Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.     

YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity

Oligoribonuclease is the only RNase in Escherichia coli that is able to degrade RNA oligonucleotides five residues and shorter in length. Firmicutes including Bacillus subtilis do not have an Oligoribonuclease (Orn) homologous protein and it is not yet understood which proteins accomplish the equivalent function in these organisms. We had previously identified oligoribonucleases Orn from E. coli and its human homolog Sfn in a screen for proteins that are regulated by 3′-phosphoadenosine 5′-phosphate (pAp). Here, we identify YtqI as a potential functional analog of Orn through its interaction with pAp. YtqI degrades RNA oligonucleotides in vitro with preference for 3-mers. In addition, YtqI has pAp-phosphatase activity in vitro. In agreement with these data, YtqI is able to complement both orn and cysQ mutants in E. coli. An ytqI mutant in B. subtilis shows impairment of growth in the absence of cysteine, a phenotype resembling that of a cysQ mutant in E. coli. Phylogenetic distribution of YtqI, Orn and CysQ supports bifunctionality of YtqI.     

A novel approach to local similarity of protein binding sites substantially improves computational drug design results

We present a novel notion of binding site local similarity based on the analysis of complete protein environments of ligand fragments. Comparison of a query protein binding site (target) against the 3D structure of another protein (analog) in complex with a ligand enables ligand fragments from the analog complex to be transferred to positions in the target site, so that the complete protein environments of the fragment and its image are similar. The revealed environments are similarity regions and the fragments transferred to the target site are considered as binding patterns. The set of such binding patterns derived from a database of analog complexes forms a cloud-like structure (fragment cloud), which is a powerful tool for computational drug design. It has been shown on independent test sets that the combined use of a traditional energy-based score together with the cloud-based score responsible for the quality of embedding of a ligand into the fragment cloud improves the self-docking and screening results dramatically. The usage of a fragment cloud as a source of positioned molecular fragments fitting the binding protein environment has been validated by reproduction of experimental ligand optimization results.     

An EPR spin label study of the quinol oxidase, E. coli cytochrome bo3: a search for redox induced conformational changes

A search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome bo3, E. coli, has been carried out using site directed nitroxide spin labeling (SDSL) of cysteine residues. These were positioned at R134 and R309, on loops that link helices II and III and VI and VII at the entrances to the D and K proton channels, respectively. The motional characteristics of both labels have been determined using X- and W-band EPR spectroscopy at room temperature in selected redox levels in the reaction sequence of QO with oxygen, namely, the mixed valence carbon monoxide form (COMV), the oxidized (O) and super-oxidized (PM) states. The O to PM step is accompanied by the uptake of protons through the K pathway. We find no evidence for changes in the motional characteristics of either label that are expected to be associated with helical motions at the entrances to the channels. Because kinetic studies of mutants show that the redox gating of protons occurs deep within the D channel close to the heme-copper site, the present study implies that no motion is transmitted to the ends of the helices.     

Cationic penetrating antioxidants switch off Mn cluster of photosystem II in situ

Photosynthesis Research - Photosystem I (PSI) generates the most negative redox potential found in nature, and the performance of solar energy conversion into alternative energy sources in...     

Reaction centers of the thermophilic microaerophile,Chloracidobacterium thermophilum (Acidobacteria) I: biochemical and biophysical characterization

Photosynthesis Research - Chloracidobacterium thermophilum is a microaerophilic, anoxygenic member of the green chlorophototrophic bacteria. This bacterium is the first characterized...