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A theory has been developed that could explain prion infection. Prions could be molecular chaperones that are required for their own assembly. The theory has been deduced from an analysis of protein folding and consequences explored by computer simulations. Thermo-kinetic analysis of protein folding shows that a misfolded chaperone gives rise to new misfolded chaperones. Consequently such a protein could behave as a new kind of informative molecule and replicate misfolding according to a process similar to infection. A quantitative model has been derived from this hypothesis that displays the characteristics of prion infections. This hypothesis satisfactorily explains the three manifestations infection, familial and sporadic — that are the characteristics features of all prion diseases.

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Under our experimental conditions, the retention time depends not on the chain length but rather on the base composition and the secondary structure of the molecule. Because of the torsional strain caused by the supercoiling of the plasmid, more of its bases are accessible for interaction with the hydrophobic stationary phase. This increases the retention time of the supercoiled DNA compared to the relaxed or linear DNA. We have exploited these properties to analyze the quality of plasmid preparations. The method is more sensitive to contaminants than common electrophoretic techniques. Furthermore, we describe a convenient and rapid procedure for purifying plasmid DNA. The highly pure plasmid is biologically more active for most of the enzymatic reactions commonly used in genetic engineering.

Large RNA molecules are retained on reversed-phase columns and can be eluted by increasing the concentration of acetonitrile. The retention behaviour depends on the base composition but not on the length of the chain, thereby providing a means for the separation of RNA molecules with different base composition. However, isocratic elution is not feasible due to the sudden changes in retention characteristics which are observed with slight variations in acetonitrile concentration. In order to interpret these results, a model has been developed called 'multiple-point interaction theory', based on the assumption that macromolecules are flexible and very large as compared to the hydrophobic phase. This model fits the experimental data and could be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. In the case of RNA, it has been observed that the retention time depends on ionic strength, pH, and temperature as expected for a true partition between two phases. Furthermore, an artifact called the 'polypeak phenomenon' appears at high flow rates which decreases the resolution of RNA. This polypeak phenomenon is controlled by the slope of the gradient d phi/dV.

Direct contact between lipids solubilized by octyl glucoside and Amberlite XAD-2 beads yielded large liposomes (240 nm diameter) with no residual detergent molecules, in less than 10 min. This extemporaneous preparation of liposomes was prepared with a detergent/bead ratio no higher than 0.12 (mumol/mg) and a phosphatidylcholine/phosphatidylserine/cholesterol molar ratio of 1:1:1. The liposomes were mainly unilamellar, as deduced from thin section and freeze-fracture electron micrographs and from measurement of calcein incorporation into the vesicles. The relatively large internal volume of these vesicles (8.9 l/mol lipid) accounts for the high percentage of entrapped material observed. The percentage increased with lipid concentration, but could not be increased above 20% corresponding to 20 mM total lipids.

A ribonucleoprotein complex whose RNA complement consists exclusively of small nuclear RNA species (snRNA) has been purified from particles containing heterogenous nuclear RNA (hnRNP) from HeLa cells. This was accomplished by taking advantage of their ability to band at a density of about 1.43 g/cm3 in plain cesium chloride as well as in cesium chloride gradients containing 0.5% sarkosyl without prior aldehyde fixation. After these two steps of equilibrium density centrifugation, these snRNPs were still largely contaminated by free proteins (and especially phosphoproteins). A final step of purification by velocity sedimentation in a sucrose gradient containing 0.5 M cesium chloride and 0.5% sarkosyl was efficient in completely eliminating all free proteins. U1, U2, U4, U5 and U6 species according to the nomenclature of Lerner et al. (Nature, (1980) 283, 220-224) were found in these purified snRNPs, while a significant part of U6 and a small amount of U2 were found in the bottom fraction. 5S species behaved entirely as free RNA and is presumably a contaminant of cytoplasmic origin. Electrophoresis of proteins from snRNP labeled in vivo with (35S) methionine, revealed four bands with migrations corresponding to molecular weights ranging between 10,000 and 14,000 daltons.

A mouse genomic library was screened for sequences complementary to U1 nuclear RNA. Out of the eight clones tested, none contained more than one copy of U1. Six of them were identical and one of those (clone 0U1-XIII) was further analyzed. This latter clone contained no other gene for discrete species of small size RNA in the 8 Kb EcoRI fragment encoding U1. A 248 bp Bg1II fragment from 0U1-XIII encompassing the full length of U1 as well as flanking regions on both sides has been subcloned and sequenced in M13 phage. Although the coding region was 96.5% homologous to rat U1a RNA, there is no direct evidence that this clone is a true gene. 3' and 5' flanking sequences of this as well as other published clones have been searched for homologies and the results of this search are discussed.

Polysomes of HeLa cells were adsorbed on Poly (U)-Sepharose columns. This adsorption is probably due to poly (A) sequences at 3′ terminus of the messenger. Stepwise elution initially removed ribosomal subunits thereafter mRNA and a set of proteins. These proteins are identical with the main components of the polysomal messenger ribonucleo-protein particles described previously. Thus, this method allows their rapid and easy separation from ribosomal and other proteins.

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.

Ribonucleoprotein Particles Experiments indicate that eggs and embryos of the North Sea sea urchin Echinus esculentus are excellent material to investigate the regulation of protein synthesis before and after fertilization. The dormant egg contains several proteins which are absent from embryos. Such additional proteins have been detected on the small ribosomal subunits, in the salt wash of whole ribosomes, and as­ sociated with cytoplasmic messenger ribonucleoprotein particles.

We have shown previously (Liautard et al., 1982, J. Mol. Biol., 162, 623-643) that digestion with micrococcal nuclease under drastic conditions of a pure U1 snRNP, as well as a mixture containing U2, U1, U4, U5 and U6 snRNPs, gives rise to resistant RNA fragments derived from all but U6 snRNAs. As an attempt to elucidate the way in which snRNPs are attached to their native structure, the same approach was applied to hnRNP which are known to contain snRNP (Guimont-Ducamp et al., 1977, Biochimie, 59, 755-758). Micrococcal nuclease digestion of hnRNPs yielded a population of 15-50 nucleotides long resistant fragments of snRNAs. Sequence analyses showed that all fragments previously identified in core snRNPs were also present. Only U2 and U5 snRNAs were further protected as a result of their association with the hnRNP complex (from the cap to nucleotide 32 for U2 and from nucleotide 22 to nucleotide 70 for U5). No additional protected fragment derived from U1, U4 and U6 snRNAs was found. This finding confirms that the 5' terminal region of U1 snRNP remains available for base-pairing interaction with the premessenger RNA, as predicted by the model of Lerner et al. (Nature, 1980, 283, 220-224).

Extensive purification of snRNPs as a subset of hnRNP from Hela cells has been previously reported (Brunelet al. (1981), Nucleic Acids Research, 9, 815). These snRNPs were shown to contain discrete RNA species comigrating in gel electrophoresis with authentic U1, U2, U4, U5 and U6 species. We now report sequence analysis data of about 50 nucleotides from the 3′-end which serve to positively establish the identity of snRNAs present in these purified snRNPs. Sequence heterogeneity was found at the 3′-end of U4 species. A minor species identical to U1 at its 3′-end but slightly shorter was identified as the U inf1 sup* described by Lerneret al. (Nature (1980) 283, 220–224) through sequencing of the 5′-end. When unfixed hnRNP are centrifuged in a CsCl gradient containing 4M guanidinium chloride instead of 0.5% sarkosyl as above, a band containing only one RNA species was observed. T1 RNAse fingerprinting and sequence analysis of the oligonucleotides produced allowed identification of this RNA as U5 snRNA.

snRNPs purified by centrifugation in density gradients (BRUNEL et al. Nucl. Acids. Res., 1981, 9, 815–830 and SRI-WIDADA et al. Biochim. Biophys. Res. Commun., 1982, 104 , 457–462) retain an important part (46%) of their antigenicity towards anti-Sm antibodies indicating that antigenic determinants are among the small 4–5 polypeptides between 9 and 14,000 daltons found to be tightly associated with snRNAs. Furthermore, digestion of a great part of the RNA moiety does not abolish this antigenicity. As to the determinant for RNP antigen, our results suggest that it could depend on the three other proteins which are lost during the purification.

The separation of ribosomal particles of differing biological properties can be achieved by electrophoresis on a sucrose gradient column.

We had previously reported the extensive purification of snRNPs (ribonucleoprotein complexes containing small nuclear RNAs) from HeLa hnRNP (ribonucleoprotein complexes containing the heterogenous nuclear RNA) by taking advantage of their ability to withstand centrifugation in cesium chloride containing 0.5% sarkosyl (C. BRUNEL et al 1981, Nucleic Acids Research, 9, 815–830). Such purified snRNPs contained the five nucleoplasmic snRNAs (U 1 , U 2 , U 4 , U 5 and U 6 ) and a very simple protein complement of 4–5 polypeptides between 10 and 14,000 daltons. The possible involvement of U 1 in messenger RNA splicing makes it crucial to obtain in pure form the individual U 1 snRNP, if an in vitro splicing system is to be reconstructed. The present work reports the purification of U 1 snRNP and shows that it contains exactly the same polypeptides as the mixture of all five snRNPs suggesting that the specific function, if any, depends on the RNA moiety.

We have cloned and determined the nucleotide sequence of a complementary DNA (cDNA) encoded by a newly isolated human tropomyosin gene and expressed in liver. Using the leastsquare method of Fitch and Margoliash, we investigated the nucleotide divergences of this sequence and those published in the literature, which allowed us to clarify the classification and evolution of the tropomyosin genes expressed in vertebrates. Tropomyosin undergoes alternative splicing on three of its nine exons. Analysis of the exons not involved in differential splicing showed that the four human tropomyosin genes resulted from a duplication that probably occurred early, at the time of the amphibian radiation. The study of the sequences obtained from rat and chicken allowed a classification of these genes as one of the types identified for humans. The divergence of exons 6 and 9 indicates that functional pressure was exerted on these sequences, probably by an interaction with proteins in skeletal muscle and perhaps also in smooth muscle; such a constraint was not detected in the sequences obtained from nonmuscle cells. These results have led us to postulate the existence of a protein in smooth muscle that may be the counterpart of skeletal muscle troponin. We show that different kinds of functional pressure were exerted on a single gene, resulting in different evolutionary rates and different convergences in some regions of the same molecule. Codon usage analysis indicates that there is no strict relationship between tissue types (and hence the tRNA precursor pool) and codon usage. G+C content is characteristic of a gene and does not change significantly during evolution. These results are in good agreement with an isochore composition of the genome, and thus suggest a similar chromosomal environment in chicken, rat, and human.

The cytoplasm of growing HeLa cells contains a certain quantity of native ribosomal subunits. They constitute a heterogeneous fraction consisting of at least two categories of particles. The first category comprises the newly emerging native 40S subunits which do not form 80S couples under any in vitro condition tried. It is postulated that these native subunits carry an anti-association factor which cannot be stripped off by 0.5 м KCl treatment. Transiently native subunits of the second category reassociate to 80S ribsomes only in the presence of an association factor.

The psychoactive component of marijuana, δ-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dosedependently inhibited in cells treated with 10–500 nM SR141716A and 1 μM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria. J. Leukoc. Biol. 67: 335–344; 2000.

The aim of this thesis is to study, under several aspects, the formation of amyloids from proteins polymerization. The mathematical modelling of these phenomena in the case of in vitro or in vivo polymerisation remains questioned. We then propose here several models, which are also investigated from theoritical and numerical point of view. In the first part we present works done in collaboration with biologists. We propose two models based on the current theory on Prion phenomena that are designed for specific experimental conditions. These models allow us to analyse the experimental data obtained in laboratory and raise phenomena that remain unexplained by the theory. Then, from these results and biophysical considerations, we introduce a model which corroborates with data and provides a new approach on the amyloid formation in the particular case of Prion. This part is ended by the mathematical analysis of the model consisting of an infinite set of differentials equations. The system analysed is a Becker-Doring system coupled to a discrete growth-fragmentation system. The second part is dedicated to the analysis of a new model for polymerization of proteins with fragmentation subject to the surrounding variations of the fluid. Thus, we propose a model which is close to the experimental conditions and introduce new measurable macroscopic quantities to study the polymerization. The first introductory chapter states the stochastic description of the problem. We give the equations of motion for each polymers and monomers as well as a general formalism to study the limit in large number. Next, we give the mathematical framework and prove the existence of solutions to the Fokker-Planck-Smoluchowski equation for the configurational density of polymers coupled to the diffusion equation for monomers. The last chapter provides a numerical method adapted to this problem with numerical simulations In the last part, we are interested in modelling Alzheimer’s disease. We introduce a model that describes the formation of amyloids plaques in the brain and the interactions between Aβ-oligomers and Prion proteins which might be responsible of the memory impairment. We carry out the mathematical analysis of the model. Namely, for a constant polymerization rate, we provide existence and uniqueness together with stability of the equilibrium. Finally we study the existence in a more general and biological relevant case, that is when the polymerization depends on the size of the amyloid, L’objectif de cette thèse est d’étudier, sous divers aspects, le processus de formation d’amyloïde à partir de la polymérisation de protéines. Ces phénomènes, aussi bien in vitro que in vivo, posent des questions de modélisation mathématique. Il s’agit ensuite de conduire une analyse des modèles obtenus. Dans la première partie nous présentons des travaux effectués en collaboration avec une équipe de biologistes. Deux modèles sont introduits, basés sur la théorie en vigueur du phénomène Prions, que nous ajustons aux conditions expérimentales. Ces modèles nous permettent d’analyser les données obtenues à partir d’expériences conduites en laboratoire. Cependant celles-ci soulèvent certains phénomènes encore inexpliqués par la théorie actuelle. Nous proposons donc un autre modèle qui corrobore les données et donne une nouvelle approche de la formation d’amyloïde dans le cas du Prion. Nous terminons cette partie par l’analyse mathématique de ce système compose d’une infinité d’équations différentielles. Ce dernier consiste en un couplage entre un système de type Becker-Doring et un système de polymérisation-fragmentation discrète. La seconde partie s’attache à l’analyse d’un nouveau modèle pour la polymérisation de protéines dont la fragmentation est sujette aux variations du fluide environnant. L’idée est de décrire au plus près les conditions expérimentales mais aussi d’introduire de nouvelles quantités macroscopiques mesurables pour l’étude de la polymérisation. Le premier chapitre de cette partie présente une description stochastique du problème. On y établit les équations du mouvement des polymères et des monomères (de type Langevin) ainsi que le formalisme pour l’étude du problème limite en grand nombre. Le deuxième chapitre pose le cadre fonctionnel et l’existence de solutions pour l équation de Fokker-Planck- Smoluchowski décrivant la densité de configuration des polymères, elle-même couplée a une équation de diffusion pour les monomères. Le dernier chapitre propose une méthode numérique pour traiter ce problème. On s’intéresse dans la dernière partie à la modélisation de la maladie d’Alzheimer. On construit un modèle qui décrit d’une part la formation de plaque amyloïde in vivo, et d’autre part les interactions entre les oligomères d’Aβet la protéine prion qui induiraient la perte de mémoire. On mène l’analyse mathématique de ce modèle dans un cas particulier puis dans un cas plus général ou le taux de polymérisation est une loi de puissance

International audience; The Rift Valley fever virus (RVFV) is the causative agent of fatal hemorrhagic fever in humans and acute hepatitis in ruminants. We found that infection by RVFV leads to a rapid and drastic suppression of host cellular RNA synthesis that parallels a decrease of the TFIIH transcription factor cellular concentration. Using yeast two hybrid system, recombinant technology, and confocal microscopy, we further demonstrated that the nonstructural viral NSs protein interacts with the p44 component of TFIIH to form nuclear filamentous structures that also contain XPB subunit of TFIIH. By competing with XPD, the natural partner of p44 within TFIIH, and sequestering p44 and XPB subunits, NSs prevents the assembly of TFIIH subunits, thus destabilizing the normal host cell life. These observations shed light on the mechanism utilized by RVFV to evade the host response.