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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nerve growth factor (NGF) can be expressed in cells by gene transfer using a defective Herpes Simplex virus type 1 (HSV-1) vector. In this report, the defective HSV-1 vector, pHSVngf, is used to infect established cell lines and cultured neurons. Infection of cell lines with pHSVngf results in gene transcription, correct RNA processing, and production of biologically active NGF. Infection of the PC12 neuronal cell line results in the production of biologically active NGF and infection of NGF-dependent neonatal sympathetic neurons in primary culture with pHSVngf leads to neuronal survival in the absence of exogenously-added NGF. NGF expressed by pHSVngf-infected cells does not appear to work through an autocrine intracellular pathway since NGF antibody added to culture media of infected cells could block NGF action. Infection with pHSVngf of cholinergic striatal or septal neurons in dissociated cell culture resulted in an increase in choline acetyltransferase activity. These studies demonstrate the efficacy of defective HSV-1 vectors for delivery and expression of neurotrophin genes in cultured neural cells.
Brain Res Mol Brain Res 1994 Jul
PMID:Transfer of the nerve growth factor gene into cell lines and cultured neurons using a defective herpes simplex virus vector. Transfer of the NGF gene into cells by a HSV-1 vector. 796 72

The present study addresses a question of differential expression for a 'plasticity' gene within neurons identified by neurotransmitter type. A method combining immunohistochemical localization of choline acetyltransferase (ChAT) with in situ hybridization histochemistry (ISHH) in the same brain sections was used to quantitate the levels of mRNA for the growth-associated protein GAP-43 (neuromodulin) in rat central cholinergic neuronal populations. We found that many cholinergic neurons in the adult rat brain express levels of GAP-43 mRNA comparable to other brain regions noted for their expression of this plasticity gene. GAP-43 expression in cholinergic cell groups appeared to be highly heterogeneous; this was often true even for cholinergic neurons within the same brain region. A dorsal-ventral gradient in GAP-43 mRNA levels was evident in the rostral basal forebrain cholinergic groups; i.e., medial septum, nucleus basalis magnocellularis and the vertical limb of the diagonal band expressed intermediate levels, while the horizontal limb of the diagonal band and the substantia innominata expressed higher levels of GAP-43 mRNA. Cholinergic interneurons of the striatum were grouped in several populations according to mRNA levels, from very low to very high expression. Similarly, GAP-43 mRNA levels in the cholinergic neurons of the nucleus basalis magnocellularis/substantia innominata were quite variable. The expression of GAP-43 mRNA in brainstem cholinergic groups (laterodorsal tegmental and pedunculopontine nuclei) was in nearly uniform populations of somewhat lower levels. While the expression of GAP-43 mRNA in cholinergic neurons was heterogeneous, virtually every ChAT-positive neuron sampled contained GAP-43 mRNA at levels significantly over background.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1994 May
PMID:Differential expression of GAP-43 mRNA in adult central cholinergic neuronal populations. 805 78

Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect. We localized several potential T3REs and characterized the most proximal T3RE (position 3280-3291) which contains two hexameric half-sites arranged as a direct repeat without a base pair spacer. An oligonucleotide containing this sequence confers T3 responsiveness to a heterologous promoter. The transcriptional response of this T3RE is markedly reduced after mutation of the first or second half-site indicating that both half-sites are required for a maximal T3 response. We have found that RAR alpha, RXR alpha and COUP-TF do not enhance T3 responsiveness and therefore they may not interact with T3R alpha in NG108-15 cells on this regulatory sequence. T3R monomer and dimer specific binding to the proximal T3RE is demonstrated by gel-retardation DNA binding assays and by methylation interference experiments. In COS-1 cells, T3R inhibits transcriptional activation by the transcription factor AP-1 whereas in NE1-115 cells T3R enhances AP-1 mediated activation in a T3 dependant fashion. It is likely that these effects involve protein-protein interactions. These results suggest that the T3 receptor can act as a positive transcriptional regulatory factor on the hChAT gene.
Brain Res Mol Brain Res 1994 May
PMID:Trans-activation by thyroid hormone receptors of the 5' flanking region of the human ChAT gene. 805 82

The minimum requirement of an enhancer for the human choline acetyltransferase (ChAT) gene was analyzed by mutagenesis and protein-DNA interaction studies. A series of deletion and site-specific mutants were expressed transiently in a rat cholinergic neuroblastoma cell, NS20Y. The results revealed that the distal region between -970 and -941 base pairs (bp) from the transcription start site is essential for efficient transcription of the human ChAT gene. Two GC box-like sequences were located within this region, and they were shown to bind purified Sp1 transcription factor by footprinting and gel retardation analyses. This sequence, however, has an orientational effect and is located approximately 1000 bp from the transcription start site, unusual for the conventional GC box sequence. Furthermore, the sequence between these GC box-like sequences is shown to bind another novel trans-acting factor by gel retardation assay and to be necessary for efficient enhancer activity. On the other hand, gel retardation assays using a nuclear extract from Drosophila SL2 cells suggested that non-Sp1 trans-acting factors could also participate in the enhancing activity of this element. Taken together, the results demonstrate that two GC box-like sequences and another trans-acting factor-binding sequence located between -970 and -941 bp act coordinately and are essential for efficient transcription of this low expressed human ChAT gene.
Brain Res Mol Brain Res 1993 Dec
PMID:Enhancer containing unusual GC box-like sequences on the human choline acetyltransferase gene. 811 17

In situ hybridization was used to compare the microscopic distribution in the rat brain of cells containing mRNA for choline acetyltransferase (ChAT) (i.e. cholinergic cells) with that of cells containing mRNA for the five subtypes of muscarinic receptors, in an attempt to establish the potential role as autoreceptors (i.e. muscarinic cholinoceptors present in cholinergic cells) of the different muscarinic receptor subtypes. [32P]alpha-dATP-labelled synthetic oligonucleotides were used as hybridization probes in serial sections. Transcripts for all five subtypes of muscarinic receptors were detected in cells co-distributing with ChAT mRNA-containing cells in one or more regions of the brain. Cells containing m2, m3, m4 or m5 mRNAs were observed in the regions of the basal forebrain where cholinergic cells are located (medial septum/diagonal band nuclei, ventral pallidum, basal nucleus of Meynert). m2, m3 and m5 mRNAs were abundant in the parabigeminal nucleus. m2, m3 and m4 transcripts were detected in the pedunculopontine and laterodorsal tegmental nuclei. m1, m2 and m3 mRNAs were present in several cranial nerve nuclei. The present results suggest that muscarinic autoreceptors belonging to the five subtypes cloned to date may exist.
Brain Res Mol Brain Res 1994 Jan
PMID:Multiplicity of muscarinic autoreceptor subtypes? Comparison of the distribution of cholinergic cells and cells containing mRNA for five subtypes of muscarinic receptors in the rat brain. 816 20

A mouse line transgenic for nerve growth factor (NGF) was developed using the mouse prepro-NGF cDNA inserted within a plasmid containing the proximal region (-10 to -550 bp) of the c-fos promoter and the transcription termination and polyadenylation signals of the rabbit beta-globin gene. No significant modification of gross behavior or central nervous system anatomy was detected in adult animals as assessed by immunohistochemistry and in situ hybridization for NGF and choline acetyltransferase. The expression of the transgene and the possible regulation of its expression by agents acting on the promoter were investigated in vitro. Despite the presence of an additional pool of NGF mRNA specific to the transgene, basal levels of NGF in the supernatant of transgenic astrocytes were similar to normal ones. On the other hand, transgenic neurons spontaneously synthesized and released levels of NGF two to three times higher than normal neurons, while mRNA levels were barely detectable by conventional Northern blotting. The tissue-specificity of NGF expression was respected, with higher levels in hippocampal than neocortical neurons. Increases of NGF mRNA by agents acting on the promoter could be observed in normal and transgenic astrocytes only after inhibition of the protein synthesis by cycloheximide, suggesting a similar rapid turnover of normal and transgenic transcripts. Cyclic AMP agonists specifically increased the secretion of NGF protein by transgenic astrocytes and neurons, while activators of the protein kinase C had a similar effect on transgenic and normal cells. Differences between amounts of NGF secreted by neurons and astrocytes with regards to their respective content in mRNA suggest that transgenic transcripts are subject to normal cell- and tissue-specific post-transcriptional regulations. Agents acting on the c-fos promoter through the protein kinase C or cyclic AMP routes differentially increased the secretion of NGF by transgenic astrocytes or neurons, supporting this hypothesis.
Brain Res Mol Brain Res 1994 Feb
PMID:Cell-type-specific expression and regulation of a c-fos-NGF fusion gene in neurons and astrocytes of transgenic mice. 817 Mar 47

The topography and fine structure of cholinergic efferent fibers were examined in the rat vestibular labyrinth using choline acetyltransferase (ChAT) immunohistochemistry. Fiber plexus of cholinergic fibers were observed beneath the sensory cell layer in all vestibular end-organs. Electron microscopic examination revealed that cholinergic fibers make synaptic contacts with chalices of vestibular nerve branches that surround type I hair cells. In situ hybridization histochemistry of nicotinic acetylcholine receptor subunit mRNAs showed that rat vestibular ganglion cells express both alpha 4 and beta 2 subunit mRNAs. These findings suggest that cholinergic efferents modulate vestibular information from type I hair cells by nicotinic receptors at the level of nerve chalices.
Brain Res Mol Brain Res 1993 Jun
PMID:Synaptic contact between vestibular afferent nerve and cholinergic efferent terminal: its putative mediation by nicotinic receptors. 832 30

We have isolated recombinant lambda (lambda) phages which contain a part of the rat choline acetyltransferase (ChAT) gene. Restriction and Southern blot analyses using synthetic oligonucleotides indicate that these clones overlap one another and contain at least four exons which reside in 16.4 kb of sequence encoding from the middle to the 3' end, but not the 5'-region, of the rat ChAT gene. Partial sequence analyses revealed that the clones contain an exon whose nucleotide sequence corresponds to a highly conserved region of ChAT during evolution. RNase protection mapping experiments show that sequences represented by this exon are expressed at high levels in the spinal cord of adult rats and at low but detectable levels in PC12 cells. By using the genomic sequences, including the exon, as a hybridization probe, we have detected ChAT mRNAs in situ in rat tissues. In situ hybridization experiments using radioactive and non-radioactive probes revealed that cholinergic motoneurons in the spinal cord, the laterodorsal tegmental nucleus as well as the hypoglossal nucleus in the brain stem were labeled, suggesting that the genomic sequence can be used as a probe to measure the ChAT mRNA levels in those cholinergic neurons. The results also indicate that the non-radioactive method gives a better resolution in localizing the expression of ChAT transcripts in the cytoplasm of cholinergic neurons.
Brain Res Mol Brain Res 1993 Jan
PMID:Partial cloning of the rat choline acetyltransferase gene and in situ localization of its transcripts in the cell body of cholinergic neurons in the brain stem and spinal cord. 838 93

The cellular distribution of choline acetyltransferase (ChAT) mRNA within the adult rat central nervous system was evaluated using in situ hybridization. In forebrain, hybridization of a 35S-labeled rat ChAT cRNA densely labeled neurons in the well-characterized basal forebrain cholinergic system including the medial septal nucleus, diagonal bands of Broca, nucleus basalis of Meynert and substantia innominata, as well as in the striatum, ventral pallidum, and olfactory tubercle. A small number of lightly labeled neurons were distributed throughout neocortex, primarily in superficial layers. No cellular labeling was detected in hippocampus. In the diencephalon, dense hybridization labeled neurons in the ventral aspect of the medial habenular nucleus whereas cells in the lateral hypothalamic area and supramammillary region were more lightly labeled. Hybridization was most dense in neurons of the motor and autonomic cranial nerve nuclei including the oculomotor, Edinger-Westphal, and trochlear nuclei of the midbrain, the abducens, superior salivatory, trigeminal, facial and accessory facial nuclei of the pons, and the hypoglossal, vagus, and solitary nuclei and nucleus ambiguous of the medulla. In addition, numerous cells in the pedunculopontine and laterodorsal tegmental nuclei, the ventral nucleus of the lateral lemniscus, the medial and lateral divisions of the parabrachial nucleus, and the medial and lateral superior olive were labeled. Occasional labeled neurons were distributed in the giantocellular, intermediate, and parvocellular reticular nuclei, and the raphe magnus nucleus. In the medulla, light to moderately densely labeled cells were scattered in the nucleus of Probst's bundle, the medial vestibular nucleus, the lateral reticular nucleus, and the raphe obscurus nucleus. In spinal cord, the cRNA densely labeled motor neurons of the ventral horn, and cells in the intermediolateral column, surrounding the central canal, and in the spinal accessory nucleus. These results are in good agreement with reports of the immunohistochemical localization of ChAT and provide further evidence that cholinergic neurons are present within neocortex but not hippocampus.
Brain Res Mol Brain Res 1993 Jan
PMID:In situ hybridization localization of choline acetyltransferase mRNA in adult rat brain and spinal cord. 838 10

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the ventral and dorsal horns and the intermediate zone of the rabbit lumbar spinal cord (L4-7) 24 and 96 h after ischemia caused by 20 or 40 min occlusion of the abdominal aorta. Changes of AChE and butyrylcholinesterase (BChE) activities were also detected histochemically by the direct thiocholine method. No significant changes were found immediately after ischemia. The most remarkable change after 20 min ischemia and 1 or 4 d of reperfusion was heterogeneous decrease in ChAT and AChE activities in the examined parts of gray matter. The highest loss of enzyme activities was found in the ventral horns and the lowest in dorsal horns. Following 40 min ischemia and reperfusion the significant depletion in enzyme activities in all investigated zones of the gray matter was accompanied with necrotic degenerative changes. There was a relatively greater decrease in ChAT and AChE activities in the ventral horns that corresponded with a more prominent morphological damage of the cholinergic neurons in this zone of the spinal cord.
Mol Chem Neuropathol 1993 Aug
PMID:Cholinergic enzymes in spinal cord infarction. Biochemical and histochemical changes. 839 88


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