A molecular phylogeny of the African widowbirds and bishops, Euplectes spp. (Aves: Passeridae: Ploceinae)

The elaborate male displays and plumage ornaments in the African widowbirds and bishops (Euplectes spp.) have inspired classic studies on mating systems and sexual selection. In order to study the extreme divergence in ornament design and expression in this group, we present and discuss a well-supported molecular phylogeny of the genus and its placement within the Ploceinae subfamily. Parsimony and Bayesian analyses were performed on 2557bp of mitochondrial DNA (ATP6, Cyt b, ND2 and ND3) and a nuclear intron (G3PDH). All 17 Euplectes species, and 31 of 51 suggested subspecies, were included, as well as eight Ploceinae outgroups from four genera (Amblyospiza, Ploceus, Quelea and Foudia). Our results show monophyly of Euplectes, but not of the intrageneric groupings of bishops and widowbirds. Most notably, the Red-collared Widowbird E. ardens belongs to a subclade of bishops, and not to the sister subclade of 'true' widowbirds. Furthermore, the two bishops E. afer and E. aureus represent lineages that branched off before this basal split, which also refutes the proposed superspecies of E. afer and E. diadematus. Also somewhat surprisingly, and despite the striking plumage similarities among the red bishops, E. franciscanus is not closely related to either E. nigroventris or E. orix (of which it until recently was considered a subspecies). Finally, the Mountain Marsh Widowbird E. psammocromius is likely closest to the Long-tailed Widowbird E. progne, and not, as previously thought, to the Marsh Widowbird E. hartlaubi.     

Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.     

Structure of a membrane-binding domain from a non-enveloped animal virus: insights into the mechanism of membrane permeability and cellular entry

The gamma(1)-peptide is a 21-residue lipid-binding domain from the non-enveloped Flock House virus (FHV). Unlike enveloped viruses, the entry of non-enveloped viruses into cells is believed to occur without membrane fusion. In this study, we performed NMR experiments to establish the solution structure of a membrane-binding peptide from a small non-enveloped icosahedral virus. The three-dimensional structure of the FHV gamma(1)-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment. The secondary and tertiary structures were evaluated in the context of the capacity of the peptide for permeabilizing membrane vesicles of different lipid composition, as measured by fluorescence assays. At both pH values, the peptide has a kinked structure, similar to the fusion domain from the enveloped viruses. The secondary structure was similar in three different hydrophobic environments as follows: water/trifluoroethanol, SDS, and membrane vesicles of different compositions. The ability of the peptide to induce vesicle leakage was highly dependent on the membrane composition. Although the gamma-peptide shares some structural properties to fusion domains of enveloped viruses, it did not induce membrane fusion. Our results suggest that small protein components such as the gamma-peptide in nodaviruses (such as FHV) and VP4 in picornaviruses have a crucial role in conducting nucleic acids through cellular membranes and that their structures resemble the fusion domains of membrane proteins from enveloped viruses.     

Quantification of protein concentration by the Bradford method in the presence of pharmaceutical polymers

We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595 nm) can be identified and corrected by recording absorption spectra in the region of 350–850 mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595 nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595 nm by measuring the sample absorbance at 850 nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595 nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.     

Efficient electroporation of peptides into adherent cells: investigation of the role of mechano-growth factor in chondrocyte culture

Peptide therapeutics are of increasing interest due to their biological specificity. We used a simple technique to study the efficacy of inducing peptides into adherent chondrocytes by transiently permeabilizing the membrane with electric pulses (in situ electroporation). Mechano-growth factor (MGF) was selected as a model peptide. FITC-labeled MGF was added to cultures of adherent primary chondrocytes grown on ITO coated glass slides. Cells were subjected to 3–9 pulses of 175–275 V and evaluated by flow cytometry. Under optimal conditions, an electroporation efficiency of close to 50% could be achieved. This technique can be used to study the functional domains of intracellular peptides, peptide inhibition of signal transduction and intracrine-mediated effects of peptides in adherent cells.     

Identification of 5-hydroxycytidine at position 2501 concludes characterization of modified nucleotides in E. coli 23S rRNA

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA—but a single modified nucleoside at position 2501 in 23S rRNA—has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine—a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.