Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library

The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.     

Effect of loading rate on the compressive mechanics of the immature baboon cervical spine

Thirty-four cervical spine segments were harvested from 12 juvenile male baboons and compressed to failure at displacement rates of 5, 50, 500, or 5000 mm/s. Compressive stiffness, failure load, and failure displacement were measured for comparison across loading rate groups. Stiffness showed a significant concomitant increase with loading rate, increasing by 62% between rates of 5 and 5000 mm/s. Failure load also demonstrated an increasing relationship with loading rate, while displacement at failure showed no rate dependence. These data may help in the development of improved pediatric automotive safety standards and more biofidelic physical and computational models.     

Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.     

Für welche Pflanzenarten hat die Schweiz eine internationale Verantwortung?

Eggenberg S. and Landolt E. 2006. For which plant species does Switzerland have an international responsibility? Bot. Helv. 116: 119 – 133. Priorities in plant species conservation are often based on national Red Lists. In an international context, however, the Red List status (threat) of a species within a limited territory may be misleading because the local disappearance of a species may or may not have serious implications for its global persistence. A second important aspect to consider in species conservation is therefore the responsibility of a country for the species, i.e. the importance of the conservation of local populations for the persistence of the species worldwide. In this contribution, we assess the responsibility of Switzerland for its vascular flora using three biogeographical criteria: (1) the Swiss portion of the species range (high responsibility for species with a large fraction of the range in Switzerland), (2) the degree of endemism (high responsibility for species with a small total range) and (3) the degree of isolation (high responsibility for isolated outposts, which may contain a large part of a species’ genetic variation). The three criteria were derived for each species from global and European distribution maps, and were then combined to an overall index of responsibility. On this basis, 397 taxa for which Switzerland has an intermediate to high international responsibility were identified. These are almost 15% of the whole vascular flora of Switzerland. Of the 397 taxa, 75% are endemic species of the Alps, and 48% are threatened taxa within Switzerland. The Responsibility List can be used together with the Red List to set priorities in plant conservation or to identify areas of particular floristic value. Manuskript angenommen am 11. September 2006     

Cloning and structural characterization of the 6-phosphogluconate dehydrogenase locus of the medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae

The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO(2) and the reduction of NADP. We have determined the genomic sequences of 6PGD of two species of Tephritidae, the medfly Ceratitis capitata and olive fruit fly Bactrocera oleae, and constructed a three-dimensional model of 6PGD of C. capitata based on the homologous known sheep structure. In a comparative study of 6PGD sequences from seven species, all the conserved and variable sites of the enzyme were analyzed and the regions of functional importance were localized, an attempt promoted also by the direct involvement of the enzyme in various human diseases. The enzymes between the two species of Tephritidae have a very high homology and further examination of the variable positions with respect to the highly conserved binding site residues enabled their grouping in three distinct categories, with possible association to dimer formation, functional specificity, and antigenicity. Moreover, placement of sequence differences on the 3-D model suggests probable sites accommodating variations appearing at the allozymic variants of both species.     

Highly dynamic exon shuffling in candidate pathogen receptors ... what if brown algae were capable of adaptive immunity?

Pathogen recognition is the first step of immune reactions. In animals and plants, direct or indirect pathogen recognition is often mediated by a wealth of fast-evolving receptors, many of which contain ligand-binding and signal transduction domains, such as leucine-rich or tetratricopeptide repeat (LRR/TPR) and NB-ARC domains, respectively. In order to identify candidates potentially involved in algal defense, we mined the genome of the brown alga Ectocarpus siliculosus for homologues of these genes and assessed the evolutionary pressures acting upon them. We thus annotated all Ectocarpus LRR-containing genes, in particular an original group of LRR-containing GTPases of the ROCO family, and 24 NB-ARC-TPR proteins. They exhibit high birth and death rates, while a diversifying selection is acting on their LRR (respectively TPR) domain, probably affecting the ligand-binding specificities. Remarkably, each repeat is encoded by an exon, and the intense exon shuffling underpins the variability of LRR and TPR domains. We conclude that the Ectocarpus ROCO and NB-ARC-TPR families are excellent candidates for being involved in recognition/transduction events linked to immunity. We further hypothesize that brown algae may generate their immune repertoire via controlled somatic recombination, so far only known from the vertebrate adaptive immune systems.     

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules.     

Quantile-based permutation thresholds for quantitative trait Loci hotspots

Quantitative trait loci (QTL) hotspots (genomic locations affecting many traits) are a common feature in genetical genomics studies and are biologically interesting since they may harbor critical regulators. Therefore, statistical procedures to assess the significance of hotspots are of key importance. One approach, randomly allocating observed QTL across the genomic locations separately by trait, implicitly assumes all traits are uncorrelated. Recently, an empirical test for QTL hotspots was proposed on the basis of the number of traits that exceed a predetermined LOD value, such as the standard permutation LOD threshold. The permutation null distribution of the maximum number of traits across all genomic locations preserves the correlation structure among the phenotypes, avoiding the detection of spurious hotspots due to nongenetic correlation induced by uncontrolled environmental factors and unmeasured variables. However, by considering only the number of traits above a threshold, without accounting for the magnitude of the LOD scores, relevant information is lost. In particular, biologically interesting hotspots composed of a moderate to small number of traits with strong LOD scores may be neglected as nonsignificant. In this article we propose a quantile-based permutation approach that simultaneously accounts for the number and the LOD scores of traits within the hotspots. By considering a sliding scale of mapping thresholds, our method can assess the statistical significance of both small and large hotspots. Although the proposed approach can be applied to any type of heritable high-volume "omic" data set, we restrict our attention to expression (e)QTL analysis. We assess and compare the performances of these three methods in simulations and we illustrate how our approach can effectively assess the significance of moderate and small hotspots with strong LOD scores in a yeast expression data set.