Breeding biology and provisioning of nestling snow buntings in the Canadian High Arctic

We examined basic breeding biology, as well as the effects of parental sex, brood size, nestling age, and nest habitat on foraging distances and parental food delivery rates of snow buntings (Plectrophenax nivalis) on Devon Island, NT, Canada, in the summers of 2003–2006. Clutch sizes and initiation dates were similar to those found in previous studies, although nest density was much higher. Feeding rates and foraging distances of buntings differed depending on the size and age of the brood, but were similar between nest habitats. Unlike in earlier studies, we found that male buntings made fewer feeding visits, but traveled longer distances to forage for food than females. Whether these differences between the sexes relate to reducing competition for prey, or to differing sex roles in parental care due to the poorly insulated nest cavities of this species, remains to be determined.     

Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO₂ chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide ⁹⁰VSELpSFR⁹⁶ (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1_P216, F2_P216, F3_P216) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1_P216, F2_P216 and F3_P216 adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae . Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.     

The Scottish Structural Proteomics Facility: targets, methods and outputs

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.     

Translational Signalling,Atrogenic and Myogenic Gene Expression during Unloading and Reloading of Skeletal Muscle in Myostatin-Deficient Mice

Skeletal muscles of myostatin null (Mstn(−/−)) mice are more susceptible to atrophy during hind limb suspension (HS) than are muscles of wild-type mice. Here we sought to elucidate the mechanism for this susceptibility and to determine if Mstn(−/−) mice can regain muscle mass after HS. Male Mstn(−/−) and wild-type mice were subjected to 0, 2 or 7 days of HS or 7 days of HS followed by 1, 3 or 7 days of reloading (n = 6 per group). Mstn(−/−) mice lost more mass from muscles expressing the fast type IIb myofibres during HS and muscle mass was recovered in both genotypes after reloading for 7 days. Concentrations of MAFbx and MuRF1 mRNA, crucial ligases regulating the ubiquitin-proteasome system, but not MUSA1, a BMP-regulated ubiquitin ligase, were increased more in muscles of Mstn(−/−) mice, compared with wild-type mice, during HS and concentrations decreased in both genotypes during reloading. Similarly, concentrations of LC3b, Gabarapl1 and Atg4b, key effectors of the autophagy-lysosomal system, were increased further in muscles of Mstn(−/−) mice, compared with wild-type mice, during HS and decreased in both genotypes during reloading. There was a greater abundance of 4E-BP1 and more bound to eIF4E in muscles of Mstn(−/−) compared with wild-type mice (P<0.001). The ratio of phosphorylated to total eIF2α increased during HS and decreased during reloading, while the opposite pattern was observed for rpS6. Concentrations of myogenic regulatory factors (MyoD, Myf5 and myogenin) mRNA were increased during HS in muscles of Mstn(−/−) mice compared with controls (P<0.001). We attribute the susceptibility of skeletal muscles of Mstn(−/−) mice to atrophy during HS to an up- and downregulation, respectively, of the mechanisms regulating atrophy of myofibres and translation of mRNA. These processes are reversed during reloading to aid a faster rate of recovery of muscle mass in Mstn(−/−) mice.     

Xylogenesis in tissue culture: Taxol effects on microtubule reorientation and lateral association in differentiating cells

Summary InZinnia elegans tissue cultures, cortical microtubules reorient from longitudinal to transverse arrays as the culture age increases and before differentiation of tracheary elements is visible. The orientation of microtubules, in the period just before visible differentiation, determines the direction of the secondary wall bands in forming tracheary elements. Taxol, applied early in culture, stabilizes the microtubules of most cells in the longitudinal direction. Tracheary elements differentiating in these taxol treated cultures show secondary wall bands parallel to the long axis of the cell while those differentiating in control cultures always have wall bands transverse to the long axis of the cell.It is proposed that, in untreatedZinnia cultures, microtubules are reoriented by a gradual shift from longitudinal to transverse and this reorientation normally occurs before differentiation becomes visible. Once initiated, tracheary element differentiation involves lateral association of microtubules to form the discrete bands typical of secondary wall patterns.     

Chemical treatment of Escherichia coli: 1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetet-raacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 453-458, 1997.     

Hydrolysis of the polysaccharides of straw by enzymes produced by a mutant strain of the fungus Penicillium pinophilum

The saccharification of the polysaccharides of barley, oat, and wheat straws and Solka Floc was studied using the extracellular enzyme system synthesized by mutant strain NTG III/6 of the fungus Penicillium pinophilum 87160iii. The enzymes obtained in cultures containing Solka Floc or barley straw as the carbon source were compared. Solka Floc at 10% (w/v) concentration was hydrolyzed to the extent of 70% in 72 h at 50 degrees C using a reaction mixture containing 7 filter paper units/mL of cellulase induced on Solka Floc, but hydrolysis was increased to 90% when the enzyme induced on barley straw was used. Under the same conditions, the polysaccharides in barley, oat, and wheat straws were hydrolyzed, respectively, in 72 h, to the extent of 42-48%, 62%, and 52%, but hydrolysis was increased to 93%, 100%, and 92%, respectively, after treatment of the substrates with alkaline-H(2)O(2) reagent at room temperature.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.