in Rat Cerebral Cortex Using a 125I‐CCK‐8 Probe Resistant to Degradation
Authors: Praissman M. , Martinez P. A. , Saladino C. F. , Berkowitz J. M. , Steggles A. W. , Finkelstein J. A.
Published: December 2006

Abstract: Specific binding sites for cholecystokinin (CCK) have been characterized in a particulate membrane fraction of rat cerebral cortex using a biologically active 125I‐labeled derivative of the C‐terminal octapeptide of CCK (CCK‐8) prepared by reaction with the iodinated form of the imidoester (125IIE), methyl‐p‐hydroxybenzimidate. The time course of binding to cortical membranes was rapid, temperature dependent, and saturable. Half‐maximal binding at 24°C was reached in 30 min and full binding at 120 min. At 37°C there was only a slight increase in 125IIE‐CCK‐8 bound after 15 min. The addition of a large excess of CCK‐8 after 30 min of binding at 24°C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of 125IIE‐CCK‐8 bound to membranes with increasing concentrations of CCK‐8 and other structurally related peptides. CCK‐8 displaced 50% of the radioligand at 4 nM, CCK‐33 at 10 nM, and gastrin (desulfated CCK‐8) at 60 nM. Secretin, a structurally unrelated peptide, was unable to displace the radioligand from cortical membranes at 1.0 μM. Finally, 125IIE‐CCK‐8 exposed to cortical membranes or to buffers that had previously contained such membranes for 60 min at 24°C bound equally as well to fresh cortical membranes as control radioligand that had not been exposed to the same conditions. Thus the 125I‐CCK‐8 radioligand used in this study was highly resistant to degradative processes in rat brain tissue.
Influence of Glucose on Somatostatin Synthesis and Secretion in Isolated Cerebral Cortical Cells
Authors: Robbins Richard J.
Published: December 2006

Abstract: Somatostatin‐producing cerebral cortical cell cultures were grown in either high‐ (33 mM) or low‐glucose (5 mM) medium and then exposed to short repetitive changes of high‐ or low‐glucose Krebs‐Ringer's bicarbonate buffer. Equivalent amounts of somatostatin were released in the high‐to‐high, the low‐to‐low, and the low‐to‐high paradigms. The high‐to‐low experiment produced a rapid rise in somatostatin release, followed by a decline. Cultures exposed to 2‐deoxyglucose after high‐glucose medium also released much greater amounts of immunoreactive somatostatin. Separate sets of cultures were grown in high‐ or low‐glucose medium for up to 19 days. Cultures grown in high‐glucose medium generally contained more somatostatin intracellularly than did those maintained in low glucose, although somatostatin in the medium was only different at day 19. These results identify extracellular glucose as an important determinant of cortical somatostatin production.
Catecholamine Glucuronidation: An Important Metabolic Pathway for Dopamine in the Rat
Authors: Wang Pin‐Chang , Kuchel Otto , Buu Nguyen T. , Genest Jacques
Published: December 2006

Abstract: In the present study, we found that large quantities of dopamine (DA) glucuronide were present in rat cerebrospinal fluid (CSF), plasma, and urine, whereas the glucuronides of norepinephrine (NE) and epinephrine (E) were almost undetectable. The high urinary excretion of DA glucuronide was in a range comparable to that of homovanillic acid (HVA). Sulfates of DA, NE, and E were measurable in all three body fluids, but only in small quantities. The measured DA glucuronide was predominantly of endogenous origin, as the feeding of sucrose instead of routine diet did not reduce the urinary output of DA glucuronide. Adrenalectomy but not peripheral sympathectomy induced by chronic guanethidine injection substantially decreased plasma DA glucuronide concentrations, indicating that the adrenals serve as an important source of endogenous DA glucuronide. The data suggest that glucuronidation constitutes an important metabolic pathway for endogenous DA of central and peripheral origin in rats; this route, however, is exclusive to DA and appears to play a negligible role for NE and E.
Turnover Rates of Amino Acid Neurotransmitters in Regions of Rat Cerebellum
Authors: Freeman Mark E. , Lane John D. , Smith James E.
Published: December 2006

Abstract: The turnover rates of aspartate, γ‐aminobutyric acid (GABA), glutamate, glutamine, alanine, serine, and glycine were measured in five regions of rat cerebellum. Turnover rates of the putative neurotransmitters (aspartate, glutamate, and GABA) were 2–20‐fold higher than those of alanine and serine, and generally consistent with the proposed neurotransmitter functions for these amino acids. However, glutamate turnover was high and similar in magnitude in the deep nuclei and granule layer, suggesting possible release, not only from parallel fibers, but from mossy fibers as well. The differential distribution of turnover rates for GABA supports its neuronal release by Purkinje, stellate, basket, and Golgi cells, whereas aspartate may be released by both climbing and mossy fibers. The distribution of glycine turnover rates is consistent with release from Golgi cells, whereas alanine may be released from granule cell parallel fibers. Turnover rates measured in two other motor areas, the striatum and motor cortex, indicated that utilization of these amino acid neurotransmitters is differentially distributed in brain motor regions. The data indicate that turnover rate measurements may be useful in identifying neurotransmitter function where content measurements alone are insufficient.
Decreased Brain Dopamine Synthesis Rate and Increased [3H]Spiroperidol Binding in Streptozotocin‐Diabetic Rats
Authors: Trulson M. E. , Himmel C. D.
Published: December 2006

Abstract: The rate of accumulation of 3,4‐dihydroxyphenylalanine following decarboxylase inhibition and of homovanillic acid following probenecid treatment were significantly decreased in streptozotocin‐diabetic rats. These changes were observed in both the striatum and limbic forebrain. The Bmax for [3H]spiroperidol receptor binding was significantly increased in both brain regions. All of these neurochemical changes were reversed by insulin replacement therapy. Whether these neurochemical changes are attributable to chronic hyperglycemia or some other aspect of the diabetic state is not known.
Testosterone 5α‐Reductase in Rat Brain
Authors: Krieger Neil R. , Scott Robert G. , Jurman Mark E.
Published: December 2006

Abstract: We describe a simple procedure for the microassay of testosterone 5α‐reductase in homogenates of rat brain. This enzyme converts testosterone to dihydrotestosterone. We have used this assay to characterize the enzymatic activity and to map its distribution. The apparent Km is 4.1 × 10−6 M and the Vmax is 85.6 pmol/mg protein/h. The pH optimum is broad and extends from pH 6.0 to 8.0. For the brain regions surveyed, testosterone 5α‐reductase activity varied over a 10‐fold range. The highest activities were observed in homogenates of the midbrain and pons (37–39 pmol/mg protein/h). The lowest were seen in homogenates of the thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, olfactory tubercle, and preoptic area (3–7 pmol/mg protein/h).
Deoxycytidine Transport and Metabolism in Choroid Plexus
Authors: Spector Reynold , Huntoon Sheryl
Published: December 2006

Abstract: In vitro, the transport into and release of [3H]deoxycytidine from the isolated choroid plexus, the anatomical locus of the blood‐cerebrospinal fluid barrier, were studied separately. By use of the ability of nitrobenzylthioinosine (NBTI) to inhibit deoxycytidine efflux from choroid plexus, the transport of 1 μM [3H]deoxycytidine into choroid plexus at 37°C was measured. Deoxycytidine was transported into choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production, but not intracellular binding or metabolism. The Michaelis‐Menten constant (KT) for the active transport of deoxycytidine into choroid plexus was 15 μM. The active transport system for deoxycytidine was inhibited by naturally occurring nucleosides and deoxynucleosides, but not by 1 mM probenecid and 2‐deoxyribose or 100 μM cytosine and cytosine arabinoside. With less than 1 μM [3H]deoxycytidine in the medium, the choroid plexus accumulated [3H]deoxycytidine against a concentration gradient. However, approximately 50% of the [3H]deoxycytidine was phosphorylated to [3H]deoxycytidine nucleotides at a low extracellular [3H]deoxycytidine concentration (6 nM) in 15‐min incubations. This accumulation process depended, in part, on saturable intracellular phosphorylation. These studies provide further evidence that the choroid plexus contains an active nucleoside transport system of low specificity for deoxynucleosides and ribonucleosides, and a separate, saturable efflux system for deoxynucleosides which is very sensitive to inhibition by NBTI.
Developmental Changes in the Biosynthesis of Sialoglycoprotein Substrates for Intrinsic Synaptic Sialidase
Authors: Cruz Tony F. , Gurd James W.
Published: December 2006

Abstract: N′‐Acetyl‐d‐[6‐3H]mannosamine was administered to 13‐ and 28‐day‐old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28‐day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13‐day‐old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins.
Increased Binding of [3H]GABA to Striatal Membranes Following Ischemia
Authors: Francis Andrew , Pulsinelli William
Published: December 2006

Abstract: Sodium‐independent binding of [3H]γ‐aminobutyric acid ([3H]GABA) to membranes prepared from ischemic‐damaged rat striatum was studied by kinetic and time‐course analysis. Three days after 40 min of ischemia, [3H]GABA binding increased fourfold over control values. Scatchard analysis of the binding showed that ischemia significantly increased the affinity (KD) and the total number of binding sites (Bmax) for the high‐affinity GABA receptor. These results support the conclusion that transient forebrain ischemia damages striatal GABAergic neurons.
Regional Energy Balance in Rat Brain After Transient Forebrain Ischemia
Authors: Pulsinelli W. A. , Duffy T. E.
Published: December 2006

Abstract: Phosphocreatine, ATP, and glucose were severely depleted, and the lactate levels were increased in the paramedian neocortex, dorsal‐lateral striatum, and CA1 zone of hippocampus of rats exposed to 30 min of forebrain ischemia. Upon recirculation of the brain, phosphocreatine, ATP, and lactate concentrations recovered to control values in the paramedian neocortex and CA1 zone of hippocampus and to near‐control values in the striatum. The phosphocreatine and ATP concentrations then fell and the lactate levels rose in the striatum after 6–24 h, and in the CA1 zone of hippocampus after 24–72 h. The initial recovery and subsequent delayed changes in the phosphocreatine, ATP, and lactate concentrations in the striatum and hippocampus coincided with the onset and progression of morphological injury in these brain regions. The results suggest that cells in these regions regain normal or near‐normal mitochondrial function and are viable, in terms of energy production, for many hours before unknown mechanisms cause irreversible neuronal injury.
Axonal Glycoproteins with Immunoglobulin‐ and Fibronectin Type III‐Related Domains in Vertebrates: Structural Features, Binding Activities, and Signal Transduction
Authors: Brümmendorf Thomas , Rathjen Fritz G.
Published: December 2006

Abstract: The L1‐ and F11‐like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III‐like domains. Their Ig‐like and fibronectin type III‐related domains are likely to be composed of seven β‐strands arranged in two opposing β‐sheets of highly similar topology. Whereas the F11‐like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 ‐like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre‐mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 ‐like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 ‐like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III‐like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.
The Peripheral‐Type Benzodiazepine Receptor Ligands [3H]Ro 5‐4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle
Authors: Giannaccini Gino , Martini Claudia , Lucacchini Antonio , Strettoi Enrica , Piccolino Marco
Published: December 2006

Abstract: The binding of [3H]flunitrazepam, [3H]RO 5‐4864, and [3H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [3H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [3H]RO 5‐4864 and [3H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (KD values of 24 ± 2.3 and 2.2 ± 0.8 nM, and Bmax values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature ‐ and protein‐dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [3H]RO 5‐4864 and [3H]PK 11195 (PK 11195 > Ro 5‐4864 > flunitrazepam > flumazenil). These results suggest that peripheral‐type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.
Effects of Specific Gangliosides on the In Vitro Proliferation of MPTP‐Susceptible Cells
Authors: Saulino Michael F. , Schengrund Cara‐Lynne
Published: December 2006

Abstract: To determine which portion of a ganglioside molecule might be necessary for the enhancement of recovery from MPTP‐induced lesions, the ability of specific gangliosides to stimulate proliferation of MPTP‐treated 140–3 cells was investigated. The results indicate that of the gangliosides tested, GM1 was the most effective. Although GD1 a and GT1 b were able to enhance the proliferation of MPTP‐treated cells, twice as much GT1b was needed to induce the same effect seen with GM1. In contrast, asialo‐GM1, GM2, and GM3 were ineffective at promoting proliferation of MPTP‐treated cells. The isolated oligosaccharide of GM1 had little effect. These results indicate that in addition to the sialosyl residue, at least the Gal(β1–3)Gal‐NAc portion of the oligosaccharide chain and the ceramide moiety are essential for the induction of proliferation of the MPTP‐treated cells. Investigation of the time of addition of GM1 on its ability to counteract the MPTP‐induced inhibition of 140–3 cell proliferation indicated that addition of GM1 before or concomitantly with MPTP resulted in a significant reduction in MPTP‐induced inhibition of cell proliferation.
Time Course of the Translocation and Inhibition of Protein kinase C During Complete Cerebral Ischemia in the Rat
Authors: Cardell Monika , Wieloch Tadeusz
Published: December 2006

Abstract: The time course for the ischemia‐induced changes in the subcellular distribution of protein kinase C (PKC) (α), (β311). and (γ) and the activity of PKC were studied in the neocortex of rats subjected to 1, 2, 3, 5, 10, and 15 min of global cerebral ischemia. In the particulate fraction, a 14‐fold increase in PKC (γ) levels was seen at 3 min of ischemia, which further increased at 5–15 min of ischemia. At 15 min of ischemia, PKC (γ) and (βll) levels had increased two‐ and six‐fold, respectively. In the cytosolic fraction, a transient early 1.4‐fold increase in PKC (βll) and PKC (γ) levels was seen, whereas no change in the levels PKC (α) was noted. PKC (γ) levels then progressively declined, reaching 50% at 15 min of ischemia. At 5 min of ischemia, a 43% decrease in PKC activity was seen in the particulate fraction, reaching 50% at 15 min of ischemia concomitant with a 27% decrease in the cytosolic fraction. There was no change in the activator‐independent PKC activity. Pretreatment with the ganglioside AGF2 prevented the redistribution of PKC (γ) in the particulate fraction at 5 min. but not at 10 min of ischemia. The observed time course for the translocation of PKC (γ) parallels the ischemia‐induced release of neurotransmitters and increased levels of diacylglycerols, arachidonate, and intra‐cellular calcium and delineates this subspecies as especially ischemia‐sensitive. Ganglioside pretreatment delayed the translocation of PKC (γ), possibly by counteracting the effects of ischemia‐induced factors that favor PKC binding to cell membranes.
Rapid Increases in Peptide Processing Enzyme Expression in Hippocampal Neurons
Authors: Bhat Ratan V. , Tausk Francisco A. , Baraban Jay M. , Mains Richard E. , Eipper Betty A.
Published: December 2006

Abstract: Recent studies have demonstrated that seizure activity causes a dramatic increase in neuropeptide expression in specific regions of the rat hippocampus. In this study we investigated the effect of electroconvulsive treatment (ECT) on the expression of three posttranslational processing enzymes involved in the production of many bioactive peptides from their inactive precursors. Peptidylglycine α‐amidating monooxygenase (PAM) converts peptidylglycine substrates into α‐amidated products and prohormone convertases 1 and 2 perform the tissue‐specific endoproteolytic cleavage of many prohormones. After a single ECT, in situ hybridization demonstrated a rapid increase in the level of PAM mRNA in the dentate granule cells of the hippocampus, reaching peak levels between 1 and 4 h and then returning to near baseline levels within 24 h. Northern blot analysis confirmed the changes in PAM mRNA expression seen by using in situ hybridization. Similar rapid changes in PAM mRNA expression were seen after repeated ECT, suggesting that chronic ECT did not affect the regulation of PAM expression in the hippocampus. Immunohistochemical staining demonstrated an increase in PAM protein in the molecular layer of the dentate gyrus at 4 and 8 h after a single ECT. Based on in situ hybridization, levels of mRNA for the prohormone convertases 1 and 2 were also increased in dentate granule cells after a single ECT. Prohormone convertase 2 mRNA levels exhibited a slower response to ECT, not reaching maximal levels until 8 h after ECT. The response of the dentate granule cells of the hippocampus to ECT provides a model system for studying the rapid, coordinate regulation of peptide‐processing enzymes.
In Vivo Production and Release of Acetylcholine from Primary Fibroblasts Genetically Modified to Express Choline Acetyltransferase
Authors: Fisher L. J. , Schinstine M. , Salvaterra P. , Dekker A. J. , Thal L. , Gage F. H.
Published: December 2006

Abstract: Primary rat fibroblasts genetically modified to express Drosophila choline acetyltransferase (dChAT) synthesize and release acetylcholine (ACh) in vitro. The ACh produced from the transduced fibroblasts was found to be enhanced by increasing amounts of choline chloride in the culture media. These dChAT‐expressing cells were then implanted into the intact hippocampus of adult rats and in vivo microdialysis was performed 7–10 days after grafting to assess the ability of the cells to produce ACh and respond to exogenous choline in vivo. Samples collected from anesthetized rats revealed fourfold higher levels of ACh around dChAT grafts than from either non‐grafted or control‐grafted hippocampi. Localized choline infusion (200 μ) through the dialysis probes was found to induce a selective twofold increase in ACh release only from the dChAT‐expressing fibroblasts. These results indicate not only that dChAT‐expressing fibroblasts continue to synthesize and secrete ACh for at least 10 days after intracerebral grafting, but that the levels of ACh can be manipulated in vivo. The ability to regulate products within genetically modified cells in vivo may provide a powerful avenue for exploring the role of discrete substances within the CNS.
The 70‐kDa Heat Shock Cognate Protein (HSC70) Is a Major Constituent of the Central Nervous System and Is Up‐Regulated Only at the mRNA Level in Acute Experimental Autoimmune Encephalomyelitis
Authors: Aquino Dennis A. , Klipfel Adam A. , Brosnan JCelia F. , Norton William T.
Published: December 2006

Abstract: The expression of the 70‐kDa heat shock cognate (HSC70) and stress‐inducible (HSP70) proteins, and their mRNAs, was examined in experimental autoimmune encephalomyelitis, a model of inflammatory demyelination in the CNS. This study was undertaken as an extension of previous work demonstrating an abrupt decline in mRNA levels of both glial fibrillary acidic protein and the low‐molecular‐weight neurofilament subunit in experimental autoimmune encephalomyelitis spinal cord at 12 days after inoculation, the height of inflammation and clinical signs. Using the same total RNA preparations as our previous study, we report here that mRNA levels for HSC70 increased approximately sixfold over control values at the same time that glial fibrillary acidic protein and low‐molecular‐weight neurofilament subunit messages decreased and were similar to controls by 21 days after inoculation. In situ hybridization experiments showed that HSC70 mRNA was predominantly expressed in neurons and that the influx of inflammatory cells into the CNS was not responsible for the large increase in HSC70 message. Despite this elevation in mRNA, only small (if any) increases in protein levels for HSC70 were detected by both western blotting and in vitro cell‐free translation systems. However, by quantitative immunoblotting, we determined that constitutive levels of HSC70 comprised a substantial portion of CNS proteins, representing 2–3% of the total protein content of spinal cord. Immunohistochemical staining illustrated that the distribution of HSC70 was consistent with that of its message. In contrast, no HSP70 mRNA or protein was detected in either control or experimental animals.
A Novel Approach to Screen for Cytokine Effects on Neuronal Gene Expression
Authors: Fann Ming‐Ji , Patterson Paul H.
Published: December 2006

Abstract: We describe an assay based on reverse transcription‐polymerase chain reaction to detect the expression of mRNAs for a variety of transmitter synthetic enzymes and neuropeptides present at low levels in primary neuronal cultures. The assay is specific for mRNA‐derived templates and is not affected by the presence of genomic DNA. Using this method, we demonstrate that cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF) induce mRNAs for choline acetyltransferase, somatostatin, substance P, vasoactive intestinal polypeptide, cholecystokinin, and enkephalin. The induction of cholecystokinin and enkephalin by CDF/LIF and CNTF had not been shown previously. These data illustrate that the assay can reproduce findings obtained with other methods, as well as provide the sensitivity necessary to produce new results. These results also extend the overlap of CDF/LIF and CNTF in controlling gene expression in cultured sympathetic neurons, supporting the idea that these cytokines may share receptor subunits and signal transduction pathways.
Relations Between Intracellular Ions and Energy Metabolism Under Acidotic Conditions: A Study with Nigericin in Synaptosomes, Neurons, and C6 Glioma Cells
Authors: Erecińska Maria , Nelson David , Dagani Fiorenzo , Deas Judith , Silver Ian A.
Published: December 2006

Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i., [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin‐stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion‐selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+], and [Na+]., The increase in [Na+], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin‐induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+], depresses both oxidative phosphorylation and glycolysis; and that the nigericin‐induced alterations in ion levels and activities of energy‐producing pathways can explain some of the deleterious effects of ischemia and hypoxia.
Regulation of Expression of Cholinergic Neuronal Phenotypic Markers in Neuroblastoma LA‐N‐2
Authors: Rylett R. J. , Goddard S. , Lambros A.
Published: December 2006

Abstract: Cholinergic neurons in PNS and CNS are identified by the presence of choline acetyltransferase and the accumulation of choline by a high‐affinity, sodium‐coupled choline transporter to be used for acetylcholine synthesis. It appears that expression of choline acetyltransferase can be altered by several physiological conditions, including hormones and trophic factors, but little is known about control of expression of the sodium‐coupled choline carrier or whether these two phenotypic markers are regulated similarly. In the present study, the cholinergic human neuroblastoma LA‐N‐2 was used to investigate regulation of expression of choline acetyltransferase and choline up take activity associated with differentiation and neurite extension. Cells grown in serum‐containing basal medium maintained a relatively undifferentiated morphology, expressed low levels of choline acetyltransferase activity, and accumulated choline by a sodium‐dependent process followed by conversion to acetylcholine. Transfer of cells to an enriched, serum‐free defined medium resulted in morphological and neurochemical differentiation, with an enhancement of cholinergic phenotype. Hemicholinium‐sensitive choline uptake activity was increased about sixfold over a 4‐day period, with no change in choline acetyltransferase or acetylcholinesterase specific activity. Acetylcholine synthesis was increased in parallel with the changes in choline accumulation; choline metabolism in the differentiated cells differed significantly from that observed in the undifferentiated cells, with proportionally less converted to phosphorylcholine and proportionally more remaining as unmetabolized choline and converted to acetylcholine. The enhanced choline accumulation appeared to be mediated by an increased number of choline carriers, demonstrated by increased binding of the affinity ligand [3H]‐choline mustard to the transporter and by an increased Vmax for the uptake process. The increased expression of the transport function appeared to be under transcriptional control. as the enhancement of uptake was blocked by the RNA polymerase II inhibitor α‐amanitin as well as by the protein synthesis inhibitor cycloheximide. These results show that expression of sodium‐coupled choline carriers and choline acetyltransferase may be regulated separately in the differentiating neuroblastoma LA‐N‐2 and that neurotransmitter synthesis is controlled by provision of precursor rather than at the level of the biosynthetic enzyme.
Glycosphingolipids of Human Peripheral Nervous System Myelins Isolated from Cauda Equina
Authors: Ogawa‐Goto K. , Ohta Y. , Kubota K. , Funamoto N. , Abe T. , Taki T. , Nagashima K.
Published: December 2006

Abstract: Compositions of neutral and sulfated glucuronyl glycosphingolipids purified from human motor and sensory nerves and myelins were studied. Higher neutral glycosphingolipids (fraction B), which were separated from GazlCer (fraction A), were analyzed by TLC and TLC‐immunostaining. Both nerve myelins contained paragloboside (nLc4Cer) and nLc6Cer dominantly as major higher glycosphingolipids and very little globoside (Gb4Cer), whereas both nerves contained Gb4Cer and nLc4Cer. Besides these major glycosphingolipids, a neutral glycolipid containing asialoGMI (Gg4Cer) epitope and other minor components such as ceramide trihexoside and ceramide dihexoside were detected in both nerves and their myelins. Furthermore, sulfated glucuronyl nLc4Cer and n Lc6Cer, which were monoclonal antibody HNK‐1 reactive glycolipids, were detected in both nerves and myelins.
Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase
Authors: Wu Donghai , Schormann Norbert , Lian Wei , Deisenhofer Johann , Hersh Louis B.
Published: December 2006

Abstract: A cDNA for Drosophila choline acetyltransferase (EC 2.3.1.6; ChAT) was fused with a polyhistidine sequence and expressed in Escherichia coli. The recombinant enzyme was purified to a specific activity of 500 μmol/min/mg of protein using metal affinity chromatography and ion exchange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular dichroism (CD) spectra revealed that the secondary structure of the enzyme is largely μ‐helical. Intrinsic fluorescence spectra of the enzyme indicate that its tryptophan residues are buried. Neither CD nor fluorescence spectra changed significantly in the presence of substrates. The cysteine content of the recombinant Drosophila ChAT was determined to be 16 in the absence and 22 in the presence of 6 M guanidine hydrochloride. Finally, crystallization of recombinant Drosophila ChAT was achieved.
Decreased Electroconvulsive Shock‐Induced Diacylglycerols and Free Fatty Acid Accumulation in the Rat Brain by Ginkgo biloba Extract (EGb 761): Selective Effect in Hippocampus as Compared with Cerebral Cortex
Authors: Rodriguez de Turco Elena B. , Droy‐Lefaix Marie Therese , Bazan Nicolas G.
Published: December 2006

Abstract: The effect of Ginkgo biloba extract (EGb 761) treatment (100 mg/kg/day, per os, for 14 days) on electroconvulsive shock (ECS)‐induced accumulation of free fatty acids (FFA) and diacylglycerols (DAG) was analyzed in rat cerebral cortex and hippocampus. EGb 761 reduced the FFA pool size by 33% and increased the DAG pool by 36% in the hippocampus. These endogenous lipids were unaffected in cerebral cortex. During the tonic seizure (10 s after ECS) the fast accumulation of FFA, mainly 20:4, was similar in sham‐ and EGb 761 ‐treated rats, in both the cerebral cortex and hippocampus. However, further accumulation of free 18:0 and 20:4, observed in the hippocampus of sham‐treated rats during clonic seizures (30 s to 2 min after ECS), did not occur in EGb 761‐treated animals. The rise in DAG content triggered in the cortex and hippocampus by ECS was delayed by EGb 761 treatment from 10 s to 1 min, when values similar to those in sham animals were attained. Moreover, in the hippocampus the size of the total DAG pool was decreased by 19% during the tonic seizure. At later times, DAG content showed a faster decrease in EGb 761‐treated rats. By 2 min levels of all DAG acyl groups decreased to values significantly lower than in sham animals in both cortex and hippocampus. This study shows that EGb 761 treatment affects, with high selectivity, lipid metabolism and lipid‐derived second messenger release and removal in the hippocampus, while affecting to a lesser extent the cerebral cortex.
Dopamine Stimulates K+ Efflux in the Chick Retina via D1 Receptors Independently of Adenylyl Cyclase Activation
Authors: Laitinen Jarmo T.
Published: December 2006

Abstract: Dopamine (DA) stimulated K+ efflux (assessed as 86Rb+ efflux) in retinal suspensions of posthatched chicken. This effect was dose dependent (EC50= 22 μM), was mimicked by the D1‐selective antagonist SKF‐38393, and reversed by the D1‐selective antagonist SCH‐23390, indicating an involvement of D1 receptors. Analogues of cyclic AMP (CAMP) did not mimic the DA action. Moreover, DA failed to affect cAMP levels, suggesting that adenylyl cyclase (AC) was not involved. In contrast, forskolin (FSK) stimulated both K+ efflux and cAMP accumulation in the retina (EC50 of 10 μM for both effects). The FSK‐elicited K+ efflux was not mimicked by 1,9‐dideoxy‐FSK (an analogue of FSK that does not activate AC), suggesting that FSK stimulated K+ efflux through the activation of AC. Both DA and FSK inhibited Na+,K+‐ATPase activity in the retina. However, the DA‐elicited K* efflux was independent of this inhibition, whereas the FSK effect on K+ efflux was largely due to the inhibitory action of the diterpene of the ion pump. A possible role of protein kinase C (PKC) in the DA action was explored. The PKC activator 4β‐phorbol 12‐myristate 13‐acetate (4β‐PMA) potently (EC50= 4 nM) stimulated K+ efflux. This action was not mimicked by the inactive isomer 4α‐PMA. When added together, DA and 4β‐PMA behaved in an additive manner, suggesting separate mechanisms of action for these two drugs. Moreover, DA failed to stimulate retinal phosphoinositide hydrolysis, a well‐known pathway leading to PKC activation. These data suggest that DA acting through D1 receptors and independently of AC can modulate its target cell excitability in the chick retina by stimulating K+ efflux pathways. The mechanism of the DA action remains to be clarified.
Toxic and Protective Effects of l‐DOPA on Mesencephalic Cell Cultures
Authors: Mytilineou Catherine , Han Shan‐Kuo , Cohen Gerald
Published: December 2006

Abstract: The autoxidation of L‐DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L‐DOPA (200 μM) for 48 h, the number of tyrosine hydroxylase‐positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [3H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron‐specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L‐DOPA results in both damaging and antioxidant actions. Ascorbate (200 μM), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [3H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L‐buthionine sulfoximine (L‐BSO), an inhibitor of GSH synthesis, l‐DOPA prevented cell death by L‐BSO.