The prion protein binds thiamine

Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction.     

Mass spectrometry imaging–based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme

Aminotransferases (ATs) catalyze pyridoxal 5′-phosphate–dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging–based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI–mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime–mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.     

The maize genetics and genomics database. The community resource for access to diverse maize data

The Maize Genetics and Genomics Database (MaizeGDB) serves the maize (Zea mays) research community by making a wealth of genetics and genomics data available through an intuitive Web-based interface. The goals of the MaizeGDB project are 3-fold: to provide a central repository for public maize information; to present the data through the MaizeGDB Web site in a way that recapitulates biological relationships; and to provide an array of computational tools that address biological questions in an easy-to-use manner at the site. In addition to these primary tasks, MaizeGDB team members also serve the community of maize geneticists by lending technical support for community activities, including the annual Maize Genetics Conference and various workshops, teaching researchers to use both the MaizeGDB Web site and Community Curation Tools, and engaging in collaboration with individual research groups to make their unique data types available through MaizeGDB.     

Training leading to repetition failure enhances bench press strength gains in elite junior athletes

The purpose of this study was to investigate the importance of training leading to repetition failure in the performance of 2 different tests: 6 repetition maximum (6RM) bench press strength and 40-kg bench throw power in elite junior athletes. Subjects were 26 elite junior male basketball players (n = 12; age = 18.6 +/- 0.3 years; height = 202.0 +/- 11.6 cm; mass = 97.0 +/- 12.9 kg; mean +/- SD) and soccer players (n = 14; age = 17.4 +/- 0.5 years; height = 179.0 +/- 7.0 cm; mass = 75.0 +/- 7.1 kg) with a history of greater than 6 months' strength training. Subjects were initially tested twice for 6RM bench press mass and 40-kg Smith machine bench throw power output (in watts) to establish retest reliability. Subjects then undertook bench press training with 3 sessions per week for 6 weeks, using equal volume programs (24 repetitions x 80-105% 6RM in 13 minutes 20 seconds). Subjects were assigned to one of two experimental groups designed either to elicit repetition failure with 4 sets of 6 repetitions every 260 seconds (RF(4 x 6)) or allow all repetitions to be completed with 8 sets of 3 repetitions every 113 seconds (NF(8 x 3)). The RF(4 x 6) treatment elicited substantial increases in strength (7.3 +/- 2.4 kg, +9.5%, p < 0.001) and power (40.8 +/- 24.1 W, +10.6%, p < 0.001), while the NF(8 x 3) group elicited 3.6 +/- 3.0 kg (+5.0%, p < 0.005) and 25 +/- 19.0 W increases (+6.8%, p < 0.001). The improvements in the RF(4 x 6) group were greater than those in the repetition rest group for both strength (p < 0.005) and power (p < 0.05). Bench press training that leads to repetition failure induces greater strength gains than nonfailure training in the bench press exercise for elite junior team sport athletes.     

Chemical-induced, nonlethal, developmental model of dissecting aortic aneurysm

BACKGROUND: A chemical-induced, nonlethal, dissecting aortic aneurysm (DAA) is described following in utero exposure to semicarbazide, an inhibitor of the vascular enzyme semicarbazide sensitive amine oxidase (SSAO). METHODS: Sprague-Dawley rat dams were given semicarbazide (0.096-49.000 mg/kg/day) by IP injection on gestation days (GDs) 14-20, a period of rapid aortic development. Newborn rats (day 1) were killed and their thoracic organs were removed en bloc for near-serial cross sections and routine histopathology, Movat stain for elastin, and immunohistochemistry to differentiate cells involved in the evolution of the DAA. In subsequent experiments, pups from treated dams (0.096-6.125 mg/kg/day) were allowed to survive for 7 or 28 days. RESULTS: DAA occurred in nearly 100% of the rats at all doses except the lowest tested (1.530, 0.096 mg/kg/day). Dissections frequently extended to the carotids and, less frequently, to the abdominal aorta. Remodeling of vascular lesions proceeded by organization of collections of blood in vascular media (the "false lumen"), proliferation of vascular smooth muscle cells, fibrosis, and formation of irregular frayed elastic lamellae in healed vascular media. Biochemical quantitation and Western blot analysis of main extracellular matrix proteins on GD 20 showed no overt difference in expression of collagen type I, fibrillin-1, or elastin. CONCLUSION: This developmental model provides investigators an opportunity to explore the pathologic mechanisms of DAA and to examine the potential long-term effects of vascular remodeling of DAA.     

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.     

Synthesis and characterization of peptide grafted porous polymer microstructures

A scanning laser system has been used to generate three-dimensional trimethylolpropane trimethacrylate (TRIM) cross-linked poly(2-hydroxylethyl methacrylate) polymer microstructures through azo-bis(isobutyro)nitrile (AIBN) photopolymerization using a 20 x 0.5 NA microscope objective and 365 nm laser excitation. Macropores are observed to form without the use of porogens in regions of highest light flux. This is attributed to phase separation, which results from differences in monomer reactivity and miscibility. The microstructures were aminated and then protected with the photolabile protective group 6-nitroveratryloxycarbonyl (NVOC). This made it possible to selectively modify the microstructures with the same scanning laser system that was used to fabricate them, resulting in peptide grafted three-dimensional porous microstructures. On the basis of the absorbance of the dibenzofulvene-piperidine, these structures have an amine site density of approximately 0.1 nmol/feature. MALDI-TOF MS was used to characterize peptide photografted microstructures. N-Tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) labeling of the peptides greatly enhanced detection and allowed post-source decay sequencing of the peptides from the microstructures. The techniques described could be used to generate three-dimensional peptide grafted porous scaffolds for tissue engineering applications.