bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin‐Like Growth Factor I
Authors: Zetterstrom Charlotta , Fang Carrie , Benjamin Ann , Rosenzweig Steven A. 
Published: October 2006

AbstractThe biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin‐like growth factor I (IGF‐I). Affinity labeling of toad retinal membrane proteins with I25I‐insulin resulted in the specific labeling of insulin receptor α‐subunits of ∼ 105 kDa. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of partially reduced (αβ‐heterodimer) receptors affinity‐labeled with I25I‐insulin indicated the presence of a disulfide‐linked β‐subunit of ∼95 kDa. Endoglycosidase F digestion of the affinity‐labeled a‐subunits increased their mobility by reducing their apparent mass to ∼83 kDa. This receptor was not detected by immunoblot analysis with a site‐specific antipeptide antibody directed against residues 657–670 of the carboxy terminal of the human insulin receptor α‐subunit, whereas this antibody did label insulin receptor a‐subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor β‐ subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose a‐subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF‐I binding activities associated with toad retinal membranes.
Insulin‐Like Growth Factor‐I Enhances Tyrosine Hydroxylase Activation in Bovine Chromaffin Cells
Authors: Dahmer Mary K. , Hart P. Michelle , Perlman Robert L. 
Published: October 2006

AbstractPrevious studies have shown that insulin‐like growth factor‐I (IGF‐I) enhances secretagogue‐stimulated Ca2+ uptake and catecholamine release in bovine chromaffin cells. This report describes the effect of IGF‐I on the activity of tyrosine hydroxylase (tyrosine 3‐monooxygenase, EC 1.14.16.2), the major regulatory enzyme in the pathway of catecholamine biosynthesis. Tyrosine hydroxylase activity was assayed by measuring 3, 4‐dihydroxyphenylalanine (Dopa) accumulation in the presence of brocresine, an inhibitor of Dopa decarboxylase. Chromaffin cells cultured in serum‐free medium produced ∼40% less Dopa when stimulated by 55 mM K+ than did cells that had been cultured in the presence of serum. Incubation of cells for 3 days in serum‐free medium containing 10 nM IGF‐I restored high K+‐stimulated Dopa accumulation to a level comparable to that seen in cells cultured continuously in serum‐containing medium. In eight experiments, IGF‐I increased high K+‐stimulated Dopa accumulation (expressed as picomoles per minute per milligram of protein) by 96 ± 13%. IGF‐I increased the protein content of chromaffin cells by ∼30%; consequently, its effect on tyrosine hydroxylase activity was even greater when Dopa synthesis was expressed as picomoles per minute per 107 cells. IGF‐I also enhanced the rate of Dopa accumulation in cells stimulated by dimethylphenyl‐piperazinium, 8‐bromo‐cyclic AMP, phorbol 12, 13‐dibutyrate, or Ba2+. The effect of IGF‐I on high K+‐stimulated tyrosine hydroxylase activity was measurable when enzyme activity was assayed in vitro, suggesting that this effect was due to a stable modification of the enzyme. Immunoblotting studies indicated that cells cultured in the presence of IGF‐I had a small selective increase (36 ±12%) in the amount of tyrosine hydroxylase per unit of protein. The effect of IGF‐I on tyrosine hydroxylase activity and catecholamine synthesis appears to be due to an integrated response of the cells to this agonist: IGF‐I increases the amount of tyrosine hydroxylase in the cells and promotes the activation of this enzyme by a variety of agents.
An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes
Authors: Barrie Anne P. , Nicholls David G. , Sanchez‐Prieto Jose , Sihra Talvinder S. 
Published: October 2006

AbstractThe mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4β‐Phorbol dibutyrate (4β‐ PDBu) enhances the Ca2+‐dependent, 4‐aminopyridine (4AP)‐evoked release of glutamate from synaptosomes, the 4AP‐evoked elevation of cytoplasmic free Ca2+, and the 4AP‐ evoked depolarization of the plasma membrane. 4β‐PDBu itself causes a slow depolarization, which may underlie the small effect of 4β‐PDBu on spontaneous, KCI‐evoked, and Ca2+‐independent/4AP‐evoked glutamate release. Because 4AP (but not KCI) generates spontaneous, tetrodotoxin‐sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.
Identification and Immunolocalization of Phospholipase C in Bovine Rod Outer Segments
Authors: Ghalayini Abdallah J. , Tarver Alan P. , Mackin William M. , Koutz Cynthia A. , Anderson Robert E. 
Published: October 2006

AbstractBovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4, 5‐bisphosphate. Approximately 60–70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10‐fold higher than that of resealed ROS enzyme activity. Peptide‐specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y‐region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of ∼ 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140‐kDa antigen from a Sephadex G‐150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140‐kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.
Membrane‐Associated Phospholipase D Activity in Rat Sciatic Nerve
Authors: Chattopadhyay Jyotiprakas , Natarajan V. , Schmid Harald H. O. 
Published: October 2006

AbstractRat sciatic nerve contains a membrane‐bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 μg of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg ot protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one‐half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.
Lan‐1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization
Authors: Fatatis A. , Bassi A. , Monsurrò M. R. , Sorrentino G. , Mita G. D. , Di Renzo G. F. , Annunziato L. 
Published: October 2006

AbstractThe LAN‐1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1 ‐1,000 μM) caused a dose‐dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to ∼70% but completely abolished the plateau phase. The muscarinic‐activated Ca2+ channel was gadolinium (Gd3+) blockable and nimodipine and ω‐conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh‐induced [Ca2+]i elevation was concentration‐dependently inhibited by pirenzepine and 4‐diphenylacetoxy‐N‐methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin‐sensitive guanine nucleotide‐binding protein or by the diacylglycerol‐protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate‐sensitive intracellular store. In addition, ryanodine did not prevent CCh‐induced [Ca2+]i mobilization, and, finally, LAN‐1 cells appear to lack caffeine‐sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 μM), or after thapsigargin.
Purification and Lectin‐Binding Properties of s‐Laminin, A Synaptic Isoform of the Laminin B1 Chain
Authors: Chiu Arlene Y. , Ugozolli Mildred , Meiri Karina , Ko Jan 
Published: October 2006

AbstractThe extracellular matrix (ECM) at the vertebrate neuromuscular junction is a repository of functionally important molecules, some of which can regulate the formation of synapses during regeneration. One candidate molecule is s‐laminin, a 185‐kDa homologue of the laminin B1 chain. Whereas several members of the laminin family are present throughout the ECM ensheathing muscle fibers, immunoreactivity for s‐laminin is found selectively at synaptic sites in adult and embryonic rats, and is detectable at a time when synaptogenesis is taking place during development. We have reported previously that a rat schwannoma cell line, D6P2T, produces and releases large amounts of s‐laminin in culture. We have now purified s‐laminin from medium conditioned by these cells by using a simple three‐step procedure. Serum‐free, conditioned medium is separated by ion‐exchange chromatography on DEAE‐Sephacel, followed by size‐exclusion chromatography on 500 HR‐Seph‐acryl. Finally, s‐laminin is dissociated from other ECM components by agarose gel electrophoresis under reducing conditions and recovered in solution by extracting slices of agarose gel. The purified preparation displays one silver‐stained band that is recognized by three monoclonal antibodies known to bind to different epitopes on s‐laminin. Lectin‐binding studies demonstrate that s‐laminin is a glycoprotein and bears many of the carbohydrate moieties present on the B1 and B2 chains of laminin. Thus, the three 185–220‐kDa members of the laminin family are related in both their protein and carbohydrate domains.
Metabolism of Lactate in the Rat Brain During the Early Neonatal Period
Authors: Vicario Carlos , Medina José M. 
Published: October 2006

AbstractThe metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2 and lipids was higher than those found for glucose and 3‐hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase‐catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase‐catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3‐hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3‐hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (‐)‐Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate and N‐acetylaspartate.
Dopamine Efflux from Striatum After Chronic Nicotine: Evidence for Autoreceptor Desensitization
Authors: Harsing Laszlo G. , Sershen Henry , Lajtha Abel 
Published: October 2006

AbstractWe examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H‐labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation‐evoked tritium overflow was higher in striatum prepared from nicotine‐treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine‐treated mice. The overflow of [3H]DA and its 3H‐metabolites exhibited similar distribution patterns in [3H]DA‐preloaded striatum dissected from untreated and chronic nicotine‐pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (−)‐Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation‐induced tritium efflux from striatum of untreated mice, whereas (±)‐sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (−)‐quinpirole and (±)‐sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (±)‐SKF‐38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)‐SCH‐23390 exerted a stimulatory action but only at a high concentration. In contrast, neither the DA receptor agonists [(−)‐quinpirole, apomorphine, and (±)‐SKF‐38393] nor the antagonists [(±)‐sulpiride and (+)‐SCH‐23390] altered the stimulation‐evoked tritium release in striatum obtained from mice repeatedly treated with nicotine. It is concluded that chronic administration of nicotine produces an increased release of DA in the striatum with a resulting elevation of synaptic DA concentrations leading to development of D2 DA autoreceptor subsensitivity. As a consequence, chronic nicotine may attenuate autoinhibition of dopaminergic neurotransmission in the striatum.
Distinct Developmental Patterns of Expression of Rat α1, α5, γ2S, andα12Lγ‐Aminobutyric AcidA Receptor Subunit mRNAs In Vivo and In Vitro
Authors: Bovolin Patrizia , Santi Maria‐Rita , Memo Maurizio , Costa Erminio , Grayson Dennis R. 
Published: October 2006

AbstractWe have quantitated the α1, α5, γ2S, and γ2Lγ‐aminobutyric acidA (GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The α1 and γ2L mRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The γ2S subunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The α5 mRNA was initially high in vivo and decreased (eightfold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of α5 GABAA receptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the α1, α5, γ2S, and γ2L GABAA receptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the γ2S and γ2L mRNAs indicates that alternative splicing of the γ2 primary RNA transcript is regulated developmentally during postnatal maturation of the rat cerebellum.
Development of an Antiserum to the Midportion of Substance P: Applications for Biochemical and Anatomical Studies of Substance P‐Related Peptide Species in CNS Tissues
Authors: Shimonaka Hiroyuki , Marchand James E. , Connelly Christopher S. , Kream Richard M. 
Published: October 2006

AbstractThis report describes the generation and biochemical characterization of a high‐affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4–10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4–10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high‐affinity COOH‐terminally directed anti‐substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P‐like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus antisubstance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P‐like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti‐substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC‐fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P‐glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohisto‐chemical analyses employing affinity‐purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.
Brain‐Derived Neurotrophic Factor Protects Dopamine Neurons Against 6‐Hydroxydopamine and N‐Methyl‐4‐Phenylpyridinium Ion Toxicity: Involvement of the Glutathione System
Authors: Spina Mary Beth , Squinto Stephen P. , Miller James , Lindsay Ronald M. , Hyman Carolyn 
Published: October 2006

AbstractBrain‐derived neurotrophic factor (BDNF) has recently been shown to enhance the survival of dopamine neurons in cultures derived from the embryonic rat mesencephalon. We now extend this study by demonstrating that, in addition to the effect of sustaining survival of dopaminergic neurons, BDNF also confers protection against the neurotoxic effects of 6‐hydroxydopamine (6‐OHDA) and N‐methyl‐4‐phenylpyridinium ion (MPP+). Exposure of mesencephalic cultures to either 6‐OHDA or MPP+ resulted in a loss of 70–80% of dopaminergic neurons, as determined by tyrosine hydroxylase (TH) immunocytochemistry. In BDNF‐treated cultures, loss of TH‐positive cells after exposure to either toxin was reduced to only 30%. To facilitate biochemical measurements, we studied SH‐SY5Y dopaminergic neuroblastoma cells. BDNF was found to protect these cells from the dopaminergic neurotoxins, 6‐OHDA and MPP+. Indicative of oxidative stress, treatment of SH‐SY5Y cells with 10 μM 6‐OHDA for 24 h caused a fivefold increase in the levels of oxidized glutathione (GSSG). Pre‐treatment with BDNF for 24 h completely prevented the rise in GSSG. Further examination revealed that BDNF increased the activity of the protective enzyme, glutathione reductase, by 100%. In contrast, BDNF had no effect on the activity of catalase. These results add further impetus to exploring the therapeutic potential of BDNF in animal models of Parkinson's disease.
Adult Rat Retina Interneurons Synthesize GD3: GD3 Expression by These Cells Is Regulated by Cell‐Cell Interactions
Authors: Daniotti J. L. , Landa C. A. , Rosner H. , Maccioni H. J. F. 
Published: October 2006

AbstractGD3, a ganglioside of the lactosyl series, is prevalent in rat retina neuronal cells. We studied here whether rat retina neurons synthesize their own surface GD3 or if they acquire it from Müller glia cells. We analyzed the activity of GD3 synthase and the in vivo labeling of gangliosides from N‐[3H]acetylmannosamine in adult rat retinas after selective destruction of Müller glia cells with the gliotoxic α‐D,L‐aminoadipate (AAA). Immunostaining of rat retina sections and western blot analysis with an antivimentin antibody confirmed the gliotoxic effect of AAA. Neither GD3 synthase activity nor the in vivo labeling of GD3 and other gangliosides was significantly affected by AAA, indicating that neuronal cells synthesize their own GD3. We next analyzed the regulation of the expression of GD3 by these neurons in culture. About 80% of freshly dissociated cells from retina of 4‐day‐old rats (R4) immunoexpress surface GD3. After 3 days in dispersed cell culture conditions, GD3 expression was under the limit of detection in 80% of neuronal cells, indicating a failure of these cells to maintain the expression of surface GD3 in these experimental conditions. Most flat Miiller glia‐derived cells present in these cultures were GD3 positive. Surface GD3 was detected in ∼60% of neuronal cells dissociated from R4 tissue that was developed in vitro as an organ culture for 3 days. Likewise, ~50% of neurites that had grown out from R4 retinal ex‐plants within 3 days in culture and whose neuronal character was indicated by immunoexpression of growth‐associated protein GAP‐43 were GD3 positive. These findings suggest that the tissue organization and/or specific interactions modulate GD3 expression in neuronal cells. Under dispersed‐cell culture conditions, c‐pathway gangliosides (GQ1c and GT1c), which are built up from the sialylation of GD3 and later completion of the oligosaccharide backbone, were detected in ~60% of neuronal cells, suggesting a maintenance of production of GD3 as an intermediate for gangliotetraosyl gangliosides.