Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

Expression of prox1, lymphatic endothelial nuclear transcription factor, in Kaposiform hemangioendothelioma and tufted angioma.

Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare tumors mainly occurring in early childhood. Our recent results showed that ectopic overexpression of human Prox1 gene, a lymphatic endothelial nuclear transcription factor, promoted an aggressive behavior in 2 murine models of KHE. This dramatic Prox1-induced phenotype prompted us to investigate immunohistochemical staining pattern of Prox1, podoplanin (D2-40), LYVE-1, and Prox1/CD34 as well as double immunofluorescent staining pattern of LYVE-1/CD31 in KHE and TA, compared with other pediatric vascular tumors. For this purpose, we examined 75 vascular lesions: KHE (n=18), TA (n=13), infantile hemangioma (n=13), pyogenic granuloma (n=18), and granulation tissue (n=13). Overall, KHE and TA shared an identical endothelial immunophenotype: the neoplastic spindle cells were Prox1, podoplanin, LYVE-1, CD31, and CD34, whereas endothelial cells within glomeruloid foci were Prox1, podoplanin, LYVE-1, CD31, and CD34. The lesional cells of all infantile hemangiomas and pyogenic granulomas were negative for Prox1 in the presence of positive internal control. These findings provide immunophenotypic evidence to support a preexisting notion that KHE and TA are closely related, if not identical. Overall, our results show, for the first time, that Prox1 is an immunohistochemical biomarker helpful in confirming the diagnosis of KHE/TA and in distinguishing it from infantile hemangioma and pyogenic granuloma.

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

INTRODUCTION Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region. METHODS To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry. RESULTS Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region. CONCLUSIONS Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.

CA88, a nuclear repetitive DNA sequence identified in Schistosoma mansoni, aids in the genotyping of nine Schistosoma species of medical and veterinary importance

CA88 is the first long nuclear repetitive DNA sequence identified in the blood fluke, Schistosoma mansoni. The assembled S. mansoni sequence, which contains the CA88 repeat, has 8,887 nucleotides and at least three repeat units of approximately 360 bp. In addition, CA88 also possesses an internal CA microsatellite, identified as SmBr18. Both PCR and BLAST analysis have been used to analyse and confirm the CA88 sequence in other S. mansoni sequences in the public database. PCR-acquired nuclear repetitive DNA sequence profiles from nine Schistosoma species were used to classify this organism into four genotypes. Included among the nine species analysed were five sequences of both African and Asian lineages that are known to infect humans. Within these genotypes, three of them refer to recognised species groups. A panel of four microsatellite loci, including SmBr18 and three previously published loci, has been used to characterise the nine Schistosoma species. Each species has been identified and classified based on its CA88 DNA fingerprint profile. Furthermore, microsatellite sequences and intra-specific variation have also been observed within the nine Schistosoma species sequences. Taken together, these results support the use of these markers in studying the population dynamics of Schistosoma isolates from endemic areas and also provide new methods for investigating the relationships between different populations of parasites. In addition, these data also indicate that Schistosoma magrebowiei is not a sister taxon to Schistosoma mattheei, prompting a new designation to a basal clade.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study.

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using (13)C- and (31)P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N(2) fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Integrating receptor interactions and dynamics and Interference with endocrine disruptors in the mechanisms of action of PPAR nuclear receptors

Abstract Peroxisome Proliferator-Activated Receptors (PPARs) form a family of three nuclear receptors regulating important cellular and metabolic functions. PPARs control gene expression by directly binding to target promoters as heterodimers with the Retinoid X Receptor (RXR), and their transcriptional activity is enhanced upon activation by natural or pharmacological ligands. The binding of PPAR/RXR heterodimers on target promoters allows the anchoring of a series of coactivators and corepressors involved in promoter remodeling and the recruitment of the transcription machinery. The transcriptional output finally depends on a complex interplay between (i) the respective expression levels of PPARs, RXRs and of other nuclear receptors competing for DNA binding and RXR recruitment, (ii) the availability and the nature of PPAR and RXR ligands, (iii) the expression levels and the nature of the different coactivators and corepressors and (iv) the sequence and the epigenetic status of the promoter. Understanding how all these factors and signals integrate and fine-tune transcription remains a challenge but is necessary to understand the specificity of the physiological functions regulated by PPARs. The work presented herein focuses on the molecular mechanisms of PPAR action and aims at understanding how the interactions and mobility of the receptor modulate transcription in the physiological context of a living cell: Such observations in vivo rely on the use of engineered fluorescent protein chimeras and require the development and the application of complementary imaging techniques such as Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS). Using such techniques, PPARs are shown to reside solely in the nucleus where they are constitutively associated with RXR but transcriptional activation by ligand binding -does not promote the formation of sub-nuclear structures as observed with other nuclear receptors. In addition, the engagement of unliganded PPARs in large complexes of cofactors in living cells provides a molecular basis for their ligand-independent activity. Ligand binding reduces receptor diffusion by promoting the recruitment of coactivators which further enlarge the size of PPAR complexes to acquire full transcriptional competence. Using these molecular approaches, we deciphered the molecular mechanisms through which phthalates, a class of pollutants from the plastic industry, interfere with PPARγ signaling. Mono-ethyl-hexyl-phthalate (MEHP) binding induces the recruitment of a specific subset of cofactors and translates into the expression of a specific subset of target genes, the transcriptional output being strongly conditioned by the differentiation status of the cell. This selective PPARγ modulation induces limited adipogenic effects in cellular models while exposure to phthalates in animal models leads to protective effects on glucose tolerance and diet-induced obesity. These results demonstrate that phthalates influence lipid and carbohydrate metabolism through complex mechanisms which most likely involve PPARγ but also probably PPARα and PPARß, Altogether, the molecular and physiological demonstration of the interference of pollutants with PPAR action outlines an important role of chemical exposure in metabolic regulations. Résumé Les PPARs (Peroxisome Proliferator-Activated Receptors) forment une famille de récepteurs nucléaires qui régulent des fonctions cellulaires et métaboliques importantes. Les PPARs contrôlent l'expression des gènes en se liant directement à leurs promoteurs sous forme d'hétérodimères avec les récepteurs RXR (Retinoid X Receptor), et leur activité transcriptionnelle est stimulée par la liaison de ligands naturels ou pharmacologiques. L'association des hétérodimères PPAR/RXR avec les promoteurs des gènes cibles permet le recrutement de coactivateurs et de corépresseurs qui vont permettre le remodelage de la chromatine et le recrutement de la machinerie transcriptionnelle. Les actions transcriptionnelles du récepteur dépendent toutefois d'interactions complexes qui sont régulées par (i) le niveau d'expression des PPARs, des RXRs et d'autres récepteurs nucléaires entrant en compétition pour la liaison à l'ADN et l'association avec RXR, (ii) la disponibilité et la nature de ligands de PPAR et de RXR, (iii) les niveaux d'expression et la nature des différents coactivateurs et corépresseurs et (iv) la séquence et le marquage épigénétique des promoteurs. La compréhension des mécanismes qui permettent d'intégrer ces aspects pour assurer une régulation fine de l'activité transcriptionnelle est un défi qu'il est nécessaire de relever pour comprendre la spécificité des fonctions physiologiques régulées par les PPARs. Ce travail concerne l'étude des mécanismes d'action moléculaire des PPARs et vise à mieux comprendre comment les interactions du récepteur avec d'autres protéines ainsi que la mobilité de ce dernier régulent son activité transcriptionnelle dans le contexte physiologique des cellules vivantes. De telles observations reposent sur l'emploi de protéines fusionnées à des protéines fluorescentes ainsi que sur le développement et l'utilisation de techniques d'imagerie complémentaires telles que le FRAP (Fluorescence Recovery After Photobleaching), le FRET (Fluorescence Resonance Energy Transfer) ou la FCS (Fluorescence Corrélation Spectroscopy). En appliquant ces méthodes, nous avons pu montrer que les PPARs résident toujours dans le noyau où ils sont associés de manière constitutive à RXR, mais que l'ajout de ligand n'induit pas la formation de structures sub-nucléaires comme cela a pu être décrit pour d'autres récepteurs nucléaires. De plus, les PPARs sont engagés dans de larges complexes protéiques de cofacteurs en absence de ligand, ce qui procure une explication moléculaire à leur activité ligand-indépendante. La liaison du ligand réduit la vitesse de diffusion du récepteur en induisant le recrutement de coactivateurs qui augmente encore plus la taille des complexes afin d'acquérir un potentiel d'activation maximal. En utilisant ces approches moléculaires, nous avons pu caractériser les mécanismes permettant aux phtalates, une classe de polluants provenant de l'industrie plastique, d'interférer avec PPARγ. La liaison du mono-ethyl-hexyl-phtalate (NERF) à PPARγ induit un recrutement sélectif de cofacteurs, se traduisant par l'induction spécifique d'un sous-ensemble de gènes qui varie en fonction du niveau de différentiation cellulaire. La modulation sélective de PPARγ par le MEHP provoque une adipogenèse modérée dans des modèles cellulaires alors que l'exposition de modèles animaux aux phtalates induit des effets bénéfiques sur la tolérance au glucose et sur le développement de l'obésité. Toutefois, les phtalates ont une action complexe sur le métabolisme glucido-lipidique en faisant intervenir PPARγ mais aussi probablement PPARα et PPARß. Cette démonstration moléculaire et physiologique de l'interférence des polluants avec les récepteurs nucléaires PPAR souligne un rôle important de l'exposition à de tels composés dans les régulations métaboliques.

Genetic consequences of the ice ages on nurseries of the bat Myotis myotis: a mitochondrial and nuclear survey.

Analyses of mitochondrial DNA (mtDNA) control region polymorphism and of variation at 10 nuclear microsatellite loci were used to investigate the mechanisms and genetic consequences of postglacial expansion of Myotis myotis in Europe. Initial sampling consisted of 480 bats genotyped in 24 nursery colonies arranged along a transect of approximately 3000 km. The phylogeographical survey based on mtDNA sequences revealed the existence of major genetic subdivisions across this area, with several suture zones between haplogroups. Such zones of secondary contact were found in the Alps and Rhodopes, whereas other potential barriers to gene flow, like the Pyrenees, did not coincide with genetic discontinuities. Areas of population admixture increased locally the genetic diversity of colonies, which confounded the northward decrease in nucleotide diversity predicted using classical models of postglacial range expansion. However, when analyses were restricted to a subset of 15 nurseries originating from a single presumed glacial refugium, mtDNA polymorphism did indeed support a northwards decrease in diversity. Populations were also highly structured (PhiST = 0.384). Conversely, the same subset of colonies showed no significant latitudinal decrease in microsatellite diversity and much less population structure (FST = 0.010), but pairwise genetic differentiation at these nuclear markers was strongly correlated with increasing geographical distance. Together, this evidence suggests that alleles carried via male bats have maintained enough nuclear gene flow to counteract the effects of recurrent bottlenecks generally associated with recolonization processes. As females are highly philopatric, we argue that the maternally transmitted mtDNA marker better reflects the situation of past, historical gene flow, whereas current levels of gene flow are better reflected by microsatellite markers.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

In vitro heat exposure induces a redistribution of nuclear matrix proteins in human K562 erythroleukemia cells.

By using both conventional and confocal laser scanning microscopy with three monoclonal antibodies recognizing nuclear matrix proteins we have investigated by means of indirect fluorescence whether an incubation of isolated nuclei at the physiological temperature of 37 degrees C induces a redistribution of nuclear components in human K562 erythroleukemia cells. Upon incubation of isolated nuclei for 45 min at 37 degrees C, we have found that two of the antibodies, directed against proteins of the inner matrix network (M(r) 125 and 160 kDa), gave a fluorescent pattern different from that observed in permeabilized cells. By contrast, the fluorescent pattern did not change if nuclei were kept at 0 degrees C. The difference was more marked in case of the 160-kDa polypeptide. The fluorescent pattern detected by the third antibody, which recognizes the 180-kDa nucleolar isoform of DNA topoisomerase II, was unaffected by heat exposure of isolated nuclei. When isolated nuclear matrices prepared from heat-stabilized nuclei were stained by means of the same three antibodies, it was possible to see that the distribution of the 160-kDa matrix protein no longer corresponded to that observable in permeabilized cells, whereas the fluorescent pattern given by the antibody to the 125-kDa polypeptide resembled that detectable in permeabilized cells. The 180-kDa isoform of topoisomerase II was still present in the matrix nucleolar remnants. We conclude that a 37 degrees C incubation of isolated nuclei induces a redistribution of some nuclear matrix antigens and cannot prevent the rearrangement in the spatial organization of one of these antigens that takes place during matrix isolation in human erythroleukemia cells. The practical relevance of these findings is discussed.

A systematic and feasible method for computing nuclear contributions to electrical properties of polyatomic molecules

An analytic method to evaluate nuclear contributions to electrical properties of polyatomic molecules is presented. Such contributions control changes induced by an electric field on equilibrium geometry (nuclear relaxation contribution) and vibrational motion (vibrational contribution) of a molecular system. Expressions to compute the nuclear contributions have been derived from a power series expansion of the potential energy. These contributions to the electrical properties are given in terms of energy derivatives with respect to normal coordinates, electric field intensity or both. Only one calculation of such derivatives at the field-free equilibrium geometry is required. To show the useful efficiency of the analytical evaluation of electrical properties (the so-called AEEP method), results for calculations on water and pyridine at the SCF/TZ2P and the MP2/TZ2P levels of theory are reported. The results obtained are compared with previous theoretical calculations and with experimental values

Nuclear relaxation contribution to static and dynamic (infinite frequency approximation) nonlinear optical properties by means of electrical property expansions: Application to HF, CH4, CF4, and SF6

Electrical property derivative expressions are presented for the nuclear relaxation contribution to static and dynamic (infinite frequency approximation) nonlinear optical properties. For CF4 and SF6, as opposed to HF and CH4, a term that is quadratic in the vibrational anharmonicity (and not previously evaluated for any molecule) makes an important contribution to the static second vibrational hyperpolarizability of CF4 and SF6. A comparison between calculated and experimental values for the difference between the (anisotropic) Kerr effect and electric field induced second-harmonic generation shows that, at the Hartree-Fock level, the nuclear relaxation/infinite frequency approximation gives the correct trend (in the series CH4, CF4, SF6) but is of the order of 50% too small

New Radicals and new applications of Dynamic Nuclear Polarization of biological systems

Dynamic Nuclear Polarization (DNP) is an emerging technique that could revolutionize the NMR study of small molecules at very low concentrations by the increase in sensitivity that results from transfer of polarization between electronic and nuclear spins. Although the underlying physics has been known for a long time, in the last few years there has been a lot of excitement on the chemistry and biology NMR community caused by the demonstration that the highly polarized nuclei that are prepared in solid state at very low temperatures (1-2 K) could be rapidly transferred to liquid samples at room temperature and studied in solution by conventional NMR techniques. In favorable cases several order of magnitude increases in sensitivity have been achieved. The technique is now mature enough that a commercial instrument is available. The efficiency of DNP depends on two crucial aspects: i) the efficiency of the nuclear polarization process and ii) the efficiency of the transfer from the initial solid state to the fluid state in which NMR is measured. The preferred areas of application (iii) will be dictated by situations in which the low concentration of the sample or its intrinsic low receptivity are the limiting factors .

Society, politcs and nuclear energy in Switzerland

After the Fukushima accident, the Swiss Federal Council opted for withdrawal from nuclear energy in the horizon 2035. Considering that this decision cannot be studied in historical isolation, this article describes the evolution of the relationship between nuclear energy and the Swiss society by highlighting four distinctive periods: the social mobilisation of the seventies and eighties, the turning point of the nineties, the nuclear revival in the late 2000s and the Fukushima accident. To describe this relationship, we will analyse social mobilisation and the results of initiatives and empirical studies related to nuclear energy in Switzerland.

A set of primers for plastid indels and nuclear microsatellites in the invasive plant Heracleum mantegazzianum (Apiaceae) and their transferability to Heracleum sphondylium

This study reports the isolation and polymorphism characterization of four plastid indels and six nuclear microsatellite loci in the invasive plant Heracleum mantegazzianum. These markers were tested in 27 individuals from two distant H. mantegazzianum populations. Plastid indels revealed the presence of five chlorotypes while five nuclear microsatellite loci rendered polymorphism. Applications of these markers include population genetics and phylogeography of H. mantegazzianum. A very good transferability of markers to Heracleum sphondylium was demonstrated.