ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010001.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We have characterised the induction of the mitogen‐inducible form of cyclooxygenase, COX‐2, in the rat cerebral cortex in response to excitotoxin injection into the nucleus basalis. This model is associated with intense stimulation of the ascending pathway to the cerebral cortex, seizure activity, and subsequent ipsilateral cortical induction of various immediate early genes (IEGs), including c‐fos, c‐jun, andzif268, and ornithine decarboxylase enzyme activity and mRNA, all of which processes are sensitive to treatment with theN‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801. In this study we show that excitotoxin injection also causes a marked induction of COX‐2 mRNA in ipsilateral cortex detectable at 1 h and peaking at 4 h, where COX‐2 mRNA levels were 19 times those in unoperated animals. Levels of COX‐2 mRNA remained significantly elevated at 24 h. The early induction of COX‐2 at 1 h was also seen in sham‐operated animals, but at 4 h the COX‐2 mRNA level was significantly increased (4.4‐fold) in animals injected with excitotoxin compared with sham‐operated animals. The induction at this time point (4 h) was explored pharmacologically and found to be significantly attenuated by treatment with MK‐801 (1.5 mg/kg), lamotrigine (10 mg/kg), which prevents presynaptic glutamate release by blocking voltage‐sensitive Na+channels, and the glucocorticoid dexamethasone (3 mg/kg), which has an indirect inhibitory effect on phospholipase A2and COX activity. These results demonstrate that the induction of COX‐2 mRNA occurs by two distinct mechanisms: the rapid and transient response to tissue damage and a second delayed and more substantial response, which is initiated by excitotoxin stimulation and is mediated by presynaptic glutamate release, NMDA receptor activation, and subsequent phospholipase A2activity. We propose a model to demonstrate the similarities between COX‐2 and IEG mRNA induction and highlight possible mechanistic differences in the nature of the i

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010006.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Aromaticl‐amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue‐specific expression of the rat AADC g

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010014.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The tyrosine hydroxylase (TH) gene is expressed exclusively in cells and neurons that synthesize and releasel‐DOPA or catecholamines. To further understand the molecular genetic mechanisms that regulate this cell‐type specific expression, a chimeric gene was prepared by linking 3.6 kb of the 5′ flanking DNA of the mouse TH gene, including the +1 initiation site for transcription, to anE. coliβ‐galactosidase reporter. This fusion gene (TH3.6LAC) was used to prepare transgenic mice, and the tissue distribution of expression of TH3.6LAC was determined by the measurement of β‐galactosidase enzymatic activity and/or by the detection of the transcription product of the chimeric gene by RNase protection assays. In two separate founder lines, TH3.6LAC expression was observed in every region of the brain that was examined, including the olfactory bulb, brainstem, cerebellum, diencephalon, hippocampus, striatum, and cerebral cortex. Expression of TH3.6LAC was observed in the adrenal gland of one founder line but not in the other. TH3.6LAC activation was undetectable in peripheral organs that were examined, including the liver, heart, salivary gland, kidney, lung, and spleen. Although 3.6 kb of the 5′ regulatory DNA of the mouse TH gene is sufficient to activate the TH fusion gene in the mouse, it is not enough to restrict its expression to catecholam

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010020.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Gangliosides are synthesized by sequential catalytic reaction of multiple glycosyltransferases. GM2/GD2 synthase and GD3 synthase are key enzymes for ganglioside synthesis, because their relative activities regulate the main profiles of ganglioside expression. Mouse GD3 synthase (EC 2.4.99.8) cDNA was cloned by eukaryotic expression cloning, and its mRNA expression as well as that of GM2/GD2 synthase gene during the development of the mouse CNS was analyzed by using northern blotting, reverse transcription‐polymerase chain reaction, and in situ hybridization. When brain tissue was analyzed as a whole mass, a typical pattern corresponding to the reported findings obtained by biochemical analyses was observed, i.e., high expression of GD3 synthase gene in the early stage and gradual increase of GM2/GD2 synthase gene expression in the late stage of the development. However, the results of in situ hybridization of these two genes revealed that the expression kinetics of these two genes were heterogeneous among various sites in the brain under development. These findings suggest that various expression patterns of the two genes reflect differences in the course of the development of individual sites, and also different ganglioside components are required in individual portions of the brain for development and maintenance of the functio

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010026.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The structure at the 5′ end of the gene encoding neural‐specific protein gene product 9.5 (PGP9.5) has been compared between two evolutionary distant species: the human andMonodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identity was found across a 1‐kb stretch of 5′ untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identified a 233‐bp CpG‐rich minimal promoter. Truncation mutagenesis revealed the presence of essential positivecis‐acting regulatory sequences within the region −182 to −123 relative to the transcription initiation site. Sequence alignment analysis of the human andMonodelphispromoter sequences showed 76% identity in this 59‐bp region of the gene. A perfectly conserved 12‐bp sequence (PSN) located within this region acts as a non‐cell‐specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detected in human neuroblastoma cell lines but is absent in nonneuronal cell lines such as HeLa. Studies on the cell type specificity of the human PGP9.5 promoter demonstrated that in contrast to the endogenous gene, the promoter is active in HeLa cells. However, the promoter displays higher levels of activity in human neuroblastoma cell lines. A conserved 16‐bp sequence located at −356 (motif 5) was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. In conclusion, we have shown that expression of the PGP9.5 gene is regulated by evolutionary conserved positive and negativecis‐acting sequences located in the u

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010035.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We describe the cloning and characterization of a human 5‐HT6serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5‐HT6cDNA encodes a 440‐amino‐acid polypeptide whose sequence diverges significantly from that published for the rat 5‐HT6receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5‐HT6amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5‐HT6receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5‐HT1Dαreceptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5‐HT6receptor also reveals anRsaI restriction fragment length polymorphism with

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010047.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To investigate the physiological role of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca2+/calmodulin‐independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP‐dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cycl

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010057.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The involvement of serotonin 5‐HT1Dβreceptor sites was investigated in the growth of rat C6 glial cells permanently transfected with a gene encoding a human 5‐HT1Dβreceptor. The 5‐HT receptor identity of control and transfected C6 glial/5‐HT1Dβcells was determined by reverse transcription‐polymerase chain reaction using primers specific for rat 5‐HT1A, rat 5‐HT1B, rat 5‐HT1Dα, human 5‐HT1Dβ, and rat 5‐HT2Areceptor genes. Constitutive mRNA for 5‐HT2Areceptors was present in control and transfected C6 glial/5‐HT1Dβcells, whereas mRNA for 5‐HT1Dβreceptor sites was only present in the transfected C6 glial/5‐HT1Dβcell line. 5‐HT inhibited forskolin‐stimulated cyclic AMP formation and promoted cell growth, in contrast to the absence of any measurable effect in pcDNA3 plasmid‐transfected and nontransfected C6 glial cells. The 5‐HT effects could be mimicked by sumatriptan (EC50= 44–76 nM) and were totally and partially blocked by methiothepin (IC50= 9 nM) and GR 127,935 {2′‐methyl‐4′‐(5‐methyl[1,2,4]oxadiazol‐3‐yl)‐biphenyl‐4‐carboxylic acid [4‐methoxy‐3‐(4‐methylpiperazin‐1‐yl)phenyl]amide; IC50= 97 pM}, respectively. No effect on cell growth was measured with the 5‐HT2receptor agonist DOI [1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane; 10 µM], suggesting that 5‐HT2Areceptors are not involved in the 5‐HT‐stimulated C6 glial/5‐HT1Dβcell growth. Dibutyryl‐cyclic AMP (0.3 mM)‐treated cultures did not show sumatriptan‐promoted cell growth, indicating an inhibitory role for cyclic AMP in the cell growth mediated by 5‐HT1Dβreceptor sites. In conclusion, 5‐HT promotes cell proliferation in transfected C6 glial/5‐HT1Dβcells by

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010065.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The effect of glial cell line‐derived neurotrophic factor (GDNF) on the growth of mesencephalic dopaminergic neurons and on their survival following exposure to the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP+) was examined in vitro. In cultures developing under normal conditions, GDNF at 1 ng/ml optimally improved the survival and stimulated the growth of dopaminergic neurons without affecting glial growth. In cultures treated with MPP+, GDNF could not prevent toxicity to dopaminergic neurons. The uptake of [3H]dopamine and the number of tyrosine hydroxylase‐positive neurons were similarly reduced by MPP+in the presence or absence of GDNF. However, after removal of MPP+, GDNF protected dopaminergic neurons from the continuous cell death and stimulated the regrowth of dopaminergic fibers damaged by MPP+. We conclude that GDNF supports the growth of normally developing dopaminergic neurons and stimulates their survival and recovery after damage. These findings suggest that GDNF could be useful in the development of therapeutic approaches to Parkinson's disease, which is characterized by dopaminergic

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66010074.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY