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  • 1
    Electronic Resource
    Electronic Resource
    Rat-1 Fibroblasts Engineered with GAD65 and GAD67 cDNAs in Retroviral Vectors Produce and Release GABA (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 61 (1993), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We have used a retroviral cDNA expression system to drive the expression of the different forms of glutamic acid decarboxylase (GAD65, GAD67, or both). Individual clones of engineered Rat-1 cells make the appropriate GAD mRNAs and GAD polypeptides, show GAD enzymatic activity, and make GABA. Clones expressing GAD65 had higher enzymatic activity than those expressing GAD67. As is the case for brain GADs and for GADs produced in engineered bacteria, the enzymatic activity of GAD66 is more responsive to added pyridoxal phosphate than that of GAD67. Immunostaining for both GADs is scattered throughout the cytoplasm. GAD65 immunostaining is less homogeneous than that of GAD67 and also appears to be associated with the surfaces of large vesicle-like structures. Cells expressing GAD65 and GAD67 showed similar immunostaining patterns with anti-GABA antibodies and contained substantial amounts of GABA (ranging from 7 to 18 pmol of GABA/106 cells), which was roughly proportional to their levels of GAD activity. GABA is released from the engineered cells into the surrounding medium under resting conditions, suggesting that cells programmed with GAD cDNAs might serve as effective sources of GABA in cell transplantation experiments.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1993.tb02186.x
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  • 2
    Electronic Resource
    Electronic Resource
    Cyclic AMP Decreases the Expression of a Neuronal Marker (GAD67) and Increases the Expression of an Astroglial Marker (GFAP) in C6 Cells (1994)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 63 (1994), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: C6 cells express proteins and mRNAs that are characteristic of both glia and neurons. Agents that increase intracellular levels of cyclic AMP (cAMP) decrease the enzymatic activity of glutamate decarboxylase (GAD), a neuronal marker, and the mRNA levels for one of the two GAD isoenzymes, GAD67. This reduction is accompanied by increased levels of glial fibrillary acidic protein (GFAP) mRNA, an astrocyte marker. Transient transfection assays, in which a 2-kb upstream regulatory region of the human GFAP gene was linked to a reporter gene, indicate that at least some of the cAMP-mediated increase of GFAP mRNA levels is due to increased transcription. Increases in intracellular cAMP appear to induce differentiation of C6 cells toward a more mature astrocyte phenotype.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1994.63041218.x
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  • 3
    Electronic Resource
    Electronic Resource
    A Rat Brain cDNA Encodes Enzymatically Active GABA Transaminase and Provides a Molecular Probe for GABA-Catabolizing Cells (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 62 (1994), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: cDNAs encoding γ-aminobutyric acid aminotransferase (GABA-T) were isolated from a λZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA-T antibody and plaque hybridization with a GABA-T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA-T from a rat brain cDNA containing the full-length GABA-T coding region. Our rat brain GABA-T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA-T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA-T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD85 and GAD87.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1994.62041267.x
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  • 4
    Electronic Resource
    Electronic Resource
    Ganglioside Modulation of Cyclic AMP-Dependent Protein Kinase and Cyclic Nucleotide Phosphodiesterase In Vitro (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 53 (1989), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Purified cyclic AMP-dependent protein kinase (cAK) catalytic subunit phosphorylated 180-, 49-, 31-, 19-, and 14-kilodalton (kDa) proteins of rabbit sciatic nerve membranes. The ability of cAK to phosphorylate these membrane substrate proteins was inhibited by gangliosides GM1, GDIa, and GTlb with half-maximal inhibitory concentration (I50) = 7–25 μM. Neutral glycolipids and lyso-phosphatidylcholine were much less effective. Cyclic AMP (cAMP) kinase phosphorylation of histone IIA was inhibited by GM1, GDla, and GTlb (I50 = 115 μM, 75 μM, and 75 μM, respectively). Inhibition by GM1 was competitive with respect to histone (K-, = 108 μM). Autophosphorylation of cAMP kinase was inhibited by GM1 (I50 = 15 μM). GTlb, GDla, and GM1 half-maximally stimulated calmodulin-de-pendent cyclic nucleotide phosphodiesterase at 0.1 μM, 0.2 μM, and 0.3 μM, respectively. Although GTlb stimulated phosphodiesterase by increasing Vmax and decreasing Km (similar to calmodulin), GDla and GM1 produced only an increase in Vmax. These results suggest that ganglioside can modulate the activity of cAMP kinase by both direct inhibition of the enzyme and indirect reduction of cAMP levels through activation of phosphodiesterase. Through these mechanisms, gangliosides may alter cAMP-dependent protein phosphorylation and cell function within the nervous system.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1989.tb07308.x
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  • 5
    Electronic Resource
    Electronic Resource
    Effect of Crush Lesion on Radiolabelling of Ganglioside in Rat Peripheral Nerve (1988)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 50 (1988), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Left sciatic nerves of adult male Sprague-Dawley rats were crushed and allowed to recover for 0, 1, 2, 4, 7, or 14 days. At each of these times both L-5 dorsal root ganglia were injected with 100 μCi of [3H]glucosamine. Two days later, dorsal root ganglia, lumbosacral trunks, and sciatic nerves were removed bilaterally. The amounts of radiolabelled ganglioside in crushed lumbosacral trunks were consistently higher than in the controls, with the largest difference occurring within 2 days from simultaneous crush and injection to killing (specimens labelled day 0). The largest difference in the amount of radiolabelled ganglioside between crushed and control sciatic nerve (4–9 days from crush to killing) occurred later than that of lumbosacral trunk, but no significant difference occurred within the first 3 days following crush. There was only a slightly higher radioactivity in gangliosides totalled from all three anatomical specimens of crushed than in control nerves. The neutral nonganglioside lipid and acid-precipitable fraction followed patterns of synthesis and accumulation similar to those of the gangliosides. These findings indicate that after nerve crush gangliosides, glucosamine-labelled neutral nonganglioside lipids, and glycoproteins accumulate close to the proximal end of the regenerating axon. This accumulation could serve as a reservoir to increase the ganglioside concentration in the growth cone membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1988.tb13255.x
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  • 6
    Electronic Resource
    Electronic Resource
    GABA Alters GABAA Receptor mRNAs and Increases Ligand Binding (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 61 (1993), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In addition to its role as an inhibitory neurotransmitter, γ-aminobutyric acid (GABA) influences the cytodifferentiation of developing neurons both in culture and in vivo. Here, we report some of the targets of GABA action and the mechanism through which GABA acts. In primary cultures of cerebellar granule cells, GABA specifically stimulates an increase in the levels of mRNAs for α 1 and β 2 GABAA receptor subunits. The GABAA agonist 4, 5, 6, 7-tetrahydroisoxazolo [5, 4-c]pyridin-3-ol (THIP) mimics this effect, and the GABAA antagonist bicuculline prevents it. In addition, GABA and THIP trigger an increase in the number of GABA binding sites. This increase parallels that seen in vivo, where the total number of GABAA receptor sites increases during postnatal cerebellar development. It is interesting that the period of the greatest increase in the number of receptor sites coincides with the development of the granule cells. Taken together, our data suggest that GABA may play an important role during maturation of cerebellar granule cells by influencing the number and composition of its own receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1993.tb07481.x
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  • 7
    Electronic Resource
    Electronic Resource
    Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Growth, Receptor Phosphorylation, and Dimerization in Neuroblastoma SH-SY5Y Cells (1995)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a KD = 0.14 ± 0.06 nM and Bmax = 7.3 ± 2.3 pM. Functionality of these receptors was demonstrated by an increased [3H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12–24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [3H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF β-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1995.65052251.x
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  • 8
    Electronic Resource
    Electronic Resource
    Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Receptor Dimerization in Human Glioma U-1242MG and Swiss 3T3 Cells (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 61 (1993), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [35S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1993.tb03581.x
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  • 9
    Electronic Resource
    Electronic Resource
    Glutamate Decarboxylases in Nonneural Cells of Rat Testis and Oviduct: Differential Expression of GAD65 and GAD67 (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 58 (1992), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: γ-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5′-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1992.tb09763.x
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  • 10
    Electronic Resource
    Electronic Resource
    Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ-Aminobutyric AcidA Receptors (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 56 (1991), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: A cDNA from a rat hippocampal cDNA library encodes an isoform of the α polypeptide of the γ-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the α2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor α polypeptides and shares 71–76% identity with previously described rat α polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the α2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined “BZ type II” receptors. Other workers have previously shown that the α polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of α2 mRNAs in rat brain suggests that the α2 subunit may indeed be involved in the BZ type II receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1991.tb02072.x
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