Protein Arginylation in Rat Brain Cytosol: A Proteomic Analysis

Arginine can be post-translationally incorporated from arginyl-tRNA into the N-terminus of soluble acceptor proteins in a reaction catalyzed by arginyl-tRNA protein transferase. In the present study, several soluble rat brain proteins that accepted arginine were identified after arginine incorporation by two dimensional electrophoresis and mass spectrometry. They were identified as: contrapsin-like protease inhibitor-3, α-1-antitrypsin, apolipoprotein E, hemopexin, calreticulin and apolipoprotein A-I. All of these proteins shared a signal sequence for the translocation of proteins across endoplasmic reticulum membranes. After losing the signal peptide, these proteins expose amino acids described as compatible for post-translational arginylation. Although the enzymatic system involved in arginylation is confined mainly in cytosol and nucleus, all the substrates described herein enter to the exocytic pathway co-translationally. Therefore, we postulate that the substrates for arginylation could reach the cytosol by retro-translocation and be then arginylated.     

Microalgal cell immobilization for the long-term storage of the marine diatom Haslea ostrearia

To preserve the characteristics of the marine diatom Haslea ostrearia during long term storage, particularly size and shape, the algal cells were immobilized in alginate beads and stored at 4 ^∘C at reduced irradiance up to 4 months. Two clones of different size (Ho34, 63 μm and Ho40, 78 μm) were studied. With Ho34, a 10.4% decrease of the size was shown after 120 days, by using the conventional storage management, while it did not exceed 2.2% with immobilized cells. Consequently, H. ostrearia would have auxosporulated after 9 months compared to 52 months. At the same time, the rate of distortion (aberrant valve structure) free Ho34 cells reached 86% while no distorted immobilized cells were observed. Chorophyll content in cells showed that all the cells were alive up to 60 days and after this time cells immobilized in the core of the beads most probably suffered from the poor light diffusion. Culturability of the immobilized cells was tested immediately after their immobilization and after 60 and 120 days of storage. The delay (at least 5) before immobilized cells released from the beads decreased with the time of storage, because of the embrittlement of the beads during the storage. Once in fresh medium, the cells actively multiplied. We concluded that immobilization strongly slowed down the decrease in frustule size with time and allowed the storage of concentrated and calibrated inocula which could be inoculated directly in liquid culture medium without needing to dissolve the beads.     

Thiazolo[5,4-f]quinazolin-9-ones, inhibitors of glycogen synthase kinase-3

In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the microwave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and GSK-3 were investigated. Several turned out to inhibit GSK-3 in the micromolar range. Molecular modeling studies suggest that the most selective GSK-3 inhibitors 7a-d bind into the ATP-binding site through a key hydrogen bond interaction with Val135 and target the specific hydrophobic backpocket of the enzyme.     

A Nomadic Subtelomeric Disease Resistance Gene Cluster in Common Bean

The B4 resistance (R) gene cluster is one of the largest clusters known in common bean (Phaseolus vulgaris [Pv]). It is located in a peculiar genomic environment in the subtelomeric region of the short arm of chromosome 4, adjacent to two heterochromatic blocks (knobs). We sequenced 650 kb spanning this locus and annotated 97 genes, 26 of which correspond to Coiled-Coil-Nucleotide-Binding-Site-Leucine-Rich-Repeat (CNL). Conserved microsynteny was observed between the Pv B4 locus and corresponding regions of Medicago truncatula and Lotus japonicus in chromosomes Mt6 and Lj2, respectively. The notable exception was the CNL sequences, which were completely absent in these regions. The origin of the Pv B4-CNL sequences was investigated through phylogenetic analysis, which reveals that, in the Pv genome, paralogous CNL genes are shared among nonhomologous chromosomes (4 and 11). Together, our results suggest that Pv B4-CNL was derived from CNL sequences from another cluster, the Co-2 cluster, through an ectopic recombination event. Integration of the soybean (Glycine max) genome data enables us to date more precisely this event and also to infer that a single CNL moved from the Co-2 to the B4 cluster. Moreover, we identified a new 528-bp satellite repeat, referred to as khipu, specific to the Phaseolus genus, present both between B4-CNL sequences and in the two knobs identified at the B4 R gene cluster. The khipu repeat is present on most chromosomal termini, indicating the existence of frequent ectopic recombination events in Pv subtelomeric regions. Our results highlight the importance of ectopic recombination in R gene evolution.In the human genome, extensive cytogenetic and sequence analyses have revealed that subtelomeres are hot spots of interchromosomal recombination and segmental duplications (Linardopoulou et al., 2005). This peculiar dynamic activity of subtelomeres has been reported in such diverse organisms as yeast and the malaria parasite Plasmodium (Louis, 1995; Freitas-Junior et al., 2000, 2005). As expected for a plastic region of the genome subject to reshuffling through recombination events, subtelomeres exhibit unusually high levels of within-species structural and nucleotide polymorphism (Mefford and Trask, 2002). In plants, this plasticity of subtelomeres has not been identified in Arabidopsis (Arabidopsis thaliana; Heacock et al., 2004; Kuo et al., 2006) and, to our knowledge, has not yet been investigated at a large scale for other plant species with full genome sequences available. Regarding Arabidopsis, the apparent lack of high subtelomeric recombination may reflect its small and simple subtelomeres, mirroring its small genome size and relative paucity of repetitive sequences (Heacock et al., 2004; Kuo et al., 2006).Repetitive sequences, such as satellite DNA and retroelements, constitute an important fraction of every eukaryotic genome and therefore constitute the environment in which genes are expressed. Satellite DNA can be defined as highly reiterated noncoding DNA sequences, organized as long arrays of head-to-tail linked repeats of 150- to 180-bp or 300- to 360-bp monomers located in the constitutive heterochromatin (Plohl et al., 2008). Despite their ubiquity in eukaryotic genomes, little is known about the mechanisms that allow these elements to accumulate. Early hypotheses considered them to be nonfunctional “selfish” or “junk” DNA segments that increase or decrease their frequency without any advantage or disadvantage for an organism (Ohno, 1972; Orgel and Crick, 1980). However, identification of satellite DNA at structurally important parts of chromosomes, such as centromeres, has suggested functional roles of satellite DNA (Ma and Jackson, 2006; Kawabe and Charlesworth, 2007). Satellite DNA can also be localized in knobs, which are cytologically visible regions of highly condensed chromatin (heterochromatin) that are distinct from pericentromeric regions in pachytene chromosomes (Fransz et al., 2000; Gaut et al., 2007; Lamb et al., 2007).The survival of most organisms depends on the presence of specific genetic systems that maintain diversity in order to respond to changing environments. Plants, like animals, are continually challenged by a large array of pathogens. To perceive and counter pathogen attack, plants have evolved disease resistance (R) genes. The largest class of R genes encodes proteins containing a central Nucleotide-Binding Site (NBS) domain, a C-terminal Leucine-Rich Repeat (LRR) domain, and a variable N-terminal domain. These R proteins detect the presence of disease-causing bacteria, oomycetes, fungi, nematodes, insects, and viruses by sensing either specific pathogen effector molecules produced during the infection process or key molecules in the plant cell that may be attacked by pathogen effectors (Dangl and McDowell, 2006). The evolution of new R genes serves to counteract the evolution of novel virulence factors from the pathogens (McDowell and Simon, 2008). Among this prevalent class of R gene, two subclasses, corresponding to two ancient lineages (Bai et al., 2002; Meyers et al., 2003; Ameline-Torregrosa et al., 2008), have been identified based on the N-terminal domain of the R protein: the Coiled-Coil (CC)-NBS-LRR (CNL) and the Toll-Interleukin receptor (TIR)-NBS-LRR (TNL). Genome studies have demonstrated that NBS-LRR (NL) sequences are abundant in any plant genome. For example, annotation of the Arabidopsis, rice (Oryza sativa), poplar (Populus trichocarpa), Medicago truncatula (Mt), grape (Vitis vinifera), Lotus japonicus (Lj), and papaya (Carica papaya) genomes identified at least 149, 480, 317, 333, 233, 229, and 55 genes encoding NL proteins, respectively (Bai et al., 2002; Meyers et al., 2003; Zhou et al., 2004; Tuskan et al., 2006; Velasco et al., 2007; Ameline-Torregrosa et al., 2008; Kohler et al., 2008; Ming et al., 2008; Sato et al., 2008). NL sequences are often located at complex loci (Smith et al., 2004), as exemplified by Arabidopsis, where two-thirds of them are organized in tightly linked clusters (Meyers et al., 2003; Leister, 2004; McDowell and Simon, 2006). Evolution of NL sequences in the Arabidopsis genome has been investigated according to their phylogenetic positions and physical locations. Although tandem duplications explain the origin of a large fraction of NLs, it seems that ectopic recombination has also played a role in Arabidopsis NL evolution, since mixed clusters comprising evolutionarily distant NL exist. Ectopic recombination is also evident when phylogenetically close R genes are physically dispersed on different chromosomes (Leister, 2004; McDowell and Simon, 2006). These results confirm pioneer macrosynteny studies between related monocot species suggesting the existence of NL movement in plant genomes. Indeed, extensive loss of collinearity between NL sequences between rice and barley (Hordeum vulgare), which diverged 50 million years ago (Mya), has suggested rapid reorganization of NL sequences (Leister et al., 1998; Leister, 2004). However, our knowledge of the molecular evolution of R genes remains limited due to the still small number of complete plant genome sequences available to date. Detailed comparative study across taxa at different evolutionary distances is needed to see how R gene clusters evolve at various time scales.Legumes (Fabaceae) constitute the third largest family of flowering plants and represent the second most important family of agronomically important plants after Poaceae (Graham and Vance, 2003). As a result of recent sequencing efforts, legumes are one of the few plant families with extensive genome sequences in different species, since the soybean (Glycine max [Gm]) genome sequence is complete (http://www.phytozome.net/soybean.php) and both Mt and Lj genome sequences are nearly complete (Young et al., 2005; Sato et al., 2008). Consequently, the legume family is extremely well adapted for comparative phylogenomic approaches, in which phylogenetic inference is combined with structural genomic analyses (Ammiraju et al., 2008). Common bean (Phaseolus vulgaris [Pv]) is the most important grain legume for direct human consumption (Broughton et al., 2003). Pv is a selfing species and has a small diploid genome (2n = 22) of 588 Mb (Bennett and Leitch, 1995). Conservation of genome macrostructure (macrosynteny) has been reported between several legumes, including common bean and the two model legume species Mt and Lj genomes (Zhu et al., 2005; Hougaard et al., 2008). However, the extent of gene order conservation at the DNA sequence level has not yet been evaluated within orthologous chromosome segments between Pv and the two model legume species.In the genome of common bean, many disease R genes are clustered at complex loci located at the ends (rather than the centers) of linkage groups (LGs; Vallejos et al., 2006; Geffroy et al., 2008). For example, Colletotrichum lindemuthianum Co-2 R specificity maps at one end of LG B11 (Adam-Blondon et al., 1994). Molecular analysis has revealed that this locus consists of a tandem array of CNL sequences (Geffroy et al., 1998; Creusot et al., 1999). Another CNL-rich region has been identified at the end of LG B4 in the vicinity of R specificities and R quantitative trait loci against a large selection of pathogens, including C. lindemuthianum, Uromyces appendiculatus, and the bacterium Pseudomonas syringae (Geffroy et al., 1998, 1999; Miklas et al., 2006). Recently, fluorescence in situ hybridization (FISH) analysis revealed that this complex R cluster is located in the subtelomeric region of the short arm of chromosome 4 and includes two knobs (Geffroy et al., 2009). In a sequencing effort focused on CNL sequences, we have previously identified 17 CNL sequences of the B4 locus (referred to as B4-CNL) from Pv genotype BAT93 (Ferrier Cana et al., 2003, 2005; Geffroy et al., 2009). In the BAT93 genotype, these B4-CNL sequences are located on both sides of the subterminal knob (Geffroy et al., 2009).To investigate the organization and the evolutionary origin of the subtelomeric B4 R gene cluster, we have sequenced approximately 650 kb of the Pv B4 R gene cluster, revealing that, in genotype BAT93, CNL are spread out in four subclusters, separated by non-CNL-encoding genes. This Pv sequence was then compared gene by gene with the sequenced portions of the three sequenced legume genomes, Mt, Lj, and Gm. Conserved microsynteny (conservation of local gene repertoire, order, and orientation) was observed, except for the CNL sequences, which appear to be completely absent in the corresponding regions of Mt and Lj. In this study, by combining genomics, phylogenetic, and cytogenetic approaches, we provide evidence that ectopic recombination in subtelomeric regions between nonhomologous chromosomes (4 and 11), involving a single CNL, gave rise to the Pv B4 R gene cluster. Chromosomal distribution of a new satellite DNA tandem repeat, referred to as khipu, suggests that ectopic recombination events in subtelomeric regions of bean nonhomologous chromosomes are frequent. Our results highlight the importance of ectopic recombination as an important evolutionary mechanism for the evolution of disease resistance genes.     

Broad-scale determinants of non-native fish species richness are context-dependent

Identifying the factors determining the non-native species richness (NNSR) in a given area is essential for preventing species invasions. The relative importance of human-related and natural factors considered for explaining NNSR might depend upon both the spatial scale (i.e. the extent of the gradients sampled) and the historical context of the area surveyed. Here, using a worldwide database of freshwater fish occurrences, we tested whether the relative influence of human and ecological determinants of non-native fish species establishment at the scale of the biogeographic realm was consistent (i) with that observed worldwide, and (ii) among the different biogeographical realms. The prominent role of human activity in shaping the global (i.e. worldwide) pattern of NNSR cannot be directly extrapolated to the biogeographic realms. Furthermore, the relationships between human and ecological determinants and NNSR vary strikingly across biogeographic realms, revealing a strong context dependency of the determinants of NNSR. In particular, the human-related factors play a predominant role in explaining the establishment of non-native species in economically developed realms, while in the other realms environmental characteristics of the river basins best explained geographical patterns of NNSR. In the face of future biological invasions, considering both the spatial scale and the historical context of the surveyed area is crucial to adopt effective conservation strategies.     

N-substituted bis-C-alkyloxadiazolones as dual effectors: Efficient intermediates to amidoximes or amidines and prodrug candidates of potent antimalarials

A convenient route to N-substituted bis-C-alkylamidines possessing antiplasmodial activity and their oxadiazolone and amidoxime prodrug candidates, is described. These three families of compounds were available after a key N-alkylation step of the parent oxadiazolone 1a. Testing of the three compound classes in vitro and in vivo is also presented.     

Localization of Nopp140 within mammalian cells during interphase and mitosis

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.     

Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4-mediated recognition through a single amino acid change

Leptosphaeria maculans is the ascomycete responsible for one of the most damaging diseases of oilseed rape ( Brassica napus ), stem canker of crucifers. Both avirulence ( AvrLm ) genes in the fungus and resistance ( Rlm ) genes in the plant are genetically clustered. Using a map-based cloning strategy, we delineated a 238 kb region containing the AvrLm7 locus. Structural features of the region were reminiscent of those previously found on another chromosome for genomic regions encompassing AvrLm1 and AvrLm6 , i.e. GC-equilibrated, gene-rich isochores alternating with AT-rich, recombination-deficient, gene-poor isochores. These latter corresponded to mosaics of degenerated and truncated transposable elements. AvrLm7 is the only gene located within a 60 kb AT-rich isochore. It induced resistance responses in plants harbouring either Rlm7 or Rlm4 , and was thus renamed AvrLm4-7 . It encodes a 143-amino-acid cysteine-rich protein, predicted to be secreted, and strongly induced during early stages of plant infection. Sequencing and restriction analyses of AvrLm4–AvrLm7 or avrLm4–AvrLm7 alleles in L. maculans field isolates, and targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine to an arginine residue, was responsible for the loss of AvrLm4 specificity whereas AvrLm7 recognition was unaltered.