Showing papers by "Centre national de la recherche scientifique published in 2000"

Mitochondrial control of cell death

Abstract: In many instances, permeabilization of mitochondrial membranes is a rate-limiting step of apoptotic or necrotic cell demise. This has important consequences for the pathophysiology of cell death, as well as for its pharmacological control.

Electronics using hybrid-molecular and mono-molecular devices

Abstract: The semiconductor industry has seen a remarkable miniaturization trend, driven by many scientific and technological innovations. But if this trend is to continue, and provide ever faster and cheaper computers, the size of microelectronic circuit components will soon need to reach the scale of atoms or molecules—a goal that will require conceptually new device structures. The idea that a few molecules, or even a single molecule, could be embedded between electrodes and perform the basic functions of digital electronics—rectification, amplification and storage—was first put forward in the mid-1970s. The concept is now realized for individual components, but the economic fabrication of complete circuits at the molecular level remains challenging because of the difficulty of connecting molecules to one another. A possible solution to this problem is ‘mono-molecular’ electronics, in which a single molecule will integrate the elementary functions and interconnections required for computation.

Systematic XPS studies of metal oxides, hydroxides and peroxides

Abstract: The results of a systematic XPS study, under high controlled conditions, of different basic oxides of transition metals, alkali and alkaline-earth metals are presented; the XPS data of some hydroxides and peroxides are also reported. Variations of the O 1s binding energies are analysed and one point of interest is the large binding energy scale obtained for O 1s peaks all associated with a ‘‘2− ’’ formal charge. Through extended Huckel theory-tight binding (EHT-TB) calculations, attempts are made to rationalize the observed variations. The results illustrate the significant differences between real charges on oxygen atoms in transition metal and alkaline-earth oxides.

Comparative Genomics of the Eukaryotes

Abstract: A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

A simple model for the absorption of starlight by dust in galaxies

Abstract: We present a new model for computing the effects of dust on the integrated spectral properties of galaxies, based on an idealized description of the main features of the interstellar medium (ISM). The model includes the ionization of H II regions in the interiors of the dense clouds in which stars form and the influence of the finite lifetime of these clouds on the absorption of radiation. We compute the production of emission lines and the absorption of continuum radiation in the H II regions and the subsequent transfer of line and continuum radiation in the surrounding H I regions and the ambient ISM. This enables us to interpret simultaneously all the observations of an ultraviolet-selected sample of nearby starburst galaxies, including the ratio of far-infrared to ultraviolet luminosities, the ratio of Hα to Hβ luminosities, the Hα equivalent width, and the ultraviolet spectral slope. We show that the finite lifetime of stellar birth clouds is a key ingredient for resolving an apparent discrepancy between the attenuation of line and continuum photons in starburst galaxies. In addition, we find that an effective absorption curve proportional to λ-0.7 reproduces the observed relation between the ratio of far-infrared to ultraviolet luminosities and the ultraviolet spectral slope. We interpret this relation most simply as a sequence in the overall dust content of the galaxies. The shallow wavelength dependence of the effective absorption curve is compatible with the steepness of known extinction curves if the dust has a patchy distribution. In particular, we find that a random distribution of discrete clouds with optical depths similar to those in the Milky Way provides a consistent interpretation of all the observations. A noteworthy outcome of our detailed analysis is that the observed mean relations for starburst galaxies can be closely approximated by the following simple recipe: use an effective absorption curve proportional to λ-0.7 to attenuate the line and continuum radiation from each stellar generation, and lower the normalization of the curve, typically by a factor of 3 after 107 yr, to account for the dispersal of the birth clouds. This recipe or our full model for absorption can be incorporated easily into any population synthesis model.

Tissue-engineered bone regeneration.

Abstract: Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.

The DNA sequence of human chromosome 21

Abstract: Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.

Abstract: Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.

Abstract: The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1–3). As Ucp2 is widely expressed in mammalian tissues4,5, uncouples respiration6 and resides within a region of genetic linkage to obesity4, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages4,5,7, suggesting a role for Ucp2 in immunity or inflammatory responsiveness4. We investigated the response to infection with Toxoplasma gondii in Ucp2−/− mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2−/− mice (63% decrease, P<0.04). Macrophages from Ucp2 −/− mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.

Hsp27 negatively regulates cell death by interacting with cytochrome c

Abstract: Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

Reconstruction of a scalar tensor theory of gravity in an accelerating universe

Abstract: The present acceleration of the Universe strongly indicated by recent observational data can be modeled in the scope of a scalar-tensor theory of gravity. We show that it is possible to determine the structure of this theory along with the present density of dustlike matter from two observable cosmological functions: the luminosity distance and the linear density perturbation in the dustlike matter component as functions of redshift. Explicit results are presented in the first order in the small inverse Brans-Dicke parameter ${\ensuremath{\omega}}^{\ensuremath{-}1}$.

Cytochromes P450: a success story

Abstract: Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.

Expression of Cd34 and Myf5 Defines the Majority of Quiescent Adult Skeletal Muscle Satellite Cells

Abstract: Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34+ve myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34+ve cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5nlacZ/+ mice, all CD34+ve satellite cells also express β-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34−ve, Myf5−ve minority may be involved in maintaining the lineage-committed majority.

Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity

Abstract: By integrating an agonist satiety signal, provided by alpha–melanocyte-stimulating hormone (α-MSH), and an antagonist signal, provided by agouti-related protein (AGRP), the melanocortin-4 receptor (MC4-R) is a key element in the hypothalamic control of food intake. Inactivation of the gene encoding this G protein–coupled receptor causes obesity in mice. In humans, frameshift mutations in MC4-R cause an early-onset dominant form of obesity in two families. In this study we find a high frequency (4%) of rare heterozygous MC4-R mutations in a large population of morbidly obese patients. No such mutations were found in controls. By analyzing the phenotypes of the probands carrying these mutations, we demonstrate that these patients display a common, nonsyndromic form of obesity. Interestingly, functional analysis of the mutant receptors indicates that obesity-associated defects in MC4-R range from loss of function to constitutive activation. Transmission of these mutations in the families of the carriers indicates a variable expressivity that is not related to the functional severity of the mutations. This variable expressivity of MC4-R–associated obesity is not due to variations in genes for α-MSH or AGRP. Taken together, these results demonstrate that MC4-R mutations are a frequent but heterogeneous genetic cause of morbid obesity.

The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP.

Abstract: Animal and plant uncoupling protein (UCP) homologues form a subfamily of mitochondrial carriers that are evolutionarily related and possibly derived from a proton/anion transporter ancestor. The brown adipose tissue (BAT) UCP1 has a marked and strongly regulated uncoupling activity, essential to the maintenance of body temperature in small mammals. UCP homologues identified in plants are induced in a cold environment and may be involved in resistance to chilling. The biochemical activities and biological functions of the recently identified mammalian UCP2 and UCP3 are not well known. However, recent data support a role for these UCPs in State 4 respiration, respiration uncoupling and proton leaks in mitochondria. Moreover, genetic studies suggest that UCP2 and UCP3 play a part in energy expenditure in humans. The UCPs may also be involved in adaptation of cellular metabolism to an excessive supply of substrates in order to regulate the ATP level, the NAD(+)/NADH ratio and various metabolic pathways, and to contain superoxide production. A major goal will be the analysis of mice that either lack the UCP2 or UCP3 gene or overexpress these genes. Other aims will be to investigate the possible roles of UCP2 and UCP3 in response to oxidative stress, lipid peroxidation, inflammatory processes, fever and regulation of temperature in certain specific parts of the body.

Nicotinic receptors at the amino acid level.

Abstract: nAChRs are pentameric transmembrane proteins into the superfamily of ligand-gated ion channels that includes the 5HT3, glycine, GABAA, and GABAC receptors. Electron microscopy, affinity labeling, and mutagenesis experiments, together with secondary structure predictions and measurements, suggest an all-β folding of the N-terminal extracellular domain, with the connecting loops contributing to the ACh binding pocket and to the subunit interfaces that mediate the allosteric transitions between conformational states. The ion channel consists of two distinct elements symmetrically organized along the fivefold axis of the molecule: a barrel of five M2 helices, and on the cytoplasmic side five loops contributing to the selectivity filter. The allosteric transitions of the protein underlying the physiological ACh-evoked activation and desensitization possibly involve rigid body motion of the extracellular domain of each subunit, linked to a global reorganization of the transmembrane domain responsible for channe...

On the properties of small-world network models

Abstract: We study the small-world networks recently introduced by Watts and Strogatz [Nature 393, 440 (1998)], using analytical as well as numerical tools. We characterize the geometrical properties resulting from the coexistence of a local structure and random long-range connections, and we examine their evolution with size and disorder strength. We show that any finite value of the disorder is able to trigger a “small-world” behaviour as soon as the initial lattice is big enough, and study the crossover between a regular lattice and a “small-world” one. These results are corroborated by the investigation of an Ising model defined on the network, showing for every finite disorder fraction a crossover from a high-temperature region dominated by the underlying one-dimensional structure to a mean-field like low-temperature region. In particular there exists a finite-temperature ferromagnetic phase transition as soon as the disorder strength is finite. [0.5cm]

Signal transduction through prion protein.

Abstract: The cellular prion protein PrPc is a glycosylphosphatidylinositol-anchored cell-surface protein whose biological function is unclear. We used the murine 1C11 neuronal differentiation model to search for PrPc-dependent signal transduction through antibody-mediated cross-linking. A caveolin-1-dependent coupling of PrPc to the tyrosine kinase Fyn was observed. Clathrin might also contribute to this coupling. The ability of the 1C11 cell line to trigger PrPc-dependent Fyn activation was restricted to its fully differentiated serotonergic or noradrenergic progenies. Moreover, the signaling activity of PrPc occurred mainly at neurites. Thus, PrPc may be a signal transduction protein.

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis

Abstract: Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale (∼50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD.fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP deple...

Two distinct pathways leading to nuclear apoptosis.

Abstract: Apaf-1−/− or caspase-3−/− cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1−/− or caspase-3−/− cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1−/− or caspase-3−/− cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.

A turbulence scheme allowing for mesoscale and large‐eddy simulations

Abstract: The paper describes the turbulence scheme implemented in the Meso-NH community research model, and reports on some validation studies. Since the model is intended to perform both large-eddy and mesoscale simulations, we have developed a full three-dimensional scheme, based on the original method of Redelsperger and Sommeria. A prognostic equation for the turbulent kinetic energy is used, together with conservative variables for moist non-precipitating processes. A particularity of the scheme is the use of variable turbulent Prandtl and Schmidt numbers, consistently derived from the complete set of second-order turbulent-moment equations. The results of three idealized boundary-layer simulations allowing detailed comparisons with other large-eddy simulation (LES) models are discussed, and lead to the conclusion that the model is performing satisfactorily. The vertical flux and gradient computation can be run in isolation from the rest of the scheme, providing an efficient single-column parametrization for the mesoscale configuration of the model, if an appropriate parametrization of the eddy length-scale is used. The mixing-length specification is then the only aspect of the scheme which differs from the LES to the mesoscale configuration, and the numerical constants used for the closure terms are the same in both configurations. The scheme is run in single-column mode for the same three cases as above, and a comparison of single-column and LES results again leads to satisfactory results. It is believed that this result is original, and is due to the proper formulation of the parametrized mixing length and of the turbulent Prandtl and Schmidt numbers. In fact, a comparison of the parametrized mixing length with the length-scale of the energy-containing eddies deduced by spectral analysis of the LES shows interesting similarity.

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

Abstract: We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.