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G G Rousseau

Publications and source records attributed to G G Rousseau.

At least 19 recordsLinked to original sources
PubMed1992

Molecular forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in rat skeletal muscle.

The rat cDNA for the muscle-type (M) isozyme of 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) contains two putative translation initiation sites. To determine whether the M isozyme expressed in rat skeletal muscle corresponds to the short (PFK2M-sf) or the long (PFK2M-lf) isoform, we have expressed them in Escherichia coli. A third construction was also expressed in which the second ATG codon was deleted (PFK2M-lf delta ATG) to ensure that initiation started at the first ATG. The properties of these recombinant proteins were compared with those of the PFK-2/FBPase-2 present in rat skeletal muscle and liver. The recombinant proteins displayed PFK-2 and FBPase-2 activities and the M(r) values of the subunits measured by SDS-polyacrylamide gel electrophoresis were compatible with the calculated ones. The purified recombinant lf form contained not only the expected lf band (54,500 M(r)) but also the sf band (52,000 M(r)), indicating that the expression system could synthesize the long and the short isoforms from the same mRNA. The kinetic properties of the recombinant sf form were not different from those of the rat muscle enzyme. By contrast, lf delta ATG PFK-2 displayed a higher Km for its substrates and a lower Vmax. Immunoblotting with an antibody directed against the long isoform revealed a 54,500 M(r) band both in the lf and the lf delta ATG recombinant, but no band in rat skeletal muscle extracts. In these extracts, one band of 52,000 and a minor one of 54,500 M(r) were detected by an anti PFK-2/FBPase-2 antibody. The 54,500 M(r) band was recognized by an antibody directed against the L isozyme, suggesting that a small amount of the latter is expressed in skeletal muscle. Thus, the M isozyme differs from the L isozyme by replacement of the first 32 amino acids of the L isozyme by an unrelated nonapeptide.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of mouse collagenase cDNA. Divergence of mouse and rat collagenases from the other mammalian collagenases.

Mouse collagenase cDNA was cloned and sequenced. The deduced amino acid sequence was compared to those of the other mammalian collagenases and related matrix metalloproteinases. These comparisons, as well as those of some enzymatic properties, show that the rodent (mouse and rat) interstitial collagenases are very similar but differ more from the other interstitial collagenases than does human neutrophil collagenase. This supports the hypothesis that the order Rodentia is an outgroup to the other eutherian (placental) mammalian orders.

Amino Acid Sequence↗
PubMed1992

Characterization of an alternative promoter in the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene depends on cis-acting elements contained within 300 base pairs of its 5'-flanking sequence. An earlier in vitro study of the transcriptional activity of this 5'-flanking region suggested that transcription can start upstream from position +1. We have investigated this phenomenon by cell-free transcription and transient transfection of chimeric constructs in cultured pituitary cells and in HeLa cells and by analysis of RNA from human pituitary glands and HeLa cells. Transcription initiation sites were identified at positions -54 and -197 by cell-free transcription assays and by RNAse mapping of human pituitary RNA. In transfection assays, the hGH gene 5'-flanking sequence upstream from position -197 displayed transcriptional activity, which critically depended on the upstream stimulatory factor-binding site located between positions -253 and -266. Transcripts initiated upstream from position +1 were detected in human pituitary RNA by polymerase chain reaction amplification and Northern blotting. These transcripts were longer than the mRNA encoding hGH. They might control initiation at position +1 or code for a novel peptide.

Base Sequence↗
PubMed1992

Characterization of an enhancer upstream from the muscle-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The muscle-type isozyme of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is encoded by a mRNA transcribed from the M promoter of a 55-kb gene, which also produces the liver-type isozyme from an alternative promoter. By transient transfection and in vitro protein-DNA binding assays we have delineated, within 4.7 kb of 5' flanking sequence, the M promoter proper and an enhancer located between -1615 and -1809. This enhancer stimulated up to 12-fold the activity of the promoter in the context of an intact 5' flanking sequence and close to 900-fold the activity of the minimal (+41 to -40) M promoter cloned directly downstream from it. A functional dissection of the enhancer by site-directed mutagenesis and use of oligonucleotides suggested that its activity involves the cooperative effect of six binding sites for trans-acting factors clustered within 150 bp. These sites contain either an EF-1A/E4TF1 motif (also known to bind the ets oncogene product) or a Sp1 motif, or both. The activity of the enhancer could be demonstrated in L6 myoblasts and myocytes and in FTO2B hepatoma cells. When left within the intact 5' flanking sequence, however, enhancer activity was inhibited upon differentiation of myoblasts into myocytes.

Animals↗
PubMed1992

The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing.

Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.

Amino Acid Sequence↗
PubMed1992

Activation of 6-phosphofructo-2-kinase by pp60v-src is an indirect effect.

6-Phosphofructo-2-kinase (PFK-2) catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), a potent stimulator of glycolysis. In chick-embryo fibroblasts, PFK-2 activity and Fru-2,6-P2 concentration increase upon transformation by Rous sarcoma virus. We show here that the increase in PFK-2 activity required more than 2 h after shifting fibroblasts infected with a thermosensitive mutant of Rous sarcoma virus from the restrictive to the permissive temperature. Pretreatment of the cells with actinomycin D prevented this increase in PFK-2 activity, suggesting a requirement for RNA synthesis. However, the increase in PFK-2 activity did not correspond to an increase in immunoprecipitable PFK-2. Moreover, the thermostability of PFK-2 and the affinity of this enzyme for its substrate fructose 6-phosphate were increased upon transformation by Rous sarcoma virus. Staurosporine, an inhibitor of protein kinase C, prevented the increase in PFK-2 activity brought about by the shift to the permissive temperature. This, together with a comparison of the effects of phorbol esters on PFK-2 activity, suggests that pp60v-src stimulates, via protein kinase C, the transcription of a gene whose products is a distinct PFK-2 isoenzyme or a protein that activates PFK-2.

Alkaloids↗
PubMed1992

Inhibition of 6-phosphofructo-2-kinase activity by mercaptopurines.

The activity of 6-phosphofructo-2-kinase (PFK-2), the enzyme that catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), was inhibited by mercaptopurines in vitro. Inhibition was observed with the purified enzyme from rat liver and bovine heart, and in extracts from rat lymphocytes and hepatoma cells, chick embryo fibroblasts, and human HeLa and lymphoblastoid cells. Half-maximal effect was obtained with 0.1-0.2 mM mercaptopurine and maximal inhibition ranged between 50 and 90% depending on the enzyme preparation. The inhibition resulted from a decrease in Vmax with no change in Km for ATP. The inhibition was relieved by treatment of the enzyme with thiol reducing agents, suggesting that it involves the formation of a mixed disulfide between mercaptopurine and thiol group(s) essential for enzyme activity. Incubation of intact lymphocytes or lymphoblastoid cells with 2- or 6-mercaptopurine resulted in a decrease in Fru-2,6-P2 content and lactate release. A decrease in Fru-2,6-P2 content but no change in lactate release was observed in HeLa cells and fibroblasts treated with 6-mercaptopurine but not with 2-mercaptopurine. Treatment of HeLa cells with 6-mercaptopurine resulted in a decreased PFK-2 activity which could be restored by treatment of the cell extract with dithiothreitol. In isolated rat hepatocytes and perfused rat hearts mercaptopurines had little or no effect on the Fru-2,6-P2 content and lactate release. These results suggest that the effect of 6-mercaptopurine of arresting growth in lymphoid cells might involve the inhibition of glycolysis in addition to the known inhibition of de novo purine nucleotide synthesis.

Animals↗
PubMed1992

Oral administration of vanadate to diabetic rats restores liver 6-phosphofructo-2-kinase content and mRNA.

Vanadate and insulin were administered to diabetic (streptozotocin) rats to compare their effects on the activity and mRNA content of 6-phosphofructo-2-kinase and L-type pyruvate kinase in the liver. The activity of 6-phosphofructo-2-kinase in livers of diabetic rats was about 40% of that found in normal rats. A similar decrease was found for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase content, measured by immunoprecipitation, and for mRNA, measured by hybridization of Northern blots. Administration of vanadate to the diabetic rats led to a progressive recovery of 6-phosphofructo-2-kinase activity, and 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase content and mRNA. This recovery, which was complete after 15 days of oral treatment, was also obtained after 60 h of insulin administration. L-type pyruvate kinase activity and mRNA were also decreased by about 70% in livers of diabetic rats. Both parameters normalized after 15 days of vanadate treatment, whereas insulin administration (60 h) raised L-pyruvate kinase mRNA three-fold above control values. Oral treatment for 15 days with vanadate can thus mimic the effect of insulin on both pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase in livers of diabetic rats.

Administration, Oral↗
PubMed1992

Growth hormone gene regulation by trans-acting factors.

The promoter sequence of the growth hormone (GH) gene binds the ubiquitous factors nuclear factor 1, activator protein-2, upstream stimulatory factor and Sp1. A pituitary-specific factor known as growth hormone factor 1 or Pit-1 also binds to the promoter sequence. This factor is responsible for somatotroph-specific expression of GH. It is also involved in the effect of GH-releasing hormone and in the ontogeny of the anterior pituitary.

Animals↗
PubMed1991

A rat gene encoding heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

There are at least 3 isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, a bifunctional enzyme which catalyzes the synthesis and degradation of fructose 2,6-bisphosphate. A 22-kb rat gene that encodes the heart isozyme has been identified and compared with the 55-kb rat gene encoding the liver and muscle isozymes which had been described earlier. Although these 2 genes include 12 successive similar exons, they contain dissimilar exons at both ends, consistent with the occurrence of different regulatory domains at the N- and C-termini in the 3 isozymes.

Amino Acid Sequence↗
PubMed1991

Human and rat chromosomal localization of two genes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization.

Two genes encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were localized in human and rat chromosomes. PFKFB1 (previously PFRX), which encodes the liver and muscle isozymes, was assigned to Xq22-q31 in the rat and to Xq27-q28 in the human by in situ hybridization using probes generated by the polymerase chain reaction. PFKFB2, which encodes the heart isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was assigned to chromosome 13 in the rat and to chromosome 1 in the human by hybridization of DNA from somatic cell hybrids. By in situ hybridization, this gene was localized to the regions 13q24-25 in the rat and 1q31 in the human.

Animals↗
PubMed1991

Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

Animals↗
PubMed1990

Nuclear factor-I and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and trans-activate the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene.

Base Sequence↗
PubMed1990

Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter.

DNase I footprinting experiments showed that binding activities of Sp1 and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Sp1 site did not affect promoter activity in cell-free transcription. Still, Sp1 could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.

Base Sequence↗
PubMed1988

RNA binding to the untransformed glucocorticoid receptor. Sensitivity to substrate-specific ribonucleases and characterization of a ribonucleic acid associated with the purified receptor.

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (Rs) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate. In contrast, the highly purified glucocorticoid receptor yielded a sharp symmetrical peak of Rs = 7.1 nm. We demonstrate that the 7.1-nm component could not result from a proteolytic degradation of the 8.3-nm receptor form. The same receptor heterogeneity was observed in thymus cytosol which contains less proteases than liver. After labeling with [3H]dexamethasone 21-mesylate and SDS/PAGE the same 94-kDa receptor band was revealed in both the 8.3-nm and 7.1-nm forms. Immunoblotting experiments showed that both the 94-kDa hormone-binding subunit and the 90-kDa heat-shock protein were present in the two different receptor forms. The 8.3-nm receptor form was converted to the 7.1-nm receptor form after treatment by ribonuclease A in the presence of molybdate and this effect was dose-dependent, being completely prevented by placental ribonuclease inhibitor (RNasin). In contrast, in the presence of molybdate, the 7.1-nm receptor form was ribonuclease-insensitive. Treatment of cytosol with RNase A in the absence of molybdate, partially shifted the untransformed receptor towards the 5.2-nm transformed receptor form. This effect was abolished by placental ribonuclease inhibitor. RNase S protein, an enzymatically inactive proteolytic fragment of RNase A, or S1 nuclease, which is specific for single-stranded nucleic acids, were ineffective when used instead of RNase A. In contrast, cobra venom endonuclease, which preferentially attacks double-stranded regions of small RNAs, caused a complete conversion of the 7-8-nm untransformed receptor to the 5.2-nm transformed receptor form. These results were not observed in the presence of molybdate. Addition of RNasin prior to heating cytosol in the absence of molybdate did not prevent the receptor from dissociating to the 5.2-nm form, suggesting that an endogenous RNase is not involved in the transformation process. The 7.1-nm receptor form was shifted to a 9.2-nm complex when incubated with an excess of GR 49 antireceptor antibody, whereas the 8.3-nm receptor form did not bind to the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Glucocorticoid receptor binding to defined regions of alpha 2u-globulin genes.

A DNA-cellulose competition assay was used to study binding of glucocorticoid receptor complexes to two alpha 2u-globulin genes, RAP 01 and RAO 01. Two binding regions were found in RAP 01, one localized between bp -642 and -452, the other between -252 and -118 from the transcriptional initiation site. Only the second region was found in RAO 01. The binding affinity was comparable to that observed using a long terminal repeat fragment of mouse mammary tumor virus. Both regions contain sequences homologous to a 17-bp consensus proposed for the glucocorticoid receptor binding. Therefore, despite evidence that glucocorticoid induction of alpha 2u-globulin may be indirect, a direct action of the receptor in the 5'-upstream region of the genes cannot be excluded.

Alpha-Globulins↗
PubMed1979

Glucocorticoid hormone action: an overview.

An overview of glucocorticoid action is presented including aspects of historic, clinical, and physiologic interest. Despite the diversity of effects, glucocorticoid hormone action at the cellular level simplifies to a rather universal model that involves binding to a soluble receptor, followed by interaction of the complex with the chromatin and modification of gene expression. This unitary concept has important implications in pathology, pharmacology, and therapeutics. Finally, a definition of a glucocorticoid in terms of its receptor is presented, which we feel is better than the traditional terminology based on specific biological effects. It is hoped that this review will also provide the reader with a framework in which the various contributions in the Monograph can be integrated.

Adrenal Cortex↗