g<Emphasis FontCategory="NonProportional">raphite</Emphasis> - a Bioconductor package to convert pathway topology to gene network

Background  

Gene set analysis is moving towards considering pathway topology as a crucial feature. Pathway elements are complex entities such as protein complexes, gene family members and chemical compounds. The conversion of pathway topology to a gene/protein networks (where nodes are a simple element like a gene/protein) is a critical and challenging task that enables topology-based gene set analyses.     

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (¹O₂) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to ¹O₂. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with ¹O₂ or superoxide (O^–₂). One sRNA was specifically induced by ¹O₂ and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by ¹O₂ and O^–₂ were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH_I and RpoH_II. The most abundant sRNA was processed in the presence of ¹O₂ but not by O^–₂. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of ¹O₂‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by ¹O₂ exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.     

Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)₂-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceA_Y51) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.     

Biogeographical and phylogenetic origins of African fig species (Ficus section Galoglychia)

Ficus section Galoglychia (subgenus Urostigma; Moraceae) includes 72 species restricted to the African floristic region (a few extending to the Arabian Peninsula and Socotra). We present the first molecular phylogenetic analysis of the section including 56 ingroup (representing 44 species) and three outgroup taxa, to investigate its monophyly, classification and evolution. We used sequence data from the nuclear ribosomal internal and external transcribed spacers (ITS and ETS). Our results suggest that section Galoglychia is paraphyletic to the neotropical section Americana, although this is not supported by bootstrap analysis and only weakly supported by Bayesian posterior probabilities. Maximum parsimony analysis conflict with maximum likelihood and Bayesian analyses with respect to the closest relatives of section Americana in Africa. The subsections of section Galoglychia proposed by Berg [Berg, C.C., 1986. Subdivision of Ficus subg. Urostigma sect. Galoglychia (Moraceae). Proc. Kon. Ned. Akad. Wetensch., Ser. C, 89, 121-127] are generally supported. We find two major clades of section Galoglychia within Africa possibly corresponding to two main centres of diversity. One clade comprises members of subsections Platyphyllae and Chlamydodorae, which are more concentrated in Eastern Africa, and extend to Madagascar and neighbouring archipelagos (Comores, Mascarenes, Aldabra Islands and Seychelles). The other main clade includes members of subsections Caulocarpae, Cyathistipulae, Crassicostae and Galoglychia, which are concentrated in West and Central Africa.     

One step cloning of defined DNA fragments from large genomic clones

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.