摘要:

Abstract:Recent studies have indicated that the blood‐brain barrier GLUT1 glucose transporter is under post‐transcriptional regulation. To begin functional mapping of the GLUT1 transcript, in the present investigation we studied the translational efficiency of capped full‐length synthetic GLUT1 mRNA, and both 5′‐ and 3′‐untranslated regions (UTRs) deleted GLUT1 mRNAs. Deletion of 5′‐ and 5′‐/3′‐UTRs markedly reduced the translation efficiency of the human (h) GLUT1 transcript in the rabbit reticulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT1 5′‐UTRcis‐acting elements was studied using the luciferase expression vector pGL2. DNA corresponding to the hGLUT1 5′‐UTR generated by PCR was subcloned at theHindIII site of the pGL2 located upstream of the luciferase 5′‐UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5′‐UTR (nucleotides 1–171) markedly increased the expression of luciferase, and disruption of luciferase‐leading sequence with an unrelated 171‐nucleotide fragment decreased its expression. Insertion of nucleotides 1–96 of the hGLUT1 5′‐UTR retained most of the stimulatory effect, and nucleotides 123–171 produced 64% of maximal induction. On the contrary, clones containing nucleotides 79–171 and 154–171 of bGLUT1 5′‐UTR had marginal effects on luciferase expression. The present data provide evidence suggesting that the 5′‐UTR of the GLUT1 mRNA containscis‐acting elements inv

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041335.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The effects of the cholinergic agonist carbachol on phenylethanolamineN‐methyltransferase promoter activity and Egr‐1 mRNA expression in PC12‐derived RS1 cells were examined to investigate the potential involvement of Egr‐1 in the neural regulation of phenylethanolamineN‐methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamineN‐methyltransferase promoter‐luciferase reporter gene construct and also elevated Egr‐1 mRNA levels in untransfected cells. Maximum induction of Egr‐1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr‐1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was medicated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr‐1 binding element in the phenylethanolamineN‐methyltransferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamineN‐methyltransferase promoter through stimulation of Egr‐1 expression, and are consistent with the potential involvement of Egr‐1 in the cholinergic activation of the phenylethanol

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041344.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In a previous study we showed that a region from −182 to +10 bp in the mouse μ‐opioid receptor (MOR) promoter exhibited strong promoter activity. To identify protein‐DNA interactions in this fragment, gel shift and DNase I footprint analyses were performed using nuclear extracts from mouse brain and the human neuroblastoma cell line, SK‐N‐SH. Two regions, nucleotide (nt) −121 to −100 and nt −42 to −22, were identified as being specific protein binding sites. The protein‐DNA interaction in the nt −42 to −22 region was characterized in detail in this study. Methylation interference analysis of this region showed that nuclear protein from SK‐N‐SH cells contacted nucleotides within the sequence ATG‐CAAAT, which is a binding motif for octamertrans‐acting factors. An octamer‐1 (Oct‐1)‐specific antibody supershifted the protein‐DNA complex in a gel shift assay. A UV cross‐linking experiment showed that a nuclear protein, whose molecular weight is similar to that of the Oct‐1 factor, bound to the octamer element in the nt −42 to −22 region. Mutagenesis of four base pairs within the octamercis‐acting element eliminated the specific protein binding in vitro. When the MOR‐luciferase reporter construct (−182 to +10 bp) with the same four base pairs mutated was transiently transfected into SK‐N‐SH cells, a 200% increase in transcriptional activity was observed. Collectively, these data suggest that Oct‐1 is binding to the octamer motif i

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041352.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Thelckgene product, p56lck, is a member of the src‐related family of protein tyrosine kinases. It is known as lymphocyte specific and involved in thymocyte development and in the immune response mediated by the T cell receptor. We report that thelckgene is also expressed in adult mouse CNS and that brain p56lckis similar to the thymus protein. In situ hybridization and immunohistochemistry show that thelckgene is expressed in neurons throughout the brain in distinct regions, including hippocampus and cerebellum. In primary cultures from fetal mouse brain, neuronal cells are immunoreactive to Lck antiserum. This suggests that thelckgene product might be involved in a new signal transduction pathway in mouse brai

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041360.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:In contrast to the intensively studied nerve growth factor (NGF)‐related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin‐6 (IL‐6). We have examined the mechanisms of IL‐6‐induced neuronal differentiation of the pheochromocytoma cell line PC12. IL‐6 independently induced the expression ofperipherin, identifying this gene as the first neuronal‐specific target of IL‐6. However, IL‐6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL‐6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL‐6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk‐1 mitogen‐activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL‐6 receptor, providing another potential mechanism of cooperativity between NGF and IL‐6 signaling. We propose that IL‐6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor‐specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling act

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041365.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The intracellular content of glutathione in astroglia‐rich primary cultures derived from the brains of newborn rats was measured to be 32.1 ± 5.4 nmol/mg of protein. During a 24‐h incubation in a minimal medium lacking amino acids and glucose, the content of glutathione in these cultures was reduced to 52% of the original content. On refeeding of glucose, glutamate, glycine, and cysteine, glutathione was resynthesized. A maximal content of glutathione was found 4 h after refeeding, exceeding the amount of glutathione of untreated cultures by 72%. Maximal glutathione synthesis was observed only if glutamate, cysteine, and glycine were present. If successively each one of these amino acids was made limiting for the synthesis of glutathione, half‐maximal contents of glutathione were found at 0.2 mMglutamate, 20 µMcysteine, or 10 µMglycine. Replacement of glutamate or glycine by other amino acids revealed the potential of astroglial cells to convert glutamine, aspartate, asparagine, proline, and ornithine into glutamate, and serine into glycine. These results demonstrate that the concentration of intracellular glutathione can serve as an indicator for the presence of metabolic pathways of amino acids in cultur

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041375.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Copper/zinc superoxide dismutase (Cu/Zn‐SOD) is a major free radical scavenging enzyme. Increased Cu/Zn‐SOD activity protects cells against oxidative stress mediated by different mechanisms. However, there is also in vitro and in vivo evidence that, in the absence of abnormal oxidative stress, chronic increased Cu/Zn‐SOD activity is detrimental to living cells. To address this issue, we examined the fate of mature midbrain neurons from transgenic mice expressing human Cu/Zn‐SOD and from their nontransgenic littermates. Midbrain from transgenic pups had about threefold higher Cu/Zn‐SOD activity than that from nontransgenic pups. Virtually all transgenic neurons were strongly immunoreactive for human Cu/Zn‐SOD protein in their cell bodies and processes. The number of midbrain neurons decreased over time in both transgenic and nontransgenic cultures, but to a significantly smaller extent in the transgenic cultures. Postnatal midbrain neurons died by either necrosis or apoptosis, and increased Cu/Zn‐SOD activity attenuated both forms of cell death. Furthermore, increased Cu/Zn‐SOD activity better prevented the loss of dopaminergic neurons than GABAergic neurons. We also found that neuronal processes were dramatically denser in transgenic cultures than in nontransgenic cultures. These results indicate that chronic increased Cu/Zn‐SOD activity does not appear to be detrimental, but rather promotes cell survival and neuronal process development in postnatal midbrain neurons, probably by providing more efficient detoxification of free radicals. They also show that increased Cu/Zn‐SOD activity does not seem to play a critical role in determining the mode of cell death in

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041383.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly thetransGolgi and thetransGolgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7‐ and 10‐day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7‐day embryos incubated with UDP‐[3H]Gal. In (a), 1 µMmonensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to ∼50 and 20% of total ganglioside labeling in 7‐ and 10‐day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of>90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited ∼30 and 70%, respectively, in 7‐ and 10‐day cells. In (b),>80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal‐labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP‐NeuAc was also present in the incubation system. Under the same conditions, however,<5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP‐[3H]NeuAc incorporated ∼20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 µMmonensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to thecis/medialGolgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to thetransGolgi as the main com

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041393.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Treatment of cultured bovine adrenal chromaffin cells with 100 nMinsulin raised [3H]saxitoxin ([3H]STX) binding in a time‐dependent manner (t1/2= 26 h). Insulin (100 nMfor 4 days) increased theBmaxof [3H]STX binding by 49% without changing theKDvalue and also augmented the maximal influx of22Na+due to 560 µMveratridine by 39% without altering the EC50value of veratridine. The stimulatory effect of insulin on22Na+influx was concentration‐dependent with an EC50of 3 nM, whereas insulin‐like growth factor (IGF)‐I had little effect at 1 nM.Ptychodiscus brevistoxin‐3 allosterically potentiated veratridine (100 µM)‐induced22Na+influx by approximately twofold in both insulin‐treated cells and untreated cells. Veratridine‐induced45Ca2+influx via voltage‐dependent Ca2+channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine‐induced22Na+influx via the nicotinic receptor‐ion channel complex and high‐K+(direct activation of voltage‐dependent Ca2+channels)‐induced45Ca2+influx. Stimulatory effects of insulin on [3H]STX binding and veratridine‐induced22Na+influx were nullified by simultaneous treatment with either 5,6‐dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na+channel α‐subunit. These results suggest that the binding of insulin to insulin (but not IGF‐I) receptors mediates the up‐regulation of functional Na+channel expression at plasma membranes; this up‐regulation may be due, at least in part, to the de novo synt

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041401.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Endothelins (ETs) and their receptors are present in high levels in the brain and have been proposed to act as neuromodulators or neurotransmitters. However, neither their role nor their precise mechanism of action in the brain is understood. In this study, c‐fosexpression was used as a marker of neuronal activation in organotypic cultures of rat cerebellum. ETs induced Fos protein expression in both granule cells and glia but not in Purkinje cells. Granule cells and glia were both very sensitive to ETs, but different receptor subtypes appeared to be involved, because granule cells did not respond to ET‐3. However, they did respond to the ETB‐selective agonist BQ3020, suggesting the possible existence of a novel neuronal ETB‐like receptor. The induction of Fos in granule cells was independent of extracellular calcium ion concentration, but the ryanodine receptor antagonist dantrolene significantly inhibited the response to ETs, suggesting that the mobilisation of calcium ions from intracellular stores is important. These data support previous evidence that ETs act directly on neurones and show that the intracellular pathways after ET receptor activation are complex. It appears likely that ETs play an important neuromodulatory role in the cer

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67041409.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY