Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.     

Inhibition of PrKX, a Novel Protein Kinase, and the Cyclic AMP-Dependent Protein Kinase PKA by the Regulatory Proteins of Adeno-Associated Virus Type 2

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, K_is of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly inhibit the trans- and autophosphorylation activities of these kinases. The biological effects of Rep78-specific inhibition of PKA-responsive genes are illustrated by the reduction of steady-state levels of cyclic AMP-responsive-element-binding protein and cyclin A protein.     

Open-Source Selective Laser Sintering (OpenSLS) of Nylon and Biocompatible Polycaprolactone

Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, high-resolution structures with SLS using diverse starting materials (including biomaterials), prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 μm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials.     

Sports and Exercise at Different Ages and Leukocyte Telomere Length in Later Life – Data from the Berlin Aging Study II (BASE-II)

Physical activity and sports have repeatedly been reported to be associated with telomere length. We studied the association of different types of sports across different stages of life on relative leukocyte telomere length (rLTL) in advanced age.815 participants (397 men) from the Berlin Aging Study II aged over 61 years were included in the analysis. rLTL was measured by real time PCR and physical activity was determined retrospectively by questionnaire, assessing type and duration of sports in the past as well as currently. Five separate multiple linear regression models adjusted for various control variables were performed. 67.3% of participants exercised currently, whereas 19.4% performed sports only between the age of 20 and 30. rLTL was higher in subjects who stated to exercise currently (N = 456), and in subjects who engaged in endurance (N = 138) or intensive activity sports (N = 32). Current physical activity was positively associated with rLTL in the risk factor adjusted regression model (β = 0.26, p < 0.001) and practicing sports for a minimum of 10 years preceding the assessment had a significant effect on rLTL (β = 0.39, p = 0.011). The highest impact was seen for intensive activity sports (β = 0.79, p < 0.001) and physical activity since at least 42 years (β = 0.47, p = 0.001). However, physical activity only between 20 and 30 years of age did not affect rLTL in old age when compared to no sports at all (β = -0.16, p = 0.21). Physical activity is clearly associated with longer rLTL. The effect is seen with longer periods of physical activity (at least 10 years), with intensive sports activities having the greatest impact on rLTL. Our data suggest that regular physical activity for at least 10 years is necessary to achieve a sustained effect on rLTL.