Quantification of oxidized levels of specific RNA species using an aldehyde reactive probe

Emerging evidence has shown that oxidation of RNA, including messenger RNA (mRNA), is elevated in several age-related diseases, although investigation of oxidized levels of individual RNA species has been limited. Recently we reported that an aldehyde reactive probe (ARP) quantitatively reacts with oxidatively modified depurinated/depyrimidinated (abasic) RNA. Here we report a novel method to isolate oxidized RNA using ARP and streptavidin beads. An oligo RNA containing abasic sites that were derivatized with ARP was pulled down by streptavidin beads, whereas a control oligo RNA was not. In vitro oxidized RNA, as well as total cellular RNA, isolated from oxidatively stressed cells was also pulled down, dependent on oxidation level, and concentrated in the pull-down fraction. Quantitative reverse transcription polymerase chain reaction (RT-PCR) using RNA in the pull-down fraction demonstrated that several gene transcripts were uniquely increased in the fraction by oxidative stress. Thus, our method selectively concentrates oxidized RNA by pull-down and enables the assessment of oxidation levels of individual RNA species. (C) 2011 Elsevier Inc. All rights reserved.

Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Accuracy of navigated surgery of the pelvis after surface matching with an a-mode ultrasound probe

Computer-aided surgery (CAS) allows for real-time intraoperative feedback resulting in increased accuracy, while reducing intraoperative radiation. CAS is especially useful for the treatment of certain pelvic ring fractures, which necessitate the precise placement of screws. Flouroscopy-based CAS modules have been developed for many orthopedic applications. The integration of the isocentric flouroscope even enables navigation using intraoperatively acquired three-dimensional (3D) data, though the scan volume and imaging quality are limited. Complicated and comprehensive pathologies in regions like the pelvis can necessitate a CT-based navigation system because of its larger field of view. To be accurate, the patient's anatomy must be registered and matched with the virtual object (CT data). The actual precision within the region of interest depends on the area of the bone where surface matching is performed. Conventional surface matching with a solid pointer requires extensive soft tissue dissection. This contradicts the primary purpose of CAS as a minimally invasive alternative to conventional surgical techniques. We therefore integrated an a-mode ultrasound pointer into the process of surface matching for pelvic surgery and compared it to the conventional method. Accuracy measurements were made in two pelvic models: a foam model submerged in water and one with attached porcine muscle tissue. Three different tissue depths were selected based on CT scans of 30 human pelves. The ultrasound pointer allowed for registration of virtually any point on the pelvis. This method of surface matching could be successfully integrated into CAS of the pelvis.