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Biomedical subjects

M Delarue

Publications and source records attributed to M Delarue.

At least 19 recordsLinked to original sources
PubMed1998

Medial cell mixing during axial morphogenesis of the amphibian embryo requires cadherin function.

A truncated form of Xenopus E-cadherin (deltaE-cad) comprising the cytoplasmic and transmembrane domains was overexpressed generating a dominant negative mutation in the urodelan amphibian embryo Pleurodeles waltl. deltaE-cad mRNA and rhodamine-lysinated-dextran (RLDx) cell lineage tracer were microinjected into 32-cell stage blastomeres which contribute principally to the notochord and central nervous system. deltaE-cad expression causes defects in forebrain and hindbrain formation coupled with the development of supernumerary vesicles. Duplication of the notochord also occurs due to the retardation of medial cell intercalation with correlated duplications of spinal cord and somites. These results emphasize the role of cadherins in mediating cell-cell adhesion in early amphibian embryogenesis. They extend to Pleurodeles the observations made in Xenopus, illustrating that despite differences in morphogenetic processes, the molecular mechanisms are conserved in these two species.

Amino Acid Sequence↗
PubMed1998

Role of fibroblast growth factor during early midbrain development in Xenopus.

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain-hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signalling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. We report that explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signalling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. We propose that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF.

Animals↗
PubMed1998

Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis.

A new auxin homeostasis gene in Arabidopsis called SUR2 has been identified. This gene, mapped to the bottom of chromosome 4, is defined by two recessive nuclear mutants designated superroot2 (sur2), which display several abnormalities reminiscent of auxin effects. A number of these characteristics are similar to the phenotype of the previously described auxin-overproducing mutant superroot1 (sur1); however, several lines of evidences reveal that the SUR2 gene defines a new key point in the regulation of endogenous auxin concentrations. The phenotype of the sur1 sur2 double mutant is additive. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of free indole-3-acetic acid correlated with a decreased level of bound auxin in the sur2 mutant. These results suggest that SUR2 may be involved in the control of auxin conjugation.

Arabidopsis↗
PubMed1998

Higher activity of an aldehyde oxidase in the auxin-overproducing superroot1 mutant of Arabidopsis thaliana.

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1-3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.

Aldehyde Oxidase↗
PubMed1997

Fates of the blastomeres of the 32-cell stage Pleurodeles waltl embryo.

Cell fate of the blastomeres at the 32-cell stage in the Pleurodeles waltl embryo was analyzed by injection of rhodamine or fluorescein lysinated-dextran (RLDx or FLDx). At the tailbud stage, the progeny of each blastomere contributed to more than one germ layer with unequal distribution along the anteroposterior and dorsoventral axis. Such a regionalized positioning of the descendants of the 32-cell blastomeres was found in the neuroectoderm, the epidermis, the notochord, the somites, the lateral plate, and the endoderm, but not in the head mesenchyme, the pronephros, or the blood islands. Results of double labeling of juxtaposed blastomeres showed that cell mixing and rearrangement take place during organ formation. Results are compared with those of the 32-cell stage fate map in Xenopus and Rana and reveal the more restricted fate of 32-cell stage blastomeres in Pleurodeles germ layers.

Animals↗
PubMed1997

Cristal mutations in Arabidopsis confer a genetically heritable, recessive, hyperhydric phenotype.

A new class of recessive Arabidopsis mutants, designated cristal (cri) has been isolated which display several abnormalities reminiscent of hyperhydric symptoms. These characteristics include translucent and wrinkled cotyledons and leaves, abnormal chloroplast organization, a reduced amount of chlorophyll, a reduced dry weight and a decreased number of palisade cells in the leaves accompanied by an increase of intercellular space, and therefore give a vitreous appearance to the aerial part. The phenotype is also dependent on the culture medium water potential. The cri1 gene was mapped on chromosome 4 close to the DHS1 marker.

Arabidopsis↗
PubMed1997

A comparison of two-dimensional electrophoresis data with phenotypical traits in Arabidopsis leads to the identification of a mutant (cri1) that accumulates cytokinins.

Total proteins extracted from developmental mutants of Arabidopsis thaliana (L.) Heyhn. and from wild-type plants cultivated in the presence of various hormones were analyzed by two-dimensional (2-D) gel electrophoresis. Computer analysis of 2-D gels followed by a statistical treatment of data allowed us to build a phenogram that describes the biochemical distances between the different genotypes. Analysis of the 2-D electrophoresis data allowed us to discriminate mutants in agreement with phenotypical and physiological traits. This biochemical analysis helped us to develop a working hypothesis which led us to show that one developmental mutant (cri1) overaccumulates cytokinins.

Arabidopsis↗
PubMed1997

The inverse protein folding problem: self consistent mean field optimisation of a structure specific mutation matrix.

The goal of the inverse folding problem is to supply a list of sequences compatible with a known protein structure. If two-body interactions are taken into account in energy calculations, an exhaustive exploration of the energy landscape in sequence space cannot be achieved because of the huge number of possible combinations. To circumvent this problem, we propose a method in which multiple copies corresponding to every possible side-chain type are attached to each C alpha position in the protein. The weights of each copy (stored in the sequence matrix SM) are refined using mean field theory: each side-chain copy interacts with the mean field generated by all possible side-chain copies at neighbouring positions, weighted by their respective probabilities. The potential energy is simply taken to be amino acid pair potentials of mean force. The method converges in a few cycles to a self-consistent solution. The refined matrix does not depend on the starting point; therefore the method succeeds in removing memory effects. Starting solely from the backbone of the known structure, and without information from the initial sequence, the final sequence matrix SM is shown to be able to retrieve significant sequence information, as observed through a series of structure-recognizes-sequence(s) computer experiments. The issue of specificity is discussed in detail.

Amino Acid Sequence↗
PubMed1997

The native sequence determines sidechain packing in a protein, but does optimal sidechain packing determine the native sequence?

Globular proteins have highly compact structures and the corresponding packing interactions are widely considered as the principal determinant of the native structure. It is therefore important that theoretical approaches to protein design explicitly take in account packing, which requires that a full atomic representation of the designed protein is maintained. As a first step towards this goal, we have developed in this report an inverse folding algorithm with the aim of specifically designing amino acid sequences which optimise sidechain packing for a given protein fold. The design is performed by a global Monte Carlo optimisation in sequence space, with constant amino acid composition and a full-atom representation of the various protein models. Packing is defined by a Lennard-Jones potential. The program was tested by designing stable sequence variants for the chymotrypsin inhibitor fold. The final protein models showed an increase in intramolecular atomic contacts and a decrease in the overall volume compared to the native structure. Starting from the backbone only of the target structure, the algorithm did gradually retrieve reliable though limited sequence information. Higher compatibility might be achieved by improving the potential, however our results suggest that packing interactions are an essential element of a yet-to-be-defined successful energy function for protein design.

Algorithms↗
PubMed1996

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Amino Acid Sequence↗
PubMed1996

Mean-field minimization methods for biological macromolecules.

Simulations of macromolecular structures involve the minimization of a potential-energy function that presents many local minima. Mean-field theory provides a tool that enables us to escape these minima, by enhancing sampling in conformational space. The number of applications of this technique has increased significantly over the past year, enabling problems with protein-homology modelling and inverted protein structure prediction to be solved.

Amino Acid Sequence↗
PubMed1996

What mechanisms drive cell migration and cell interactions in Pleurodeles?

Embryogenesis implies a strict control of cell interaction and cell migration. The spatial and temporal regulation of morphogenetic movements occurring during gastrulation is directly dependent on the early cell interactions that take place in the blastula. The newt Pleurodeles waltl is a favorable model for the study of these early morphogenetic events. The combination of orthotopic grafting and fluorescent lineage tracers has led to precise early gastrula mesoderm fate maps. It is now clear that there are no sharp boundaries between germ layers at the onset of gastrulation but rather diffuse transition zones. The coordination of cell movements during gastrulation is closely related to the establishment of dorsoventral polarity. Ventralization by U.V. irradiation or dorsalization by lithium treatment modifies the capacity for autonomous migration on the fibronectin coated substratum of marginal zone cells accordingly. It is now firmly established that mesodermal cells need to adhere to a fibrillar extracellular matrix (ECM) to undergo migration during gastrulation. Extracellular fibrils contain laminin and fibronectin (FN). Interaction of cells with ECM involves receptors of the beta 1 integrin family. A Pleurodeles homolog of the alpha v integrin subunit has been recently identified. Protein alpha v expression is restricted to the surface of mesodermal cells during gastrulation. Integrin-mediated interactions of cells with FN are essential for ECM assembly and mesodermal cell migration. Intracellular injection of antibodies to the cytoplasmic domain of beta 1 into early cleavage embryos causes inhibition of FN fibril formation. Intrablastocoelic injections of several probes including antibodies to FN or integrin alpha 5 beta 1, competitive peptides to the major cell binding site of FN or the antiadhesive protein tenascin all block mesodermal cell migration. This results in a complete arrest of gastrulation indicating that mesodermal cell migration is a major driving force in urodele gastrulation. It is now possible to approach the role of fibroblast growth factor (FGF) during cell interactions taking place in urodele embryos. Four different FGF receptors (FGFR) have been cloned in Pleurodeles. Each of them has a unique mRNA expression pattern. FGFR-1, FGFR-3, and the variant of FGFR-2 containing the IIIb exon are maternally expressed and might be involved in mesodermal induction. During gastrulation, FGFR-3 and FGFR-4 have a restricted pattern of expression, whereas FGFR-1 mRNA is nearly uniformly distributed. Splicing variants FGFR-2IIIb and FGFR-2IIIc have exclusive expression patterns during neurulation. IIIb is expressed in epidermis and IIIc in neural tissue, suggesting a function in the differentiation of ectodermal derivatives.

Animals↗
PubMed1995

Atomic environment energies in proteins defined from statistics of accessible and contact surface areas.

Atomic contact potentials are derived by statistical analysis of atomic surface contact areas versus atom type in a database of non-homologous protein structures. The atomic environment is characterized by the surface area accessible to solvent and the surface of contacts with polar and non-polar atoms. Four types of atoms are considered, namely neutral polar atoms from protein backbones and from protein side-chains, non-polar atoms and charged atoms. Potential energies delta Ej(E) are defined from the preference for an atom of type j to be in a given environment E compared to the expected value if everything was random; Boltzmann's law is then used to transform these preferences into energies. These new potentials very clearly discriminate misfolded from correct structural models. The performance of these potentials are critically assessed by monitoring the recognition of the native fold among a large number of alternative structural folding types (the hide-and-seek procedure), as well as by testing if the native sequence can be recovered from a large number of randomly shuffled sequences for a given 3D fold (a procedure similar to the inverse folding problem). We suggest that these potentials reflect the atomic short range non-local interactions in proteins. To characterise atomic solvation alone, similar potentials were derived as a function of the percentage of solvent-accessible area alone. These energies were found to agree reasonably well with the solvation formalism of Eisenberg and McLachlan.

Animals↗
PubMed1995

Partition of aminoacyl-tRNA synthetases in two different structural classes dating back to early metabolism: implications for the origin of the genetic code and the nature of protein sequences.

We describe, on the molecular level, a possible fuzzy and primordial translation apparatus capable of synthesizing polypeptides from nucleic acids in a world containing a mixture of coevolving molecules of RNA and proteins already arranged in metabolic cycles (including cofactors). Close attention is paid to template-free systems because they are believed to be the immediate ancestors of this primordial translation apparatus. The two classes of aminoacyl-tRNA synthetases (aaRSs), as seen today, are considered as the remnants of such a simple imprecise translation apparatus and are used as guidelines for the construction of the model. Earlier theoretical work by Bedian on a related system is invoked to show how specificity and stability could have been achieved automatically and rather quickly, starting from such an imprecise system, i.e., how the encoded synthesis of proteins could have appeared. Because of the binary nature of the underlying proto-code, the first genetically encoded proteins would then have been alternating copolymers with a high degree of degeneracy, but not random. Indeed, a clear signal for alternating hydrophobic and hydrophilic residues in present-day protein sequences can be detected. Later evolution of the genetic code would have proceeded along lines already discussed by Crick. However, in the initial stages, the translation apparatus proposed here is in fact very similar to the one postulated by Woese, only here it is given a molecular framework. This hypothesis departs from the paradigm of the RNA world in that it supposes that the origin of the genetic code occurred after the apparition of some functional (statistical) proteins first. Implications for protein design are also discussed.

Amino Acid Sequence↗
PubMed1995

Aminoacyl-tRNA synthetases.

Detailed mechanisms for each step of the reaction catalyzed by both class I and class II aminoacyl-tRNA synthetases have been proposed on the basis of crystallographic data of aminoacyl-tRNA synthetases in complex with their different substrates. Despite the very different topologies of the two classes, there are striking and unanticipated chemical similarities between their active sites and proposed mechanisms.

Amino Acid Sequence↗
PubMed1995

A self consistent mean field approach to simultaneous gap closure and side-chain positioning in homology modelling.

A new computational procedure which simultaneously provides gap closure and side-chain positioning in homology modelling is described. It uses a database search scheme to generate fragments to model gaps, a rotamer library to define side-chain conformations, and iteratively refines a conformational matrix CM, such that its elements CM(i,j,o) and CM(i,j,k) give the probabilities that the backbone of residue i adopts the conformation described by fragment j and that its side-chain adopts the conformation of its possible rotamer k. Each residue experiences the average of all possible environments, weighted by their respective probabilities. The method converges, thereby deserving the name of 'self consistent mean field' approach.

Algorithms↗