Flow dichroism of DNA: a new apparatus and further studies

A new apparatus for the study of flow dichroism of macromolecules is described. The flow is down a long, narrow channel and an unpolarized light beam propagates along the flow direction. For a molecule such as DNA, in which the transition moments of the chromophores are perpendicular to the axis of orientation, an increase of absorbance is observed during flow. The apparatus is best suited for macromolecules which are readily orientable or at high shear gradients so that the extinction angle is close to 0°. The apparatus has the following advantages: dilute macromolecule solutions can be used; high shear gradients are easily obtained; only small volumes of solution are needed. The flow can be stopped rapidly so that relaxation times for disorientation can be studied. The flow dichorism of native, two-stranded DNA has been measured for the molecular weight range of 0.6 × 10⁶ to 125 × 10⁶, and for the shear gradient range (in aqueous solution at 25°C) from 200 sec^?1 to 21000 sec^?1. At a fixed gradient the dichroism increases with molecular weight, but the curve is concave downwards. At a given molecular weight the dichroism increases with increasing shear gradient, but the curve is concave downwards. When the solvent viscosity and temperature are varied, the dichroism is a function of η〈G〉/T showing that the orientation is due to hydro-dynamic shear stress and that the flexibility of DNA in a flow field is not due to local denaturation. The Zimm-Rouse theory with no parameters taken from flow optical data predicts the correct order of magnitude of the dichroism but the experimentally observed shear gradient and molecular weight dependence do not fit the theory. This is an expected result, since the theory is believed to be applicable only at small distortions and extensions of the macromolecule.     

Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum

The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.     

IL-28 Supplants Requirement for Treg Cells in Protein σ1-Mediated Protection against Murine Experimental Autoimmune Encephalomyelitis (EAE)

Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (pσ1), may circumvent these shortcomings based upon the recent finding that when reovirus pσ1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP_130–151) was genetically fused to OVA to pσ1 (PLP:OVA-pσ1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10⁺ forkhead box P3 (FoxP3)⁺ CD25⁺CD4⁺ T_reg and IL-4⁺CD25⁻CD4⁺ Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-pσ1, and adoptive transfer of Ag-specific T_reg or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-pσ1''s protective capacity, triggering TGF-β-mediated inflammation; however, concomitant inactivation of TGF-β and CD25 reestablished PLP:OVA-pσ1-mediated protection by IL-28-producing FoxP3⁺CD25⁻CD4⁺ T cells. Thus, pσ1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.     

Does small-sided-games’ court area influence metabolic,perceptual, and physical performance parameters of young elite basketball players?

The purpose of this study was to investigate the effect of court size on physiological responses and physical performance of young elite basketball players. Twelve male basketball players (18.6 ± 0.5 years; 88.8 ± 14.5 kg; 192.6 ± 6.5 cm) from an under-19 team performed two small-sided games (matches) with different court areas (28x15 m and 28x9 m; 28x15 and 28x9 protocols). The number of players (3x3) was kept the same in each protocol. The players performed a repeated-sprint ability (RSA) test before and after each match. Blood lactate concentration was collected before (pre) and after (post) the matches, and the session rating of perceived exertion (session-RPE) was determined 30 minutes after the match. Best and mean time in the RSA test were not different between the 28x15 and the 28x9 match protocols (p > 0.05). A significant difference was observed for lactate concentration from pre- to post-match (p < 0.05) in both protocols (28x15 and 28x9); however, there was no significant interaction between protocols. A similar session-RPE mean score (28x15: 7.2 ± 1.4 and 28x9: 6.6 ± 1.4) was detected for both protocols (p > 0.05, ES=0.41). In summary, the results of the current study suggest that the different court areas induced similar responses. Although there was no significant difference in effort perception, players tended to perceive a greater effort in the larger court size.     

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.