Biomedical subjects

M Delarue

Publications and source records attributed to M Delarue.

At least 55 records · Page 3Linked to original sources

PubMed1994

Polar and nonpolar atomic environments in the protein core: implications for folding and binding.

Hydrophobic interactions are believed to play an important role in protein folding and stability. Semi-empirical attempts to estimate these interactions are usually based on a model of solvation, whose contribution to the stability of proteins is assumed to be proportional to the surface area buried upon folding. Here we propose an extension of this idea by defining an environment free energy that characterizes the environment of each atom of the protein, including solvent, polar or nonpolar atoms of the same protein or of another molecule that interacts with the protein. In our model, the difference of this environment free energy between the folded state and the unfolded (extended) state of a protein is shown to be proportional to the area buried by nonpolar atoms upon folding. General properties of this environment free energy are derived from statistical studies on a database of 82 well-refined protein structures. This free energy is shown to be able to discriminate misfolded from correct structural models, to provide an estimate of the stabilization due to oligomerization, and to predict the stability of mutants in which hydrophobic residues have been substituted by site-directed mutagenesis, provided that no large structural modifications occur.

Chemical Phenomena↗

PubMed1994

Superficial cells in the early gastrula of Rana pipiens contribute to mesodermal derivatives.

We have studied the fate of presumptive mesodermal cells in the early Rana pipiens gastrula. We labeled superficial cells of the early gastrula with 125I. We also labeled all cells in a gastrula with rhodamine-lysine-dextran cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the notochord and somite regions are both derived from the superficial and deep portions of circumblastoporal regions. In contrast, pronephros, lateral plate, cardiac anlagen, and blood cells only arise from deep cells in circumblastoporal regions. Such observations indicate that the fate map for R. pipiens is different from that of Xenopus laevis, where mesodermal derivatives appear to be restricted to the deep layers, and from that of Pleurodeles waltl, where all mesodermal derivatives are formed both from superficial and deep layers. We also have shown that from the neurula stage there is substantial mixing between cells due to the ingression of cells in the dorsal region so that superficial labeled cells, initially located in the roof of the archenteron, contribute to ventral regions of both the somites and notochord.

Animals↗

PubMed1994

Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases.

The sequence of Rift Valley fever virus L segment that we published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA molecule. Here, we have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 negative-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called 'polymerase module', we identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented negative-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the 'polymerase module', we have determined the position of the conserved residues in these polymerases and discuss their possible functions in light of the available structural information.

Amino Acid Sequence↗

PubMed1993

Crystal structure of cleaved bovine antithrombin III at 3.2 A resolution.

The crystal structure of cleaved antithrombin III (ATIII) has been determined to 3.2 A resolution by single isomorphous replacement, real space density modification and phase extension protocols. The heavy-atom sites and the first molecular envelope were determined owing to the molecular replacement solution previously reported and partially refined. Refinement of the two molecules of the asymmetric unit led to a crystallographic R-factor of 0.212 for all reflections between 8.0 and 3.2 A, without inclusion of water molecules. The root-mean-square deviation from ideal values is, respectively, 0.015 A and 3.6 degrees for bond lengths and bond angles. The topology of the molecule closely resembles that of cleaved serpins inhibitors with the two residues forming the reactive bond at opposite ends of the molecule. The most significant difference between ATIII and alpha 1-antitrypsin lies in the 45 residue N-terminal extension in ATIII which contribute to the definition of the heparin binding site. This loop region at the surface of the molecule is held by two disulphide bridges to the protein core and exhibits high temperature factor values. It forms a valley which restrains the possibilities for binding of heparin. Docking of the pentasaccharide unit which represents the minimum fragment of heparin able to bind to ATIII indicates a possible role for arginine 14 in the interaction of heparin and the protein.

Amino Acid Sequence↗

PubMed1993

Identification of potential active-site residues in the human poly(ADP-ribose) polymerase.

The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J. (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.

Amino Acid Sequence↗

PubMed1993

The aminoacyl-tRNA synthetase family: modules at work.

The combined use of molecular and structural biology techniques has proved very efficient in elucidating structure-function relationships in aminoacyl-tRNA synthetases. Our present understanding of this family of enzymes is based on two main unifying principles: (i) division into two different classes, corresponding to two different modes of ATP binding and attachment of the activated amino acid to the last nucleotide of tRNA (either 2'OH or 3'OH of the ribose) by two different catalytic mechanisms and two structural domains with completely different folding, and (ii) the modular organization into separate and additional domains that we are just beginning to understand. Sequence analysis complements very nicely existing structural, biochemical and genetic results and makes them more general, leading to verifiable predictions.

Amino Acid Sequence↗

PubMed1992

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.

Chemical Phenomena↗

PubMed1992

A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl.

We have determined the fate of presumptive mesodermal cells in the early Pleurodeles waltl gastrula. We labeled all cells in a gastrula with RLDx cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the presumptive regions are generally distributed according to classical fate maps for urodeles but that the boundaries between presumptive regions are indistinct, due to extensive intermingling between cells at the edges of grafted regions. We have shown that there is a high dorsal to low ventral gradient of mixing between superficial and deep cells.

Animals↗

PubMed1990

Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs.

The aminoacyl-transfer RNA synthetases (aaRS) catalyse the attachment of an amino acid to its cognate transfer RNA molecule in a highly specific two-step reaction. These proteins differ widely in size and oligomeric state, and have limited sequence homology. Out of the 18 known aaRS, only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display two short common consensus sequences ('HIGH' and 'KMSKS') which indicate, as observed in three crystal structures, the presence of a structural domain (the Rossman fold) that binds ATP. We report here the sequence of Escherichia coli ProRS, a dimer of relative molecular mass 127,402, which is homologous to both ThrRS and SerRS. These three latter aaRS share three new sequence motifs with AspRS, AsnRS, LysRS, HisRS and the beta subunit of PheRS. These three motifs (motifs 1, 2 and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in two classes is found to be strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.

Amino Acid Sequence↗

PubMed1990

Antithrombin III: structural and functional aspects.

Antithrombin III is a plasma glycoprotein responsible for thrombin inhibition in the blood coagulation cascade. The X-ray structure of its cleaved form has been determined and refined to 3.2 A resolution. The overall topology is similar to that of alpha 1-antitrypsin, another member of the serpin (serine protease inhibitor) superfamily. The biological activity of antithrombin III is mediated by a polysaccharide, heparin. The binding site of this effector is described. A possible structural transition from the native to the cleaved structure is discussed.

Amino Acid Sequence↗

PubMed1990

An attempt to unify the structure of polymerases.

With the great availability of sequences from RNA- and DNA-dependent RNA and DNA polymerases, it has become possible to delineate a few highly conserved regions for various polymerase types. In this work a DNA polymerase sequence from bacteriophage SPO2 was found to be homologous to the polymerase domain of the Klenow fragment of polymerase I from Escherichia coli, which is known to be closely related to those from Staphylococcus pneumoniae, Thermus aquaticus and bacteriophages T7 and T5. The alignment of the SPO2 polymerase with the other five sequences considerably narrowed the conserved motifs in these proteins. Three of the motifs matched reasonably all the conserved motifs of another DNA polymerase type, characterized by human polymerase alpha. It is also possible to find these three motifs in monomeric DNA-dependent RNA polymerases and two of them in DNA polymerase beta and DNA terminal transferases. These latter two motifs also matched two of the four motifs recently identified in 84 RNA-dependent polymerases. From the known tertiary architecture of the Klenow fragment of E. coli pol I, a spatial arrangement can be implied for these motifs. In addition, numerous biochemical experiments suggesting a role for the motifs in a common function (dNTP binding) also support these inferences. This speculative hypothesis, attempting to unify polymerase structure at least locally, if not globally, under the pol I fold, should provide a useful model to direct mutagenesis experiments to probe template and substrate specificity in polymerases.

Amino Acid Sequence↗

PubMed1990

Fibronectin-rich fibrillar extracellular matrix controls cell migration during amphibian gastrulation.

We have reviewed the evidence supporting the notion that the fibrillar extracellular matrix on the basal surface of the blastocoel roof in amphibian embryos directs and guides mesodermal cell migration during gastrulation. Based on extensive experimental evidence in several different systems, we conclude the following: (i) the fibrillar extracellular matrix contains fibronectin (FN) and laminin. (ii) The fibrils are oriented in such a way as to promote directional migration of mesodermal cells during migration. (iii) We have used several different probes to disrupt the interaction between migrating mesodermal cells and the fibrillar extracellular matrix. These probes include: (a) nucleocytoplasmic and interspecific hybridization. Such embryos have defects in FN synthesis and gastrulation. (b) Fab' fragments of anti-FN and anti-integrin VLA-5 IgGs prohibit mesodermal cell adhesion both in vitro and in vivo and gastrulation is arrested. (c) Peptides containing the RGDS sequence specifically inhibit interactions between migrating mesodermal cells and the FN-fibrillar matrix. (d) Tenascin blocks cell adhesion to FN in vitro and gastrulation in vivo. (e) Antibodies against the cytoplasmic domain of beta 1 integrin, when injected into blastomeres, prevent FN-fibrillogenesis in progeny of injected blastomeres and delay mesodermal cell migration selectively in the progeny of injected blastomeres but not in the uninjected blastomere progeny.

Amphibians↗

PubMed1989

Crystallization and preliminary crystallographic data for bovine antithrombin III.

Crystals of bovine antithrombin III were obtained in the presence of metal ions with ammonium sulphate as precipitating agent. Crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with cell parameters a = b = 91.4 A, c = 383.1 A; there are two molecules per asymmetric unit. Electrophoresis experiments and amino acid sequence analysis of the N-terminal part of redissolved crystals suggest that the protein molecules are cleaved at the active site.

Animals↗

PubMed1989

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.

Four consensus sequences are conserved with the same linear arrangement in RNA-dependent DNA polymerases encoded by retroid elements and in RNA-dependent RNA polymerases encoded by plus-, minus- and double-strand RNA viruses. One of these motifs corresponds to the YGDD span previously described by Kamer and Argos (1984). These consensus sequences altogether lead to 4 strictly and 18 conservatively maintained amino acids embedded in a large domain of 120 to 210 amino acids. As judged from secondary structure predictions, each of the 4 motifs, which may cooperate to form a well-ordered domain, places one invariant amino acid in or proximal to turn structures that may be crucial for their correct positioning in a catalytic process. We suggest that this domain may constitute a prerequisite 'polymerase module' implicated in template seating and polymerase activity. At the evolutionary level, the sequence similarities, gap distribution and distances between each motif strongly suggest that the ancestral polymerase module was encoded by an individual genetic element which was most closely related to the plus-strand RNA viruses and the non-viral retroposons. This polymerase module gene may have subsequently propagated in the viral kingdom by distinct gene set recombination events leading to the wide viral variety observed today.

Amino Acid Sequence↗

PubMed1988

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis.

A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-1/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.

Animals↗

PubMed1987

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae.

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.

Animals↗

PubMed1986

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos.

Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.

Animals↗

PubMed1986

Cyclic regulation of cytokinesis in amphibian eggs.

The effects of amphibian egg cytoplasm extracted at different times after activation and during the first four cleavages on cytokinesis were examined. Extracts of artificially activated or fertilized Xenopus or Pleurodeles eggs taken at the time of activation (T = 0) provoked precocious cleavage furrows in Pleurodeles eggs. Between T = 0.25 and T = 0.75 of the first cell cycle, the period corresponding to interphase, an inhibitory effect was found, and the division of injected eggs was delayed up to 30%. After T = 0.75, that is during mitosis, the cleavage induction effect was observed again. These enhancing and inhibitory effects were also found in the two fractions obtained following gel filtration of the cytoplasmic extracts. These experiments support the hypothesis that two antagonistic factors control cytokinesis. The inhibitory factor is active only during interphase, while the positive factor is present during mitosis and appears to regulate cytokinesis.

Animals↗