Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Design, synthesis and biological evaluation of substituted dioxodibenzothiazepines and dibenzocycloheptanes as farnesyltransferase inhibitors

A new series of FTase inhibitors containing a tricyclic moiety--dioxodibenzothiazepine or dibenzocycloheptane--has been designed and synthesized. Among them, dioxodibenzothiazepine 18d displayed significant inhibitory FTase activity (IC(50)=17.3 nM) and antiproliferative properties.     

Architecture of the pruned tree: impact of contrasted pruning procedures over 2 years on shoot demography and spatial distribution of leaf area in apple (Malus domestica)

BACKGROUND AND AIMS: Demography and spatial distribution of shoots are rarely studied on pruned trees. The present 2-year study deals with the effect of pruning strategies on shoot demography and development, and consequences on the spatial distribution of leaf area in three architecturally contrasted - from type II to IV - apple cultivars: 'Scarletspur Delicious', 'Golden Delicious' and 'Granny Smith'. METHODS: All trees were initially subjected during 5 years to Central Leader training with winter heading on all long shoots. For 2 years, half of the trees were further trained with Centrifugal training, where removal of flowering shoots - called extinction pruning - was carried out along the trunk and at the bottom of branches at flowering time. During these 2 years, shoot type (vegetative, inflorescence) and length, and the three-dimensional spatial distribution of all shoots were assessed with an electromagnetic digitizer. KEY RESULTS: Shoot demography, frequency of transitions toward an inflorescence from either an inflorescence (bourse-over-bourse) or a vegetative shoot (trend toward flowering), and the number of bourse-shoots per bourse were strongly affected by cultivar, with little influence of tree manipulation. In contrast, the proportion of vegetative long shoots developing from previous year latent buds was significantly lower in Centrifugal-trained trees for the three cultivars. Canopy volume showed large variations between cultivars, but only that of 'Granny Smith' was affected by tree manipulation in the 2 years. Spatial distribution of shoots varied significantly according to cultivar and manipulation. In 'Scarletspur Delicious' and, to a lesser extent 'Golden Delicious', the distribution of vegetative and flowering shoots in the outer and the inner parts, respectively, was not affected by tree manipulation. In contrast, in 'Granny Smith', vegetative shoots were stimulated in the periphery of Central Leader trees, whereas flowering shoots were stimulated in the periphery of Centrifugal-trained trees. CONCLUSIONS: In apple, the variability of responses to contrasted pruning strategies partly depends on the genetically determined growth and flowering habit of the cultivar.     

Molecular systematics, character evolution, and pollen morphology of Cistus and Halimium (Cistaceae)

Pollen analysis and parsimony-based phylogenetic analyses of the genera Cistus and Halimium, two Mediterranean shrubs typical of Mediterranean vegetation, were undertaken, on the basis of cpDNA sequence data from the trnL-trnF, and trnS-trnG regions, to evaluate limits between the genera. Neither of the two genera examined formed a monophyletic group. Several monophyletic clades were recognized for the ingroup. (1) The ??white and whitish pink Cistus??, where most of the Cistus sections were present, with very diverse pollen ornamentations ranging from striato-reticulate to largely reticulate, sometimes with supratectal elements; (2) The ??purple pink Cistus?? clade grouping all the species with purple pink flowers belonging to the Macrostylia and Cistus sections, with rugulate or microreticulate pollen. Within this clade, the pink-flowered endemic Canarian species formed a monophyletic group, but with weak support. (3) Three Halimium clades were recovered, each with 100% bootstrap support; all Halimium species had striato-reticulate pollen. Two Halimium clades were characterized by yellow flowers, and the other by white flowers.     

Netrin-1 and axonal guidance: signaling and asymmetrical translation

More than 10 years after its initial discovery, netrin-1 - the first described chimioattractive molecule controlling the guidance of the commissural axons - has recently known a unsuspected wave of interest because of its implication in the development of the nervous system but also, more recently, fot its role in angiogenesis and tumorigenesis. Because, of a series of recent publications on netrin-1 signaling, we propose here to describe the recent insight in netrin-1 signaling via its main receptor DCC (deleted in colorectal cancer), and the recent discovery that netrin controls the assymetric distribution of beta-actin in the growth cone. Thus, it seems that netrin-1, but also the neurotrophic factor BDNF, controls acute growth cone responses such as collapse and turning by the regulation of localized protein translation, such as beta-actin. This process involves both transport of beta-actin mRNA, bound to Vg1RBP, to specific locations, and mRNA translation upon stimulation by local activation of the translation initiation regulator eIF-4E-binding protein 1. Indeed, Netrin-1 induces the movement of Vg1RBP granules into filopodia, and triggers a polarized increase in beta-actin translation on the near side of the growth cone before growth cone turning. The binding of BDNF to its receptor Trk has a similar effect for growth cone attraction, althought it is differentially regulated. Thus, this asymetrically synthesized beta-actin may direct actin polymerization and consequently the migration of the growth cone toward the cue.     

Halophytes--an emerging trend in phytoremediation

Halophytic plants are of special interest because these plants are naturally present in environments characterized by an excess of toxic ions, mainly sodium and chloride. Several studies have revealed that these plants may also tolerate other stresses including heavy metals based on the findings that tolerance to salt and to heavy metals may, at least partly, rely on common physiological mechanisms. In addition, it has been shown that salt-tolerant plants may also be able to accumulate metals. Therefore, halophytes have been suggested to be naturally better adapted to cope with environmental stresses, including heavy metals compared to salt-sensitive crop plants commonly chosen for phytoextraction purposes. Thus, potentially halophytes are ideal candidates for phytoextraction orphytostabilization of heavy metal polluted soils and moreover of heavy metal polluted soils affected by salinity. Some halophytes use excretion processes in order to remove the excess of salt ions from their sensitive tissues and in some cases these glandular structures are not always specific to Na+ and Cl- and other toxic elements such as cadmium, zinc, lead, or copper are accumulated and excreted by salt glands or trichomes on the surface of the leaves--a novel phytoremediation process called "phytoexcretion". Finally, the use of halophytes has also been proposed for soil desalination through salt accumulation in the plant tissue or dissolution of soil calcite in the rhizosphere to provide Ca2+ that can be exchanged with Na+ at cation exchange sites.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.