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Biomedical subjects

C Brunel

Publications and source records attributed to C Brunel.

At least 19 recordsLinked to original sources
PubMed2003

[Correction of the congenital malformations of the front chest wall by the modelling technique of sternochondroplasty: technique and results on a series of 14 cases].

In the congenital malformations of the front chest wall showing depression or protrusion, the cardiorespiratory disorders are inconstant and often not linked with the distortion. On the other hand psychoaffective repercussions are major in adolescents and young adults justifying recourse to surgery with morphological and aesthetic aiming. The authors present the technique of modelling sternochondroplasty by raising with osteosynthesis by slide fastener-handle of Martin-Borrelly, applied to the correction of congenital malformation of the front chest wall in young adult, both of protrusion (pectus carinatum), of thoraxes in funnel (pectus excavatum) types or of mixed types (pectus arcuatum). This surgical replanning intervention is carried out by under-mammary way and aims at reestablishing a normal anatomy of the front chest wall. It leads to a genuine wide sternochondrocostal volet, which is stabilised by a specific, multipurpose, adaptable and reliable osteosynthesis equipment. The results obtained on a series of 14 cases are good or excellent in 86% of the cases and stable over time in the absence of major complications. A good cooperation of the patient is essential to obtain a good result in particular via the continuation of musculation exercises after assembly consolidation, possibly associated with remote mammary plastic surgery in the event of hypoplasia or of associated mammary agenesia.

Adolescent↗
PubMed2002

Host manipulation by Ligula intestinalis: a cause or consequence of parasite aggregation?

Previous investigations suggest that the infection of the cyprinid roach, Rutilus rutilus, with the larval plerocercoid forms of the cestode, Ligula intestinalis, creates behavioural and morphological changes in the fish host, potentially of adaptive significance to the parasite in promoting transmission to definitive avian hosts. Here we consider whether these behavioural changes are important in shaping the distribution of parasite individuals across the fish population. An examination of field data illustrates that fish infected with a single parasite were more scarce than expected under the negative binomial distribution, and in many months were more scarce than burdens of two, three or more, leading to a bimodal distribution of worm counts (peaks at 0 and >1). This scarcity of single-larval worm infections could be accounted for a priori by a predominance of multiple infection. However, experimental infections of roach gave no evidence for the establishment of multiple worms, even when the host was challenged with multiple intermediate crustacean hosts, each multiply infected. A second hypothesis assumes that host manipulation following an initial single infection leads to an increased probability of subsequent infection (thus creating a contagious distribution). If manipulated fish are more likely to encounter infected first-intermediate hosts (through microhabitat change, increased ingestion, or both), then host manipulation could act as a powerful cause of aggregation. A number of scenarios based on contagious distribution models of aggregation are explored, contrasted with alternative compound Poisson models, and compared with the empirical data on L. intestinalis aggregation in their roach intermediate hosts. Our results indicate that parasite-induced host manipulation in this system can function simultaneously as both a consequence and a cause of parasite aggregation. This mutual interaction between host manipulation and parasite aggregation points to a set of ecological interactions that are easily missed in most experimental studies of either phenomenon.

Animals↗
PubMed2001

Dimerization of the 3'UTR of bicoid mRNA involves a two-step mechanism.

The proper localization of bicoid (bcd) mRNA requires cis-acting signals within its 3' untranslated region (UTR) and trans-acting factors such as Staufen. Dimerization of bcd mRNA through intermolecular base-pairing between two complementary loops of domain III of the 3'UTR was proposed to be important for particle formation in the embryo. The participation in the dimerization process of each domain building the 3'UTR was evaluated by thermodynamic and kinetic analysis of various mutated and truncated RNAs. Although sequence complementarity between the two loops of domain III is required for initiating mRNA dimerization, the initial reversible loop-loop complex is converted rapidly into an almost irreversible complex. This conversion involves parts of RNA outside of domain III that promote initial recognition, and dimerization can be inhibited by sense or antisense oligonucleotides only before conversion has proceeded. Injection of the different bcd RNA variants into living Drosophila embryos shows that all elements that inhibit RNA dimerization in vitro prevent formation of localized particles containing Staufen. Particle formation appeared to be dependent on both mRNA dimerization and other element(s) in domains IV and V. Domain III of bcd mRNA could be substituted by heterologous dimerization motifs of different geometry. The resulting dimers were converted into stable forms, independently of the dimerization module used. Moreover, these chimeric RNAs were competent in forming localized particles and recruiting Staufen. The finding that the dimerization domain of bcd mRNA is interchangeable suggests that dimerization by itself, and not the precise geometry of the intermolecular interactions, is essential for the localization process. This suggests that the stabilizing interactions that are formed during the second step of the dimerization process might represent crucial elements for Staufen recognition and localization.

3' Untranslated Regions↗
PubMed2001

Cyanide stimulated dissociation of chloride from the catalytic center of oxidized cytochrome c oxidase.

A comparison of bovine cytochrome c oxidase isolated in the presence and the absence of chloride salts reveals that only enzyme isolated in the presence of chloride salts is a mixture of a complex of oxidized enzyme with chloride (CcO.Cl) and chloride-free enzyme (CcO). Using a spectrophotometric method for chloride determination, it was shown that CcO.Cl contains one chloride ion that is released into the medium by a single turnover or by cyanide binding. Chloride is bound slowly within the heme a(3)-Cu(B) binuclear center of oxidized enzyme in a manner similar to the binding of azide. The pH dependence of the dissociation constant for the formation of the CcO.Cl complex reveals that chloride binding proceeds with the uptake of one proton. With both forms of the enzyme the dependence of the rate of reaction for cyanide binding upon cyanide concentration asymptotes a limiting value indicating the existence of an intermediate. With CcO.Cl this limiting rate is 10(3) higher than the rate of the spontaneous dissociation of chloride from the binuclear center and we propose that the initial step is the coordination of cyanide to Cu(B) and in this intermediate state the rate of dissociation of chloride is substantially enhanced.

Animals↗
PubMed2001

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting.

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.

Age Factors↗
PubMed2001

Selection of genomic target RNAs by iterative screening.

A growing number of proteins are known to exert their regulatory or biological functions via RNA binding. In some cases genetic interactions allow us to infer candidate targets for RNA directed regulation, but in many other cases identification of potential regulatory targets is problematic. We have developed an in vitro biochemical screen, SETIS (SElection of < > Target RNAs by Iterative Screening) that allows screening of a major portion of the genome for identification of potential targets for RNA binding proteins.

Animals↗
PubMed2000

H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation.

H19 is a paternally imprinted gene whose expression produces a 2.4 kb RNA in most tissues during development and in mammalian myoblastic cell lines upon differentiation. Deletion of the active maternal allele of H19 and its flanking regions in the mouse leads to biallelic methylation and loss of imprinting of the neighbouring Igf2 gene. The function of H19 RNA remains unknown and, although polysome-associated, the absence of a conserved open reading frame suggests that it does not encode a protein product. We describe a novel post-transcriptional regulation of H19 gene expression which, in spite of this lack of coding capacity, is dependent on translational activity. We show that stabilization of the RNA is solely responsible for its accumulation during in vitro muscle cell differentiation. This conclusion is based on the finding that inhibition of protein synthesis results in a dramatic destabilization of H19 RNA in proliferating mouse C2C12 myoblastic cells but not in differentiated cells, and on run-on experiments which showed that the rate of transcription of H19 RNA remains constant during muscle cell differentiation. This mechanism could also be involved in H19 gene expression during mouse development in addition to its transcriptional activation which we have shown to occur.

Animals↗
PubMed1998

Molecular cloning and bacterial expression of a general odorant-binding protein from the cabbage armyworm Mamestra brassicae.

A cDNA clone encoding a general odorant-binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (>90%) was found to be insoluble. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. The refolded rGOBP2 was cross-reactive with a serum raised against the GOBP2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEF, N-terminal sequencing, and two-dimensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of production and purification fulfil the requirements of structural studies.

Amino Acid Sequence↗
PubMed1996

The expression of E.coli threonyl-tRNA synthetase is regulated at the translational level by symmetrical operator-repressor interactions.

Threonyl-tRNA synthetase from Escherichia coli represses the translation of its own mRNA by binding to the operator region located upstream from the ribosome binding site. The operator contains two stemloop structures which interact specifically with the homodimeric enzyme. Here, we provide in vitro and in vivo evidence that these two stem-loop structures are recognized by the enzyme in an analogous way and mimic the anticodon arm of E.coli tRNA(Thr). Determination of the stoichiometry of the different RNA-threonyl-tRNA synthetase complexes reveals that two tRNA(Thr) molecules bind to the enzyme whereas only one thrS operator interacts with the homodimeric enzyme. A model is presented in which the two anticodon-like domains of the operator bind symmetrically to the two tRNA(Thr) anticodon recognition sites (one per subunit) of the dimeric threonyl-tRNA synthetase. Although symmetrical operator-repressor interactions in transcriptional control are widespread, this report stresses the importance of such interactions in translational regulation of gene expression.

Anticodon↗
PubMed1996

Involvement of U1 small nuclear ribonucleoproteins (snRNP) in 5' splice site-U1 snRNP interaction.

U1 small nuclear ribonucleoprotein (snRNP) is an important ribonucleoprotein involved early in the spliceosome formation to commit pre-mRNAs to the splicing pathway. We have determined the association and dissociation kinetics of the 5' splice site-U1 snRNP interaction using purified U1 snRNP and a short RNA oligonucleotide comprising the 5' splice site (5'-SS) consensus sequence of pre-mRNAs (5'-SS RNA oligo). The association is rapid, does not require ATP, and is almost irreversible. Surprisingly, oligonucleotide-directed cleavage of the U1 small nuclear RNA (snRNA) 5' end sequence with RNase H has no significant effect on the rate of association of the 5'-SS RNA oligo, but it does lead to rapid dissociation. This provides evidence that U1-specific snRNP proteins are critical for the 5' splice site recognition while base pairing ensures the stability of the interaction. The recognition of the 5' splice site by U1 snRNP does not result from the individual action of one or more proteins but rather from their organization around U1 snRNA. A consequence of this organization is that the U1-C protein makes direct contacts with the site, as it becomes cross-linked to the RNA oligo upon exposition of the reactions to shortwave UV light.

Cell-Free System↗
PubMed1996

Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

Adenosine Triphosphate↗
PubMed1996

Structural features of U6 snRNA and dynamic interactions with other spliceosomal components leading to pre-mRNA splicing.

In the spliceosome, the pre-mRNA, U2 and U6 snRNAs fold into a catalytic structure exhibiting striking similarities with domain V and VI of group II introns. Building of this tripartite structure implies that an evolutionary conserved base pairing between U4 and U6 snRNAs should be disrupted to allow potentially U6 catalytic residue to interact with U2 snRNAs and the pre-mRNA. The steps leading to U4/U6 disruption have been recently discovered and have been shown to involve a modification of the 3' end of U6 snRNA and the hnRNP C protein.

Base Composition↗
PubMed1995

Stabilised secondary structure at a ribosomal binding site enhances translational repression in E. coli.

The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase is negatively autoregulated at the translational level. The negative feedback is due to the binding of the synthetase to an operator site on its own mRNA located upstream of the initiation codon. The present work describes the characterisation of operator mutants that have the rare property of enhancing repression. These mutations cause (1) a low basal level of expression, (2) a temperature-dependent expression, and (3) an increased capacity of the synthetase to repress its own expression at low temperature. Surprisingly, this enhancement of repression is not explained by an increase of affinity of the mutant operators for the enzyme but by the formation, at low temperature, of a few supplementary base-pairs between the ribosomal binding site and a normally single-stranded domain of the operator. Although this additional base-pairing only slightly inhibits ribosome binding in the absence of repressor, simple thermodynamic considerations indicate that this is sufficient to increase repression. This increase is explained by the competition between the ribosome and repressor for overlapping regions of the mRNA. When the ribosomal binding site is base-paired, the ribosome cannot bind while the repressor can, giving the repressor the advantage in the competition. Thus, the existence of an open versus base-paired equilibrium in a ribosomal binding site of a translational operator amplifies the magnitude of control. This molecular amplification device might be an essential component of translational control considering the low free repressor/ribosome ratio of the low affinity of translational repressors for their target operators.

Base Composition↗
PubMed1995

Disruption of base-paired U4.U6 small nuclear RNAs induced by mammalian heterogeneous nuclear ribonucleoprotein C protein.

Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size. The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end. They are labeled in the presence of [alpha-32P]UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts. That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously. Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches. This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein. Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli. In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP.

Base Composition↗
PubMed1993

Trimethylphosphine binding to horse-heart and sperm-whale myoglobins. Kinetics, proton magnetic resonance assignment and nuclear Overhauser effect investigation of the heme pocket.

Two-dimensional nuclear magnetic resonance techniques have been used to assign resonances corresponding to the heme pocket and several other residues of horse heart and sperm whale myoglobins ligated by trimethylphosphine. The assignment procedure was based mainly on the nuclear Overhauser effect connectivities with the ligand and the heme substituents. For quantitative measurements of Overhauser effects, application of truncated driven techniques between a proton from distal residues and methyl groups from the ligand was used to determine internuclear distances. These new results have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between horse heart and sperm whale myoglobins. The interproton distances between distal amino acid residues and trimethylphosphine were found to be longer in horse heart myoglobin relative to those in sperm whale myoglobin. This result suggests that the size of the heme pocket is larger in horse heart myoglobin. Association and dissociation rate constants were measured for trimethylphosphine binding to myoglobins. Both values were four times larger for horse heart myoglobin than those for sperm whale myoglobin. This observation confirms the structural results obtained with NMR studies and is rationalized by a greater stabilization of a larger pocket in horse heart myoglobin relative to sperm whale myoglobin.

Animals↗
PubMed1993

Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing.

The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.

Adenosine Triphosphate↗