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Biomedical subjects

C Chabert

Publications and source records attributed to C Chabert.

At least 19 recordsLinked to original sources
PubMed2001

Assessing implications between genotypic and phenotypic variables through lattice analysis.

A previous paper assessed a "Molecular Mapping of Twenty-Four Features of Down Syndrome on Chromosome 21" (Delabar et al., 1993), by analyzing the genotypes/phenotypes of patients suffering from partial trisomy. The mapping was defined through implications--each feature was mapped to the conjunction of cytogenetic bands that were shared by all patients having that feature. In the present paper, we extend that approach to determine how far those implications depart from defining equivalences. Finding equivalences is important. Local equivalences permit a genetic characterization of a feature. And if global equivalences held for all features, that set of bands would be sufficient to characterize the various phenotypes observed in individuals with partial trisomy 21. To extend the earlier approach, we examine the structure of equivalences as well as the structure of implications. We examine both conjunctions of bands and conjunctions of features. The use of Galois lattices permits simultaneous evaluation of both kinds of structures. Each Galois lattice is labeled with a basis (minimal generating set) of implications going from conjunctions of features into bands and those going from conjunctions of bands into features. Analysis reveals that about half of the conjunctions of bands that characterize the genetic structure embody equivalences. This allows us to improve the genetic description of features and to specify minimal sets of questions that need to be investigated to make the global genetic description more precise.

Chromosome Banding↗
PubMed2000

Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3.

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

Amino Acid Sequence↗
PubMed2000

Loss of aggression, after transfer onto a C57BL/6J background, in mice carrying a targeted disruption of the neuronal nitric oxide synthase gene.

Phenotypic differences among mice with disrupted genes and those with wild-type alleles have not provided the necessary evidence for desired gene/phenotype correlations. These differences could be due to "passenger genes" from the donor 129 strains that are used to produce stem cells. Three variations of attack behavior were measured, using mice carrying a disruption of the neural nitric oxide synthase gene. In the first population, the disrupted gene had been maintained on a mixed background including C57BL/6J and 129 alleles. We have developed a second population in which the disrupted gene was transferred onto a C57BL/6J background during five backcross generations. On the mixed C57BL/6J-129 background, mice homozygous for disrupted Nos1 alleles attacked more frequently, had shorter attack latencies, and presented a greater number of attacks than mice carrying nondisrupted alleles. On the C57BL/6J background, no significant difference persisted between the carriers of the disrupted gene and their noncarrier siblings. The noncarriers on the mixed C57BL/6J-129 background, and the carriers or noncarriers on the C57BL/6J background, did not differ from C57BL/6J. The frequency of attacking males was identical in the homozygous carriers of the disrupted gene, in the mixed C57BL/6J-129 background, and in the 129/SvPas, which approximates the 129/SvJae strain from which the stem cells were derived to produce the disrupted Nos1 gene. These results suggest that Nos1 disruption was not implicated in attack behavior. A possible passenger-gene effect from the 129 donor strain is discussed.

Aggression↗
PubMed1998

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase.

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.

Amino Acid Sequence↗
PubMed1998

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity.

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.

Amino Acid Sequence↗
PubMed1998

Induction of the mitogen-activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12.

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)-dependent differentiation. We have recently cloned MKP-3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP-3 undergoes powerful and specific up-regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF-stimulated MKP-3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF-stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9-cis-retinal, two additional differentiation factors found to induce powerfully MKP-3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non-nuclear compartments during essential stages of NGF-mediated PC12 differentiation.

Animals↗
PubMed1997

Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1.

We have studied the phosphorylation of the Bcl-2 family of proteins by different mitogen-activated protein (MAP) kinases. Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta, and this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen by both 32PO4 labeling and the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4.

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

Amino Acid Sequence↗
PubMed1995

Mapping regions of G alpha q interacting with PLC beta 1 using multiple overlapping synthetic peptides.

The heterotrimeric G-protein alpha-chain G alpha q plays a critical role mediating receptor-linked activation of the beta isoforms of PLC which hydrolyse membrane inositol-containing phospholipids to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Despite knowledge of the three-dimensional structure of two G-protein alpha-chains (G alpha t and G alpha i1) as well as high regional amino acid conservation between members of the G-protein alpha-chain family, the precise molecular domains of G alpha q mediating activation of PLC beta 1 are unknown. To map sites responsible for effector interaction we employed 188 peptides each of 15 residues and corresponding to overlapping regions of the complete G alpha q sequence. These were tested for their ability to inhibit G alpha q-dependent activation of recombinant PLC beta 1 using an in vitro reconstitution assay. Peptides from two regions of G alpha q mediated up to 100% inhibition of GTP gamma S-stimulated PLC beta 1 activity, and representative peptides from each of these regions were half-maximally effective at 69.3 +/- 27.4 microM (n = 4) (G alpha q: 251-265) and 110.0 +/- 41.9 microM (n = 4) (G alpha q: 306-319). G alpha q regions described by inhibitory peptides are conserved selectively in other G-protein alpha-chains linked to PLC beta 1 activation (G alpha 11, G alpha 14) and correspond spatially to sites of effector interaction identified in G alpha s by scanning mutagenesis and in transducin using site-specific antibodies and peptides. Computer transducin using site-specific antibodies and peptides. Computer homology modelling of G alpha q based on the crystal structure of transducin indicates that regions interacting with PLC beta 1 form two parallel alpha-helices lying at the surface of the G alpha q structure. These observations provide the first description of two regions within G alpha q critically important for activating PLC beta 1, and moreover, indicate that effector binding domains identified in transducin and G alpha s are also conserved spatially in G alpha q.

Amino Acid Sequence↗
PubMed1995

["I non-exist". Paradoxes of death in adolescence].

This text presents, from the case study of the analytical treatment of a young girl after a very serious suicide attempt, a theoretical reflection on the question of death during adolescence and its place and function in the psyche. The hypothesis suggested is that resorting to the "idea of death" constitutes an attempt to neutralise the excitement created by the excess of representational activities. A parallel is suggested between the way death and pain are used as a protective shield.

Adolescent↗
PubMed1994

Palmitoylation but not the extreme amino-terminus of Gq alpha is required for coupling to the NK2 receptor.

Gq alpha and G11 alpha differ from other G protein alpha subunits in that they have unique, conserved 6 residue amino-terminal extensions. Wild-type and amino-terminal mutants of Gq alpha expressed in COS cells were analyzed for their ability to functionally couple with co-expressed neurokinin NK2 receptor. Wild-type, T2A and delta 2-7 Gq alpha were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino-terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with G beta gamma subunits and GTP gamma S-induced trypsin resistance are also similar to the wild-type alpha subunit. This demonstrates that the NK2 receptor is able to functionally interact with the alpha subunit of Gq and that the first seven amino-acids of Gq alpha are not required for any of the alpha subunit functions tested. In contrast to the T2A and delta 2-7 mutants, a C9,10A Gq alpha mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high-affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTP gamma S-induced trypsin-resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild-type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino-terminus of Gq alpha results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.

Aluminum Compounds↗
PubMed1994

Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system.

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinant Drosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by the Drosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.

Animals↗
PubMed1994

Characterization of the functional activity of dopamine ligands at human recombinant dopamine D4 receptors.

The human D4 dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [35S]GTP gamma S binding assay. By measuring D4 receptor stimulation of [35S]-GTP gamma S binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D4 receptor, dopamine, quinpirole, and dp-2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (-)-apomorphine appeared to be a partial agonist. No increase in [35S]GTP gamma S binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [35S]GTP gamma S binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D4 receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [3H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.

Animals↗
PubMed1993

Expression and characterization of human D4 dopamine receptors in baculovirus-infected insect cells.

The human D4 dopamine receptor has been genetically engineered for expression in insect cells using the baculovirus system. A D4 cDNA gene fusion construct [(1991) Nature 350, 610-614] was synthetically modified to remove two introns from the coding region, and expressed in S. frugiperda (Sf9) cells as a fusion with a short sequence from the polyhedrin protein. Binding assays with [3H]spiperone indicated high levels of D4 receptor binding 90 h after infection and a pharmacological profile identical to that reported for D4 receptors expressed in COS-7 cells using the cDNA gene hybrid. We also show that the agonist binding affinity of D4 receptors expressed in Sf9 cells can be shifted by GTP-gamma-S, indicating coupling to G-proteins.

Animals↗
PubMed1990

[Evaluation of the change process during treatment of adolescent psychotics].

This study rests on work comparing the processes of change in the mental functioning of eleven psychotic teenagers through two perspectives: a psychological perspective centered on the comparison of two psychological examinations carried out at intervals of one and a half and three years; a psychiatric perspective taking into account elements supplied by the doctor and the nursing staff in the facility and concerning the evolution of each patient between the first and second psychological examination. The evolution was judged according to three factors liable to explain the therapeutic effects: the ability of psychic movement through the appearance or the reappearance of a function in the primal process; setting up limits and defenses of a narcissistic order; the improvement in the ability to make bonds. In spite of the difficulties in regrouping the subjects, the comparative results between the tests and the clinical setting generally confirmed our initial hypotheses.

Adolescent↗
PubMed1987

The relationship between HDL-, LDL-, liposomes-free cholesterol, biliary cholesterol and bile salts in the rat.

In order to study the relationship between bile cholesterol and free cholesterol carried by high and low density lipoproteins (HDL and LDL), 10 male Wistar rats, 11 weeks old and fed with a standard diet were divided into 3 groups which received an intravenous infusion (jugular vein) of either LDL, HDL or liposomes. Liposomes were used for comparison because they are assimilated by hepatocytes, but are not recognized by specific receptors. HDL isolated from rat sera were labeled with [14C]cholesterol by molecular exchange and LDL were labeled by exchange with [14C]cholesterol incorporated into phosphatidyl choline/cholesterol liposomes. The peaks of radioactivity appeared in bile 30 min after the HDL or liposome injection and after 210 min for the LDL injection. The kinetic behavior of the cholesterol carried by the liposomes was quite similar to that of cholesterol carried by HDL. Cholesterol carried by HDL was metabolized in bile salts faster than that carried by LDL: cholesterol-HDL or cholesterol-liposomes contributed to the same extent to the secretion of bile cholesterol (15 and 11%, respectively, of the injected dose), LDL (20% of the injected dose). However, the main part of [14C]cholesterol from HDL, LDL or liposomes was metabolized in bile salts. Thus, cholesterol from an exogenous source seemed to be used mainly as a substrate for bile salts. Our study revealed a difference between the hepatic metabolism of HDL, liposomes and LDL in the rat: the kinetic difference between the secretions of the radioactive compounds in bile may be explained by differences in assimilation, intracellular pathways or bile secretion.

Animals↗