Sagittal profile of the spine prominent part of the pelvis.
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Animals↗
Biomedical subjects
F Barthel
Publications and source records attributed to F Barthel.
Transcriptional regulation of the GluR2 gene: neural-specific expression, multiple promoters, and regulatory elements.
To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.
Animals↗
GABAB receptors negatively regulate transcription in cerebellar granular neurons through cyclic AMP responsive element binding protein-dependent mechanisms.
GABAB receptors affect short-term signalling in various cell types. However, nothing is known about possible long-term effects on transcription. To analyse such effects in the CNS, we studied GABAB receptor-mediated gene regulation in primary cultures of cerebellar granule neurons. Transcription was followed using a chloramphenicol acetyl transferase reporter gene driven by the minimal cyclic AMP-responsive element (TGACGTCA). Transcription was stimulated by activation of both the cyclic AMP (forskolin: 5 x 10(-6) M) and the Ca2+ dependent (KCl: 30 mM) pathways (-)-Baclofen (10(-6) M to 10(-4) M), a specific GABAB receptor agonist, reduced by 50-70% the transcriptional stimulation evoked by both forskolin and KCl, whereas isoguvacine, a GABAA receptor agonist, was without effect. Moreover, the GABAB antagonist CGP 35348 abrogated the inhibitory effects of both GABA and baclofen, indicating that GABAB receptors were specifically implicated in this response. Measurements of cyclic AMP levels suggested that (-) baclofen inhibits forskolin-initiated transcription by reducing cyclic AMP production. Direct transcriptional activation, via the cyclic AMP pathway, by overexpression of the catalytic subunit of the cyclic AMP-dependent protein kinase, was not significantly altered by (-) baclofen. This indicates again that (-) baclofen-dependent inhibitory mechanisms operate upstream of cyclic AMP-dependent protein kinase at the level of second messenger formation. Further, we used a yeast transcriptional activator GAL4-cyclic AMP-responsive element binding protein to analyse whether GABAB receptor-mediated inhibition of cyclic AMP-responsive element transcription implicated the transacting factor cyclic AMP-responsive element binding protein. We show that the negative effects of (-) baclofen implicate this transcription factor and this holds good for both the forskolin and KCl-stimulated pathways. The results indicate that GABAB receptors negatively regulate cyclic AMP-responsive element binding protein-mediated transcription in the CNS.
Animals↗
Fine tuning of calcium entry into neurons regulates adenosine 3',5'-monophosphate-dependent transcription by several distinct mechanisms.
Activation of both calcium and AMP-dependent regulatory pathways promotes survival of cerebellar neurons in vitro. Complex cellular programs such as survival must involve precise genetic responses. We show here, at the genomic level, that depolarization potentiates AMP-driven transcription of a variety of genes including the c-fos and c-jun proto-oncogenes, and the gene for somatostatin, proenkephalin and nerve growth factor. We used a reporter gene driven by the minimal AMP-responsive element (TGACGTCA) as a model system for studying this class of genes. In primary neurons, this reporter construct is co-activated in a synergistic manner by forskolin and KCl. We show that, in contrast to AMP, calcium-driven transcription does not require functional AMP-dependent protein kinase. Thus, when calcium and AMP levels are increased, these two second messengers stimulate transcription through different kinases which converge at the level of the AMP-responsive element. In addition, lower levels of intracellular free calcium can potentiate AMP-dependent transcription. This effect results from increased cyclic AMP accumulation and is strictly mediated by the AMP/AMP-dependent protein kinase pathway. In summary, low and high calcium concentrations potentiate AMP-dependent transcription via distinct mechanisms. Low calcium increases AMP production, whereas high calcium activates a non-cyclic AMP-dependent protein kinase, which in turn synergizes with AMP-activated transcription. These distinct mechanisms are likely to operate under specific physiological conditions within the neuronal network.
Animals↗
Beta 2-adrenoreceptors stimulate c-fos transcription through multiple cyclic AMP- and Ca(2+)-responsive elements in cerebellar granular neurons.
Neurons from the granular layer of the cerebellum express functional beta 2-adrenoreceptors (beta 2-ARs). We show that stimulation of beta 2-ARs with isoprenaline increases cyclic AMP (cAMP) production and stimulates transcription of genes containing the cAMP-responsive element (CRE; TGACGTCA). This effect is mediated by cAMP-dependent protein kinase and the trans-acting factor CRE binding protein. Transcriptional regulation by the beta 2-AR was investigated by using the c-fos protooncogene as a model system. We show that beta 2-ARs stimulate c-fos mRNA accumulation, increase AP1 binding activity, and stimulate transcription through the phorbol ester-responsive element (TGACTCA). The transcriptional regulation of c-fos itself was studied with reporter constructs driven by c-fos promoter sequences. Deletion studies revealed that beta 2-ARs stimulate c-fos transcription through at least three distinct regulatory sequences: (a) the CRE located at -60 bp 5' to the initiation site, (b) the fos AP1-like element (-291 to -297), and (c) the serum-responsive element (-297 to -317). The regulation of these elements by the two putative second messengers of the beta 2-AR, cAMP and Ca2+, was analyzed. We report that all three of these regulatory sequences are coregulated by both second messengers. These results indicate that beta 2-ARs stimulate c-fos transcription by multiple cAMP- and Ca(2+)-dependent regulatory elements in neurons.
Animals↗
Characterization and genetic analysis of functional corticotropin-releasing hormone receptors in primary cerebellar cultures.
We demonstrate that granular cerebellar neurons express functional corticotropin-releasing hormone (CRH) receptors. Activation of these receptors with CRH receptor agonists leads to a dose-dependent increase in cyclic AMP (cAMP) levels with an apparent EC50 close to 10(-9) M. Using the c-fos protooncogene as a system to evaluate genomic effects of CRH, we show that activation of CRH receptors regulates gene expression at the transcriptional level. CRH rapidly induced c-fos mRNA accumulation. Genetic studies, using chimera genes containing human c-fos promoter sequences coupled to a chloramphenicol acetyltransferase (CAT) reporter gene, confirmed and extended this observation. When protein kinase A (PKA) was specifically inactivated by gene transfer of a mutated regulatory subunit of PKA lacking cAMP binding sites, CRH-stimulated c-fos transcription was suppressed but the increase in cAMP level was not affected, indicating a key role of PKA in mediating CRH-stimulated transcription. As CRH clearly modulates gene expression via the cAMP pathway, we analyzed the genomic effect of this neurohormone on a deleted c-fos-CAT construct containing only the cAMP-responsive element (CRE) and on a heterologous promoter construct bearing the minimal palindromic consensus CRE (core sequence TGACGTCA). These minimal cAMP-responsive genes are induced by CRH. These inductions are dependent on functional PKA. Taken together, our results demonstrate the presence of functional CRH receptors in primary cerebellar cultures. Activation of these receptors stimulates gene expression via the cAMP/PKA pathway and the transacting factor CREB (cAMP-responsive element binding protein).
Animals↗
Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.
Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants.
Animals↗
Genetic analyses in neurons and neural crest-derived post mitotic cells.
Cationic lipids are shown to be efficient DNA carriers for gene transfer into neurons and chromaffin cells. Chimeric genes containing the coding sequence for the bacterial gene chloramphenicol acetyl transferase (CAT), under various promoter sequences were used to estimate transfection efficiency and to analyse transcriptional mechanisms. Using a ubiquitously expressed construct (RSV-CAT), and an anti-CAT antibody to identify CAT positive cells transfection efficiency was found to be approximately 70% in cultures of hypothalamic neurons. Proenkephalin gene expression was studied in chromaffin cells by employing a chimeric gene containing the rat pro-enkephalin promoter fused to the CAT reporter gene. Using this model we show that the transfected gene is inducible by neurotransmitters and second messengers. The contribution of a specific kinase (protein kinase A, PKA) to proenkephalin gene regulation is analysed using this model system, and expression vectors coding for the catalytic subunit of PKA.
Animals↗
Striatal proenkephalin turnover and gene transcription are regulated by cyclic AMP and protein kinase C-related pathways.
Preproenkephalin metabolism, in the rat, was studied in primary striatal neurons maintained in a chemically defined medium. Acute treatment (30 min) with forskolin (10(-5) M) or phorbol 12 myristate 13 acetate (10(-7) M) resulted, respectively, in a two- and seven-fold increase in methionine-enkephalin secretion. Chronic treatment with forskolin or phorbol 12 myristate 13 acetate (24 h) induced a 100% increase in methionine-enkephalin content (forskolin) and on the other hand a 50% decrease in methionine-enkephalin (phorbol 12 myristate 13 acetate). Both treatments increased preproenkephalin mRNA levels in a time-dependent manner, this augmentation being observable after 180 min by Northern blot analysis and in situ hybridization. These data indicate that under chronic stimulation, with either forskolin or phorbol 12 myristate 13 acetate, proenkephalin turnover is accelerated. However, after stimulation with phorbol 12 myristate 13 acetate, the more potent methionine-enkephalin secretagogue, increased peptide synthesis is not sufficient to replenish methionine-enkephalin intracellular stores. Preproenkephalin gene transcription was analysed by introducing the preproenkephalin gene promoter fused to the bacterial acetyl chloramphenicol transferase reporter gene into primary neurons. Chronic stimulation (48 h) by forskolin (10(-5) M) or phorbol 12 myristate 13 acetate (10(-7) M) of striatal neurons transfected with this fusion gene increased chloramphenicol acetyltransferase activity six-fold and the two effects were additive. These data suggest that the cyclic AMP and the protein kinase C pathways directly activate preproenkephalin gene transcription.
Animals↗
Lipopolyamine-mediated transfection allows gene expression studies in primary neuronal cells.
A simple and efficient transfection technique based on lipopolyamine-coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans-acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.
Amino Acid Sequence↗
[Growth in scoliotics].
The growth of a group of 151 French children suffering from scoliosis were studied and compared with the growth of similar children in Sweden and Germany. The aim was to establish whether or not French children had an acceleration of growth before and during the first phase of puberty as occurs in Germans and Swedes. It was demonstrated that the scoliotic group were significantly taller than a group of normal children. The excess of height was temporary in the upper segments of the trunk and seem to be related to an accelerated maturation but it was permanent in the lower segments of the trunk and seem to be related to a genetic causation. The pathology of this discussed.
Adolescent↗
Gene transfer optimization with lipospermine-coated DNA.
Designed synthetic DNA carriers represent an attractive alternative to the widely used calcium phosphate gene transfer technique. In this context, we developed a class of nucleic acid binding lipids, the lipopolyamines, which spontaneously condense DNA on a cationic lipid layer. The resulting nucleolipidic particles transfect most animal cells efficiently. However, compaction depends on many experimental factors, some of which have been varied here to give optimal transfection efficiency. When plasmid condensation by the lipospermine is performed in the absence of competing polyions or serum proteins, or when the gene of interest is diluted into carrier DNA, transfection efficiency is increased by 2-3 orders of magnitude. With these improvements, chloramphenicol acetyl transferase activity resulting from transfection of as little as 25 ng could easily be detected by a nonradioactive ELISA test.
Animals↗
[Congenital radio-ulnar synostosis of children (author's transl)].
29 congenital radio-ulnar synostosis have been observed in 16 childrens. The authors review the clinical patterns of this affection: most often bilateral, it results in impossibility of pronosupination of the wrist which has but little functional consequence, if the hand is in an intermediary position. On the X-ray its almost always a superior radio-ulnar synostosis but the inferior radio-ulnar joint is abnormal and non functional. Only the children severely handicapped by a hand fixed in pronation should be operated upon. No good result can be hoped from a surgery that tries to restore pro-supination. The best surgical technique seems to be a simple horizontal osteotomy through the synostosis itself which allows a derotation of the forearm into the functional intermediary position. Severe complications can occur. Indications and technique must be very careful since this congenital abnormality is very well tolerated.
Child↗