raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences

Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences. However, such arguments are based upon the examination of a limited number of protein folds. To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior. To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members. As demonstrated for mUbi [Krantz, B. A., and Sosnick, T. R. (2000) Biochemistry 39, 11696-11701], we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD. We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue. These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence.     

Equivalence of multibreed animal models and hierarchical Bayes analysis for maternally influenced traits

Background

It has been argued that multibreed animal models should include a heterogeneous covariance structure. However, the estimation of the (co)variance components is not an easy task, because these parameters can not be factored out from the inverse of the additive genetic covariance matrix. An alternative model, based on the decomposition of the genetic covariance matrix by source of variability, provides a much simpler formulation. In this study, we formalize the equivalence between this alternative model and the one derived from the quantitative genetic theory. Further, we extend the model to include maternal effects and, in order to estimate the (co)variance components, we describe a hierarchical Bayes implementation. Finally, we implement the model to weaning weight data from an Angus × Hereford crossbred experiment.

Methods

Our argument is based on redefining the vectors of breeding values by breed origin such that they do not include individuals with null contributions. Next, we define matrices that retrieve the null-row and the null-column pattern and, by means of appropriate algebraic operations, we demonstrate the equivalence. The extension to include maternal effects and the estimation of the (co)variance components through the hierarchical Bayes analysis are then straightforward. A FORTRAN 90 Gibbs sampler was specifically programmed and executed to estimate the (co)variance components of the Angus × Hereford population.

Results

In general, genetic (co)variance components showed marginal posterior densities with a high degree of symmetry, except for the segregation components. Angus and Hereford breeds contributed with 50.26% and 41.73% of the total direct additive variance, and with 23.59% and 59.65% of the total maternal additive variance. In turn, the contribution of the segregation variance was not significant in either case, which suggests that the allelic frequencies in the two parental breeds were similar.

Conclusion

The multibreed maternal animal model introduced in this study simplifies the problem of estimating (co)variance components in the framework of a hierarchical Bayes analysis. Using this approach, we obtained for the first time estimates of the full set of genetic (co)variance components. It would be interesting to assess the performance of the procedure with field data, especially when interbreed information is limited.     

Recoil effect of the ice hockey stick during a slap shot

The purpose of this study was to examine the "recoil" effect of the ice hockey stick shaft during a stationary slap shot. Nine male adult subjects (four elite and five recreational) were tested. Their performances were evaluated by simultaneously recording stick movement and internal bending from high-speed digital video (1,000 Hz) and puck acceleration from a triaxial accelerometer positioned inside the puck. In addition, an electrical circuit measured blade-puck contact time. Data were analyzed with a one-way MANOVA for several dependent variables, including final puck velocity, puck acceleration, maximum stick shaft bending (angle and distance deflection), stick shaft angular velocities, blade-puck contact time, and corresponding time events. The results indicate the following. First, blade-puck contact time was greater for the elite than for recreational players (38 +/- 9 ms and 27 +/- 5 ms); however, measures for puck acceleration were essentially the same (63.8 g +/- 9.9 and 61.8 g +/- 19.5). Two, the elite players were able to generate greater puck velocities (120 +/- 18 km/h and 80.3 +/- 11.6 km/h). Three, the recoil timing was found to be reater for elite players (59.8% of blade-puck contact).     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Design and evaluation of dihydroisoquinolines as potent and orally bioavailable human cytomegalovirus inhibitors

Following the identification of first pass metabolism issues with our recently described anti-HCMV compounds, the naphthyridines and isoquinolines, we have designed a class of novel metabolically stable and orally bioavailable anti-HCMV agents, the dihydroisoquinolines.     

Genome-wide location analysis reveals an important overlap between the targets of the yeast transcriptional regulators Rds2 and Adr1

A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of tetrahydrobiopterin (BH4) and dopamine in rat brain using epsilon-acetamidocaproic acid (AACA) as an internal standard. Proteins in the samples were precipitated with acetonitrile and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 × 100 mm, i.d., 3 μm) column using a mixture of 10mM ammonium formate in acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 300 μl/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) and positive ion mode from m/z 242.1 → 166.0 for BH4, m/z 154.1 → 90.0 for dopamine and m/z 174.1 → 114.0 for AACA (IS). The total chromatographic run time was for 5.5 min. The method was validated for the analysis of samples: the limit of detection was 10 ng/g. The calibration curve was linear between 10-2000 ng/g for BH4 (r(2)=0.995) and 10-5000 ng/g for dopamine (r(2)=0.997) in the rat brain. Thus, good correlated LC-ESI/MS/MS results were obtained and found to be a powerful tool for the quantitative analysis of BH4 and dopamine in the rat brain.     

Motor protein Myo5p is required to maintain the regulatory circuit controlling WOR1 expression in Candida albicans

The Candida albicans MYO5 gene encodes myosin I, a protein required for the formation of germ tubes and true hyphae. Because the polarized growth of opaque-phase cells in response to pheromone results in mating projections that can resemble germ tubes, we examined the role of Myo5p in this process. We localized green fluorescent protein (GFP)-tagged Myo5p in opaque-phase cells of C. albicans during both bud and shmoo formation. In vegetatively growing opaque cells, Myo5p is found at sites of bud emergence and bud growth, while in pheromone-stimulated cells, Myo5p localizes at the growing tips of shmoos. Intriguingly, cells homozygous for MTLa in which the MYO5 gene was deleted failed to switch efficiently from the white phase to the opaque phase, although ectopic expression of WOR1 from the MET3 promoter can convert myo5 mutants into mating-competent opaque cells. However, when WOR1 expression was shut off, the myo5-defective cells rapidly lost both their opaque phenotype and mating competence, suggesting that Myo5p is involved in the maintenance of the opaque state. When MYO5 is expressed conditionally in opaque cells, the opaque phenotype, as well as the mating ability of the cells, becomes unstable under repressive conditions, and quantitative real-time PCR demonstrated that the shutoff of MYO5 expression correlates with a dramatic reduction in WOR1 expression. It appears that while myosin I is not directly required for mating in C. albicans, it is involved in WOR1 expression and the white-opaque transition and thus is indirectly implicated in mating.