Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.     

Trapping of Aethina tumida Murray (Coleoptera: Nitidulidae) from Apis mellifera L. (Hymenoptera: Apidae) colonies with an in-hive baited trap

The effectiveness of two lures for trapping the small hive beetle, Aethina tumida, by means of in-hive traps was tested by field trials in apiaries located in Florida, Delaware, and Pennsylvania during 2003-2005. Both lures included a mixture (pollen dough) consisting of bee pollen and commercial pollen substitute formulated with or without glycerol and honey. Before it was used in the traps, the dough was conditioned either by the feeding of adult small hive beetles or by inoculation with the yeast Kodamaea ohmeri (NRRL Y-30722). Traps baited with conditioned dough captured significantly more beetles than unbaited traps, and traps positioned under the bottom board of a hive captured significantly more beetles than traps located at the top of a hive. In fact, baited in-hive bottom board traps nearly eliminated the beetles from colonies at a pollination site in Florida. However, when these honey bee colonies were moved to an apiary, trap catch increased markedly over time, indicating a resurgence of the beetle population produced by immigration of beetles from nearby hives or emerging from the soil. In tests at three Florida apiaries during 2006, yeast-inoculated dough baited bottom board traps captured significantly more beetles than unbaited traps, showing the effectiveness of yeast-inoculated dough as a lure and its potential as a tool in managing the small hive beetle.     

Evolution of the TOR Pathway

The TOR kinase is a major regulator of growth in eukaryotes. Many components of the TOR pathway are implicated in cancer and metabolic diseases in humans. Analysis of the evolution of TOR and its pathway may provide fundamental insight into the evolution of growth regulation in eukaryotes and provide a practical framework on which experimental evidence can be compared between species. Here we performed phylogenetic analyses on the components of the TOR pathway and determined their point of invention. We find that the two TOR complexes and a large part of the TOR pathway originated before the Last Eukaryotic Common Ancestor and form a core to which new inputs have been added during animal evolution. In addition, we provide insight into how duplications and sub-functionalization of the S6K, RSK, SGK and PKB kinases shaped the complexity of the TOR pathway. In yeast we identify novel AGC kinases that are orthologous to the S6 kinase. These results demonstrate how a vital signaling pathway can be both highly conserved and flexible in eukaryotes.     

Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.     

Consistent growth of black cottonwoods despite temperature variation across elevational ecoregions in the Rocky Mountains

Globally, water and temperature provide the dominant environmental determinants of tree distribution and growth. In riparian or streamside zones, groundwater is abundant, and we consequently predicted that temperature would limit the growth of riparian cottonwoods in a cool climate northern mountain region. To investigate this association, we analyzed tree rings of 167 black cottonwoods, Populus trichocarpa, along two adjacent Rocky Mountain creeks in Alberta. Cottonwoods were sampled from 1700 m, near their upper elevational limit, down 500 m through three progressively warmer ecoregions, the montane, aspen parkland, and fescue prairie. Across these zones, June through September mean temperatures rose from 12.4 to 16.2°C (lapse rate = 0.67°C/100 m), and there was subsequently a 42% increase in growing degree days (base 5°C, GDD₅) from 900 GDD₅ at the trees’ upper limit. Despite this variation, growth rate of most trees was fairly consistent across the ecoregions; trunk diameter versus age associations were relatively similar (r ² = 0.85) with an estimated 14% increase in trunk sizes of 50 year-old trees with decreasing elevation. In all ecoregions, developmental patterns were prominent as annual radial increments increased up to about 20 years, and then progressively declined to an apparent lethal threshold of about 0.4 mm/year at about 100 years. Basal area increments also increased through the juvenile phase, but remained fairly constant thereafter. The weak association between growth and temperature suggests that other environmental factors limited growth rates or there were differences in temperature adaptation across these elevational ecoregions. The results suggest that predicted regional climate warming may not substantially promote the growth rates of these Rocky Mountain trees.     

Detection of cytogenetic regions influencing RNA polymerase activity in Drosophila melanogaster by segmental aneuploid mapping

In a search for loci with RNA polymerase related function, we have screened the Drosophila melanogaster genome for regions effective in eliciting a dosage response for total RNA polymerase activity using segmental aneuploids generated by the use of Y-autosome translocations. From this screen we have identified a total of six cytogenetically defined regions which elicit significant dosage response: a single X-chromosome region, 9C-11A, within which resides a known RNA polymerase locus and five noncontiguous regions on chromosome 3 with no previously identified RNA polymerase related function.     

A rolling-gliding wear simulator for the investigation of tribological material pairings for application in total knee arthroplasty

Background  

Material wear testing is an important technique in the development and evaluation of materials for use in implant for total knee arthroplasty. Since a knee joint induces a complex rolling-gliding movement, standardised material wear testing devices such as Pin-on-Disc or Ring-on-Disc testers are suitable to only a limited extent because they generate pure gliding motion only.     

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.     

Protein complex evolution does not involve extensive network rewiring

The formation of proteins into stable protein complexes plays a fundamental role in the operation of the cell. The study of the degree of evolutionary conservation of protein complexes between species and the evolution of protein-protein interactions has been hampered by lack of comprehensive coverage of the high-throughput (HTP) technologies that measure the interactome. We show that new high-throughput datasets on protein co-purification in yeast have a substantially lower false negative rate than previous datasets when compared to known complexes. These datasets are therefore more suitable to estimate the conservation of protein complex membership than hitherto possible. We perform comparative genomics between curated protein complexes from human and the HTP data in Saccharomyces cerevisiae to study the evolution of co-complex memberships. This analysis revealed that out of the 5,960 protein pairs that are part of the same complex in human, 2,216 are absent because both proteins lack an ortholog in S. cerevisiae, while for 1,828 the co-complex membership is disrupted because one of the two proteins lacks an ortholog. For the remaining 1,916 protein pairs, only 10% were never co-purified in the large-scale experiments. This implies a conservation level of co-complex membership of 90% when the genes coding for the protein pairs that participate in the same protein complex are also conserved. We conclude that the evolutionary dynamics of protein complexes are, by and large, not the result of network rewiring (i.e. acquisition or loss of co-complex memberships), but mainly due to genomic acquisition or loss of genes coding for subunits. We thus reveal evidence for the tight interrelation of genomic and network evolution.