Metabolic effects of adenosine on regional myocardial ischemia by phosphorus 31 nuclear magnetic resonance spectroscopy

The metabolic effects of adenosine on regionally ischemic myocardium were investigated in an open-chest rabbit model by means of phosphorus 31 nuclear magnetic resonance (NMR) spectroscopy. Sixteen anesthetized New Zealand white rabbits were subjected to thoracotomy; a reversible snare occluder was placed around a large branch of the left circumflex coronary artery, and an NMR surface coil was positioned adjacent to the myocardium perfused by this vessel. The animals were placed in a 2.0 T CSI spectrometer (GE Medical Systems, Fremont, Calif.), and baseline spectra were acquired. Eight animals were treated with intravenous adenosine (25 mg/kg), and eight rabbits served as control subjects. All animals were subjected to a 10-minute period of ischemia followed by a period of reperfusion. NMR spectra were acquired during both intervals. During the occlusion period, expected increases in inorganic phosphate levels and decreases in phosphocreatine levels were observed in both groups; however, inorganic phosphate increased less in adenosine-treated animals (adenosine: 33 +/- 2.8% total spectral area during occlusion vs control: 41+/-3.1%) and phosphocreatine diminished less with adenosine (adenosine: 26+/-3% vs control: 13+/-1.2%; p<0.002). No significant differences were seen in [beta]-adenosine triphosphate levels. In both groups the metabolite levels during reperfusion recovered to near baseline values, although phosphocreatine remained slightly higher in the treated group during early reperfusion. An apparent cardioprotective effect of adenosine on relative phosphocreatine and inorganic phosphate levels can be observed in intact rabbits by means of phosphorus 31 NMR spectroscopy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29773/1/0000112.pd

The Influence of Transcription Factor Competition on the Relationship between Occupancy and Affinity

Transcription factors (TFs) are proteins that bind to specific sites on the DNA and regulate gene activity. Identifying where TF molecules bind and how much time they spend on their target sites is key to understanding transcriptional regulation. It is usually assumed that the free energy of binding of a TF to the DNA (the affinity of the site) is highly correlated to the amount of time the TF remains bound (the occupancy of the site). However, knowing the binding energy is not sufficient to infer actual binding site occupancy. This mismatch between the occupancy predicted by the affinity and the observed occupancy may be caused by various factors, such as TF abundance, competition between TFs or the arrangement of the sites on the DNA. We investigated the relationship between the affinity of a TF for a set of binding sites and their occupancy. In particular, we considered the case of the transcription factor lac repressor (lacI) in E.coli, and performed stochastic simulations of the TF dynamics on the DNA for various combinations of lacI abundance and competing TFs that contribute to macromolecular crowding. We also investigated the relationship of site occupancy and the information content of position weight matrices (PWMs) used to represent binding sites. Our results showed that for medium and high affinity sites, TF competition does not play a significant role for genomic occupancy except in cases when the abundance of the TF is significantly increased, or when the PWM displays relatively low information content. Nevertheless, for medium and low affinity sites, an increase in TF abundance (for both cognate and non-cognate molecules) leads to an increase in occupancy at several sites. © 2013 Zabet et al

Distinguishing closely related amyloid precursors using an RNA aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ?2-microglobulin (h?2m) can co-polymerize with its N-terminally truncated variant (?N6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using h?2m and its amyloidogenic counterpart, ??6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds h?2m with an EC50 of ?200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-h?2m complex, revealing that the aptamer binds to the face of h?2m containing the A, B, E, and D ?-strands. In contrast, binding of B6 to ?N6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of h?2m and ?N6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (? 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ? 2.8-3.6 ?(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources