Biomedical subjects

P Robin

Publications and source records attributed to P Robin.

At least 55 records · Page 3Linked to original sources

PubMed1994

Expression of the phosphoenolpyruvate carboxykinase gene in 3T3-F442A adipose cells: effects of retinoic acid and differentiation.

3T3-F442A adipocytes express the gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK). Retinoic acid (RA) caused a 5-fold induction of PEPCK mRNA within 6 h in these cells with a half-maximal effective concentration of approximately 75 microM. This effect was independent of cycloheximide and inhibited by actinomycin D. In vitro run-on experiments using isolated nuclei confirmed that the RA-induced increase was mainly due to an increased rate of transcription of the gene. Stable transfectants bearing either the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) or -600 to +69 fused to CAT (pPL9-CAT) were used to study PEPCK gene regulation during differentiation. The same transfected cells were used to analyse the RA effect. Preadipocytes containing pPL1-CAT expressed a much lower level of CAT activity than did adipocytes. pPL9-CAT was not expressed in either preadipocytes or adipocytes. RA induced the expression of CAT activity in preadipocytes and adipocytes transfected with pPL1-CAT, but had no effect in cells transfected with pPL9-CAT. These results suggest that one or more DNA sequences located between -2100 and -600 bp of the PEPCK promoter is required for adipocyte-specific expression of this gene. RA action is independent of the state of differentiation and appears to require different elements in fat cells from those required in liver.

3T3 Cells↗

PubMed1994

Stimulation of phosphoenolpyruvate carboxykinase gene expression by fatty acids.

We used the 3T3-F442A adipocytes and the FAO hepatoma cells to analyze the effect of oleate on phosphoenolpyruvate carboxykinase (PEPCK) gene expression. In serum-deprived, glucose-free medium, 1 mM oleate, bound to albumin in a 6:1 ratio, specifically stimulated PEPCK mRNA. In 3T3-F442A adipocytes, the maximum 5-fold increase occurred in 4 hours then rapidly declined to reach the basal level 20 hours later. This increase was cycloheximide-independent and actinomycin D-dependent, suggesting a direct, transcriptional effect of oleate. FAO cells also responded to oleate with a transient induction of PEPCK mRNA, although the extent of stimulation was lower. Thus, the PEPCK gene provides a useful molecular tool for studying the mechanisms by which fatty acids stimulate gene expression.

3T3 Cells↗

PubMed1994

Are microtubules essential for the secretory process in rat parotid gland?

The role of microtubules in the exocrine secretory process is not yet well established, and their disruption by anti-microtubule drugs leads to variable effects on intracellular transit and protein secretion. We investigated the involvement of microtubules in the regulated secretory process of rat parotid glands using microscopic techniques and pulse-chase experiments. We showed that 10 microM colchicine or nocodazole destroys the microtubule network in parotid acinar cells but only weakly reduces the release of newly synthesized proteins. The half-effect was obtained with 0.22 microM colchicine. Moreover, this small reduction was found to be independent of the nature of the drug (colchicine, colcemid, or nocodazole) and of the nature of the stimulation (beta-adrenergic or cholinergic pathways). Using nocodazole, we have been able to determine that the steps affected by the drug are very early events in the secretory pathway. Finally, we showed by kinetic analysis that microtubule disruption slows protein release only moderately but does not reduce the total amount of secreted protein. We conclude from this study that microtubule integrity is not essential for protein secretion in rat parotid gland.

Animals↗

PubMed1994

Failures in outpatient tonsillectomy policy in children: a retrospective study in 311 children.

The literature suggests that outpatient tonsillectomy in children is a safe and cost-effective procedure. These conclusions have been based on the low rate of post-operative complications. Recent papers suggest contra-indications for ambulatory surgery in some patients. A retrospective study involving 311 children was performed in our Department. We defined two groups in which the out-patient policy had failed. The first group (43 children) comprised inpatient children scheduled because of an unhealthy preoperative state (12 patients), a sleep-apnea syndrome (5 children), a major associated procedure (3 patients), a social or family environment not reliable enough for postoperative supervision (19 patients) or because of parental refusal (4 patients). The second group (268 patients) was constituted of scheduled outpatients. In this group, the outpatient policy failed in 31 and children had to be kept overnight, because complications occurred. The main short-term complication was bleeding (13 patients). In 8, delayed complications were observed. Thus, according to the literature, children with concomitant heavy medical problems or with a poor social environment have to be managed as inpatients. For the others outpatient procedures were possible but parents should previously be informed of the possible overnight hospital supervision which is needed in 11.6% of cases. When comparing the youngest patients under 4 years of age with the others, although the preexisting medical and social conditions are important factors that may contraindicate ambulatory surgery, once the latter has been decided on, there is no significant difference between the two age groups regarding the number of children requiring overnight hospital supervision.

Adolescent↗

PubMed1994

Glucocorticoids antagonize retinoic acid stimulation of PEPCK gene transcription in 3T3-F442A adipocytes.

Cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK) is a key glyceroneogenic enzyme in adipose tissue. The regulation of PEPCK gene expression by retinoic acid (RA) and dexamethasone (DEX) was studied in 3T3-F442A adipocytes maintained in a serum-free medium. RA induced whereas DEX reduced PEPCK mRNA steady-state level. RA stimulation was about 4-fold and DEX repression was of 80% in 4 hrs. In addition to reducing basal mRNA level, DEX was able to counteract RA induction in a dominant manner. The use of the glucocorticoid antagonist RU 38486 indicated that the DEX effect was mediated by the glucocorticoid receptor. Stable transfectants bearing the region of the PEPCK promoter from -2100 to +69 fused to the chloramphenicol acetyltransferase (CAT) gene (pPL1-CAT) were used to study PEPCK gene regulation in differentiated adipocytes. In such cells, RA stimulated CAT expression 3 to 5.5 fold. DEX had no effect on basal CAT activity whereas it inhibited the stimulation induced by RA. Thus, in adipocytes, the PEPCK gene regulatory region between -2100 and +69 bp mediates both stimulation by RA and repression by DEX of RA action.

3T3 Cells↗

PubMed1994

A fall of the base of the tongue considered as a new cause of nasopharyngeal respiratory impairment: Pierre Robin sequence, a translation. 1923.

In 1923, Pierre Robin, in a classic article "A Fall of the Base of the Tongue Considered as a New Cause of Nasopharyngeal Respiratory Impairment" introduced the term glossoptosis and emphasized the well-known respiratory complications of the condition that now bears his name. He stated that glossoptosis and the resulting respiratory obstruction was due to the "dysmorphic atresia of the mandible" and proposed a prosthetic device for its correction. Here, an English translation of this classic article by Dr. Robin is presented. Figures 1 and 2 are included as they appeared in the original text. There was no reference in the original text to these figures.

France↗

PubMed1993

The serum unresponsive Rous sarcoma virus promoter sustains a high serum response factor-dependent transcription in vitro.

CArG boxes are cis-regulatory elements which are represented both in serum responsive and unresponsive promoters. Here we show that the RSV Long Terminal Repeat contains two CArG boxes, which were efficiently recognised by purified Serum Response Factor, although they remained unresponsive to serum in transient transfection assays. However, RSV CArG boxes were as efficient as c-fos Serum Response Element in mediating a Serum Response Factor-dependent transcription in vitro. Thus, the fact that a CArG box is able to bind Serum Response Factor in an active form is insufficient for serum responsiveness in vivo.

3T3 Cells↗

PubMed1993

Repression of c-fos promoter by MyoD on muscle cell differentiation.

Terminal differentiation and cell proliferation are in many cases, as in muscle cells, mutually exclusive processes. While differentiating myoblasts are withdrawn from the cell cycle, myogenesis is inhibited by some mitogens and overexpression of some oncogenes, including proto-oncogene c-fos (which expresses a growth-associated protein constituting the regulatory factor AP-1 in conjunction with c-Jun). MyoD, a muscle-specific transcription factor of the basic helix-loop-helix family, acts at both levels because it triggers a muscle differentiation programme in non-muscle cells, and induces a complete block of cell proliferation. Antagonistic interaction between MyoD and c-Jun has been demonstrated. We here show that c-fos expression greatly decreases upon muscle cell differentiation, concomitant with MyoD-induced activity. We have identified a MyoD-binding site overlapping with the serum-responsive element in the c-fos promoter. We demonstrate that MyoD can act as a negative regulator for c-fos transcription by blocking serum responsiveness through this binding site. These data suggest that the MyoD negative effect on cell growth could be partly mediated by transcriptional inactivation of growth-responsive genes.

3T3 Cells↗

PubMed1993

Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites.

Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner. We have recently designed psoralen-oligonucleotide conjugates, which, upon binding to double-stranded DNA sequences via triple helix formation, may be cross-linked in vitro to both strands of the DNA following UV irradiation. A psoralen-oligonucleotide conjugate was targeted to the promoter of the alpha subunit of the interleukin 2 receptor (IL-2R alpha) gene. The triple helix site overlaps the binding site for the transcription factor NF-kappa B, which activates transcription from the IL-2R alpha promoter. After UV irradiation, the oligonucleotide conjugate becomes cross-linked to the target site and inhibits transcription of reporter plasmids transfected in live cells. Inhibition is observed when UV-induced cross-linking occurs both in vitro (before transfection) and in vivo (after transfection). We directly demonstrate that this inhibitory effect is due to triple helix formation at the target site, since a mutant of the promoter, to which oligonucleotide binding was inhibited, was not affected by the psoralen-oligonucleotide conjugate after UV irradiation. In addition, we demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.

Base Sequence↗

PubMed1993

A top-down control analysis in isolated rat liver mitochondria: can the 3-hydroxy-3-methylglutaryl-CoA pathway be rate-controlling for ketogenesis?

We incubated isolated liver mitochondria with palmitoyl-CoA, 2,4-dinitrophenol and malonate. Under these conditions all the flux of carbon from palmitoyl-CoA was directed towards acetoacetate synthesis. We measured the rate of acetyl-CoA formation from palmitoyl-CoA (by measuring the rate of oxygen consumption) and the rate of acetoacetate production from acetyl-CoA at three different acetyl-CoA/CoA ratios. Using the top-down approach of metabolic control analysis we calculated the control over ketogenesis exerted by (a) the conversion of extramitochondrial palmitoyl-CoA to intramitochondrial acetyl-CoA and by (b) the conversion of acetyl-CoA to acetoacetate (the 'HMG-CoA pathway'). The overall flux control coefficients of the groups of enzymes involved in (a) and (b) over ketogenesis were 0.28 and 0.72, respectively. Our results show that it is possible for significant control to be exerted over ketogenesis by the enzymes of the HMG-CoA pathway.

Acetoacetates↗

PubMed1993

Inhibition of interleukin-2 receptor alpha-subunit gene expression by oligonucleotide-directed triple helix formation.

Oligonucleotide-directed triple helix formation represents a promising approach to block gene expression at the transcriptional level. We have previously shown [10] that a triple-helix-forming oligonucleotide was able to inhibit promoter function of reporter constructs in live cells, provided that the oligonucleotide was covalently linked to an intercalating agent which stabilizes triple-helical complexes. In order to demonstrate that this inhibitory effect was due to triple helix formation, we have mutated the oligonucleotide target site in the promoter of the interleukin-2 receptor alpha-chain gene. The mutated version of the promoter does not bind, and is not inhibited by the oligonucleotide, demonstrating that the observed inhibition of the wild-type promoter is indeed due to triple helix formation within cells.

Gene Expression↗

PubMed1992

A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence.

Oligonucleotide-directed triplex formation within upstream regulatory sequences is envisioned as a potential tool for gene inhibition. However, this approach requires that triple helix-forming oligonucleotides are chemically modified, so that the triplex is stable under physiological conditions. Here, we have compared several chemical modifications of an oligonucleotide, targeted to a natural 15-base pair homopyrimidine.homopurine sequence located in the upstream regulatory region of the gene encoding the interleukin-2 receptor alpha chain (p55, IL-2 R alpha). Methylation of the cytosines strongly stabilized the triplex. Further attachment of an intercalating agent (acridine) dramatically increased the stability of the triplex, as assessed by Tm measurements or by band shift assays. Furthermore, the acridine-derivatized oligonucleotide was more efficient in competing away high affinity DNA-binding proteins, as assessed by restriction enzyme inhibition assays. Using a novel footprinting assay, we have further shown that the interaction of the methylcytosine-substituted, acridine-derivatized oligonucleotide with a plasmidic target, harboring the IL-2 R alpha regulatory region, remains highly sequence specific, occurs at physiological pH and is independent of the superhelicity of the plasmid. Acridine derivatization did not impair the exquisite target specificity of triplex formation, since the derivatized oligonucleotide inhibited the binding of nuclear proteins to the overlapping NF kappa B enhancer sequence on an IL-2 R alpha target and not on the related human immunodeficiency virus long terminal repeat target. Finally, the oligonucleotide inhibited the NF kappa B-dependent tax-induced transcriptional activation of the IL-2 R alpha chloramphenicol acetyltransferase construct in live cells, whereas it did not have any effect on a human immunodeficiency virus long terminal repeat chloramphenicol acetyltransferase construct. We conclude that this modified oligonucleotide acts as a transcriptional repressor for the IL-2 R alpha gene via triple helix formation with regulatory sequences.

Acridines↗

PubMed1992

Haematopoietic stem cell lines activate novel enhancer-dependent expression of reporter DNA immediately after transfection by mechanisms involving interleukin 3 and protein kinase C.

Approaches to analysing gene regulation in haematopoietic stem cells are limited by their low concentration and rapid cell death outside of a trophic marrow environment. We have used interleukin 3 (IL3)-dependent cell lines as stem-cell models to investigate gene regulation during signal transduction by growth factors. We report that expression of the bacterial chloramphenicol acetyl transferase reporter gene linked via the weak thymidine kinase promoter to known upstream enhancer regions required for expression of the proliferation-dependent proto-oncogene c-fos occurs almost immediately (within 2 h) after transfection. Expression is stimulated by IL3 or activation of protein kinase C. Our findings indicate that IL3-dependent cell lines possess an extremely rapid transcription mechanism for introduced DNA, which if also present in normal cells may be usefully used to analyse gene regulation during signal transduction leading to growth and differentiation by haematopoietic growth factors.

Alkaloids↗

PubMed1991

Universality of c-fos transcriptional regulation: the Dyad Symmetry Element mediates activation by PMA in T lymphocytes.

We here have delineated the regulatory sequences responsible for c-fos transcriptional activation in human primary T lymphoblasts and in a human tumor T cell line (Jurkat), using transient transfection assays. Our results indicate that, as it has been demonstrated for fibroblastic or epithelial cells, the Dyad Symmetry Element is necessary and sufficient to confer responsiveness to an heterologous promoter in both cell types. Protein binding to this element was constitutive, as assessed by gel shift assays. These results suggest that c-fos transcriptional regulation occurs through a widely conserved mechanism in highly differentiated tissues.

Base Sequence↗

PubMed1991

c-fos transcriptional activation by IL-2 in mouse CTL-L2 cells is mediated through two distinct signal transduction pathways converging on the same enhancer element.

The c-fos protooncogene is suspected to play a major role during the activation of cells from different lineages. In particular, c-fos transcription is induced upon entry into a proliferation cycle in a wide variety of cell types. In this study, we have transfected an IL-2-dependent murine T cell line with chloramphenicol acetyl transferase (CAT) reporter constructs, harboring various regions of the human c-fos promoter. We show that IL-2 induces activation of fos CAT reporter constructs in these cells. Furthermore, the induction by IL-2 is mediated through a dyad symmetry element, the serum response element, which is also responsible for fos CAT reporter constructs activation by PMA, a pharmacologic activator of the protein kinase C (PKC). To assess any involvement of PKC in signal transduction for fos CAT reporter activation by IL-2, CTL-L2 cells were PKC-depleted by treatment with high doses of PMA. Such a treatment abolished the transcriptional response of fos CAT reporter constructs to PMA. In contrast, IL-2 was still able to activate fos CAT transcription, albeit with a lower efficiency. These results suggest that PMA-sensitive PKC might be part of intracellular transduction pathways leading to c-fos transcriptional activation by IL-2, and that at least one alternate pathway participates in the complete response. However, these distinct signal transduction pathways have the same DNA target on c-fos promoter, the serum response element.

Animals↗

PubMed1991

Control of hepatic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase during the foetal/neonatal transition, suckling and weaning in the rat.

(1) We assayed active and total (i.e. active plus succinylated) 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase in mitochondria isolated from foetal, neonatal, suckling or weaned rats. (2) HMG-CoA synthase was substantially succinylated and inactivated in mitochondria isolated from term-foetal, (1-h-old, 6-h-old, 1-day-old) neonatal, suckling and high carbohydrate/low-fat (hc)-weaned rats. Succinylation of HMG-CoA synthase was very low in mitochondria isolated from the livers of foetal, 30-min-old neonatal and high-fat/carbohydrate-free (hf)-weaned rats. (3) There was a negative correlation between active HMG-CoA synthase and succinyl-CoA content in mitochondria isolated from term-foetal, suckling and hc-weaned rats. (4) Differences in active enzyme could not be entirely accounted for by differences in succinylation and inactivation of the synthase. Immunoassay confirmed that the absolute amounts of mitochondrial HMG-CoA synthase increased during the foetal/neonatal transition and decreased with hc weaning. The levels remained elevated with hf weaning. (5) From these data we propose that mitochondrial HMG-CoA synthase is controlled by two different mechanisms in young rats. Regulation by succinylation provides a mechanism for rapid modification of existing enzyme in response to changing metabolic states. Changes in the absolute amounts of HMG-CoA synthase provide a more long-term control in response to nutritional changes.

Acyl Coenzyme A↗

PubMed1991

The dyad symmetry element is the molecular target for c-fos induction and inhibition during K 562 differentiation along mutually exclusive lineages.

The c-fos proto-oncogene seems to play an important role during differentiation and activation of cells from the hematopoietic lineage. Therefore, it is of interest to investigate the mechanism underlying its transcriptional activation in these cells. To delineate the sequences and factors involved in c-fos transcriptional activation during the course of myeloid cell differentiation, we have used the K 562 chronic leukemic cell line as a model. K 562 cells were transfected with chloramphenicol transacetylase (CAT) reporter constructs, including various regions of the human c-fos promoter, and induced to differentiate by two distinct agents: 12-O-tetradecanoyl phorbol-13-acetate (TPA), which activates a differentiation program along the megakaryoblastic pathway; and hemin, which induces erythroid differentiation. We show here that TPA treatment of K 562 cells induces fos CAT reporter constructs activation, whereas treatment with hemin does not. Furthermore, predifferentiation of the cells with hemin blocks a subsequent induction by TPA, in correlation with the inhibition by hemin of megakaryoblastic differentiation markers appearance. Both the induction by TPA and the inhibition by hemin are mediated by a dyad symmetry element (DSE) located in the upstream regulatory region, between -318 and -296. These results suggest that the protein complex binding to the DSE regulatory element is the target for c-fos activation by TPA and inhibition by hemin in K 562 cells. However, no modulation of protein affinity for the DSE sequence was detected by gel shift assay during the course of induction or inhibition, suggesting that the structural change responsible for the transcriptional modulation is too unstable or too subtle to be detected by this method.

Base Sequence↗

PubMed1991

Effect of insulin on the properties of liver carnitine palmitoyltransferase in the starved rat: assessment by the euglycemic hyperinsulinemic clamp.

The effect of insulin on the properties of liver carnitine palmitoyltransferase I (CPT I) was assessed in conscious starved rats with the euglycemic hyperinsulinemic clamp. A 24-hour clamp was necessary to fully reverse the effect of starvation on liver malonyl-CoA concentration, CPT I maximal activity, and apparent km and Ki for malonyl-CoA. Since glucagon was not decreased during the clamp, insulin is the major factor involved in the regulation of CPT I.

Animals↗