Computing the geometry of a molecule in dihedral angle space using n.m.r.-derived constraints. A new algorithm based on optimal filtering.

We have developed a method based on optimal filtering to determine the three-dimensional structure of a protein from n.m.r.-derived constraints, using the dihedral angle internal representation of the molecule. It differs from currently proposed methods in that it directly produces estimates of errors on the parameters that are refined, hence providing an image of the minimum that has been found. A similar algorithm had already been proposed using cartesian co-ordinates as independent parameters, encoded in PROTEAN2. We found that using dihedral angles significantly reduces the computational burden of the technique, and provides better control over a priori informations that can be used, such as geometric restrictions for proline residues and informations from vicinal coupling constants. Performance of the method, encoded in FILMAN, is demonstrated by application to the folding of a ten-residue alanine polypeptide, to the geometric cyclization of an 11-residue peptide, as well as on the folding of a medium size protein, i.e. tendamistat. The validity of the error estimates on the dihedral angles produced by FILMAN is discussed.     

Role of plant parasitic nematodes and abiotic soil factors in growth heterogeneity of sugarcane on a sandy soil in South Africa

A feature of many sugarcane fields is the patchy growth. This is often thought to be due to physical or chemical differences in the soil. In this paper we investigate the causes of growth heterogeneity of sugarcane on a sandy soil in KwaZulu-Natal, South Africa. To identify the factors that were associated with the good and poor areas, soil texture, pH, organic matter content and a number of soil chemical elements and nematode community data were subjected to principal component analysis (PCA). The numbers of each of the nematode species (Meloidogyne sp., Pratylenchus zeae, Helicotylenchus dihystera, Xiphinema elongatum and Paratrichodorus sp.) were first converted to relative proportions of ectoparasites and endoparasites. The data were collected from the 2nd ratoon crop of a nematicide trial, where half of the plots had been treated with aldicarb in the preceding two crops. Yields of control plots varied from 34.7 to 126.8 t cane ha^–1 and from 85.4 to 138.7 for the treated plots. The yield data were centred and normalised separately for the treated and control plots and the values projected on the trial map to study spatial distribution. Plots with above-average yields, whether treated or untreated, occurred in the lower part of the trial site. The PCA factorial values were also projected onto the map of the trial. According to the first factor of the analysis of the abiotic soil characteristics in the 0–20 cm surface layer, the trial site could be divided into two areas, one on the left and one on the right. PCA of the soil data from the 0–20 and 20–40 cm layers showed that there was little or no difference between the two that might explain the two growth areas. However, analysis of the nematode community distinguished two main areas that largely corresponded to the distribution of the plots of low and high yielding cane. Correlation analysis confirmed the relationship between nematodes and yield. H. dihystera was positively correlated with yield of cane whereas the reverse was true for the Meloidogyne species.     

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.     

Hair analysis for nordiazepam and oxazepam by gas chromatography-negative-ion chemical ionization mass spectrometry

A procedure is presented for the identification of nordiazepam and its metabolite, oxazepam, in human hair. The method involves decontamination of hair with dichloromethane, incubation in phosphate buffer (pH 7.6) in the presence of deuterated internal standards, liquid-liquid extraction, derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane and gas chromatography-mass spectrometry using negative-ion chemical ionization with methane. Among thirty samples obtained from polydrug abusers, thirteen tested positive for nordiazepam, in the range 0.25–18.87 ng/mg. Five samples were also positive for oxazepam, in the range 0.11–0.50 ng/mg.     

Constitutive autophagy contributes to resistance to TP53-mediated apoptosis in Epstein-Barr virus-positive latency III B-cell lymphoproliferations

The Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and lymphomas. We have previously demonstrated that treating wild-type TP53-expressing B cell lines with the TP53 pathway activator nutlin-3 induced apoptosis in EBV-negative and EBV-positive latency I cells whereas EBV-positive latency III cells remained much more apoptosis-resistant. Here, we report a constitutively high level of autophagy in these resistant cells which express high levels of the proautophagic protein BECN1/Beclin 1 based, at least in part, on the activation of the NFKB signaling pathway by the viral protein LMP1. Following treatment with nutlin-3, several autophagy-stimulating genes were upregulated both in EBV-negative and EBV-positive latency III cells. However the process of autophagy was only triggered in the latter and was associated with an upregulation of SESN1/sestrin 1 and inhibition of MTOR more rapid than in EBV-negative cells. A treatment with chloroquine, an inhibitor of autophagy, potentiated the apoptotic effect of nutlin-3, particularly in those EBV-positive cells which were resistant to apoptosis induced by nutlin-3 alone, thereby showing that autophagy participates in this resistant phenotype. Finally, using immunohistochemical staining, clinical samples from various B cell lymphoproliferations with the EBV-positive latency II or III phenotype were found to harbor a constitutively active autophagy.     

Mitochondrial morphodynamics alteration induced by influenza virus infection as a new antiviral strategy

Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.