Identification of a novel 7-cis-11-trans-lipoxin A4 generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of lipoxins A4 and B4

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.     

Early effects of temperate agroforestry practices on soil organic matter and microbial enzyme activity

Plant and Soil - A field experiment was conducted to evaluate the effects of alley cropping systems on microbial activity and soil organic matter (SOM) pools. We hypothesized that enzyme activity...     

BlackOPs: increasing confidence in variant detection through mappability filtering

Identifying variants using high-throughput sequencing data is currently a challenge because true biological variants can be indistinguishable from technical artifacts. One source of technical artifact results from incorrectly aligning experimentally observed sequences to their true genomic origin (‘mismapping’) and inferring differences in mismapped sequences to be true variants. We developed BlackOPs, an open-source tool that simulates experimental RNA-seq and DNA whole exome sequences derived from the reference genome, aligns these sequences by custom parameters, detects variants and outputs a blacklist of positions and alleles caused by mismapping. Blacklists contain thousands of artifact variants that are indistinguishable from true variants and, for a given sample, are expected to be almost completely false positives. We show that these blacklist positions are specific to the alignment algorithm and read length used, and BlackOPs allows users to generate a blacklist specific to their experimental setup. We queried the dbSNP and COSMIC variant databases and found numerous variants indistinguishable from mapping errors. We demonstrate how filtering against blacklist positions reduces the number of potential false variants using an RNA-seq glioblastoma cell line data set. In summary, accounting for mapping-caused variants tuned to experimental setups reduces false positives and, therefore, improves genome characterization by high-throughput sequencing.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Creating porcine biomedical models through recombineering

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to 'forward genetics', in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the 'reverse genetics' approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a 'genetic model' organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide 'clones' of genetically modified animals.     

Fine-mapping of the type 1 diabetes locus (IDDM4) on chromosome 11q and evaluation of two candidate genes (FADD and GALN) by affected sibpair and linkage-disequilibrium analyses

Previous studies have identified a susceptibility region for insulin-dependent (type 1) diabetes mellitus on chromosome 11q13 (IDDM4). In this study, 15 polymorphic markers were analyzed for 382 affected sibpair (ASP) families with type 1 diabetes. Our analyses provided additional evidence for linkage for IDDM4 (a peak LOD score of 3.4 at D11S913). The markers with strong linkage evidence are located within an interval of approximately 6 cM between D11S4205 and GALN. We also identified polymorphisms in two candidate genes, Fas-associated death domain protein (FADD) and galanin (GALN). Analyses of the data by transmission/disequilibrium test (TDT) and extended TDT (ETDT) did not provide any evidence for association/linkage with these candidate genes. However, ETDT did reveal significant association/linkage with the marker D11S987 (P=0.0004) within the IDDM4 interval defined by ASP analyses, suggesting that IDDM4 may be in the close proximity of D11S987.     

Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Nitrile hydratase genes are present in multiple eukaryotic supergroups

Background

Nitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens.

Methodology/Principal Findings

Here we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade.

Conclusions/Significance

The evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.