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.

Using transcranial magnetic stimulation to probe decision-making and memory

Decision-making and memory are fundamental processes for successful human behaviour. For eye movements, the frontal eye fields (FEF), the supplementary eye fields (SEF), the dorsolateral prefrontal cortex (DLPFC), the ventrolateral frontal cortex and the anterior cingulum are important for these cognitive processes. The online approach of transcranial magnetic stimulation (TMS), i.e., the application of magnetic pulses during planning and performance of saccades, allows interfering specifically with information processing of the stimulated region at a very specific time interval (chronometry of cortical processing). The paper presents studies, which showed the different roles of the FEF and DLPFC in antisaccade control. The critical time interval of DLPFC control seems to be before target onset since TMS significantly increased the percentage of antisaccade errors at that time interval. The FEF seems to be important for the triggering of correct antisaccades. Bilateral stimulation of the DLPFC could demonstrate parallel information-processing transfer in spatial working memory during memory-guided saccades.

Comparison of multiplex ligation-dependent probe amplification and real-time PCR accuracy for gene copy number quantification using the beta-defensin locus

The reliable quantification of gene copy number variations is a precondition for future investigations regarding their functional relevance. To date, there is no generally accepted gold standard method for copy number quantification, and methods in current use have given inconsistent results in selected cohorts. In this study, we compare two methods for copy number quantification. beta-defensin gene copy numbers were determined in parallel in 80 genomic DNA samples by real-time PCR and multiplex ligation-dependent probe amplification (MLPA). The pyrosequencing-based paralog ratio test (PPRT) was used as a standard of comparison in 79 out of 80 samples. Realtime PCR and MPLA results confirmed concordant DEFB4, DEFB103A, and DEFB104A copy numbers within samples. These two methods showed identical results in 32 out of 80 samples; 29 of these 32 samples comprised four or fewer copies. The coefficient of variation of MLPA is lower compared with PCR. In addition, the consistency between MLPA and PPRT is higher than either PCR/MLPA or PCR/PPRT consistency. In summary, these results suggest that MLPA is superior to real-time PCR in beta-defensin copy number quantification.

Occurrence of vasospasm and infarction in relation to a focal monitoring sensor in patients after SAH: placing a bet when placing a probe?

INTRODUCTION Vasospastic brain infarction is a devastating complication of aneurysmal subarachnoid hemorrhage (SAH). Using a probe for invasive monitoring of brain tissue oxygenation or blood flow is highly focal and may miss the site of cerebral vasospasm (CVS). Probe placement is based on the assumption that the spasm will occur either at the dependent vessel territory of the parent artery of the ruptured aneurysm or at the artery exposed to the focal thick blood clot. We investigated the likelihood of a focal monitoring sensor being placed in vasospasm or infarction territory on a hypothetical basis. METHODS From our database we retrospectively selected consecutive SAH patients with angiographically proven (day 7-14) severe CVS (narrowing of vessel lumen >50%). Depending on the aneurysm location we applied a standard protocol of probe placement to detect the most probable site of severe CVS or infarction. We analyzed whether the placement was congruent with existing CVS/infarction. RESULTS We analyzed 100 patients after SAH caused by aneurysms located in the following locations: MCA (n = 14), ICA (n = 30), A1CA (n = 4), AcoA or A2CA (n = 33), and VBA (n = 19). Sensor location corresponded with CVS territory in 93% of MCA, 87% of ICA, 76% of AcoA or A2CA, but only 50% of A1CA and 42% of VBA aneurysms. The focal probe was located inside the infarction territory in 95% of ICA, 89% of MCA, 78% of ACoA or A2CA, 50% of A1CA and 23% of VBA aneurysms. CONCLUSION The probability that a single focal probe will be situated in the territory of severe CVS and infarction varies. It seems to be reasonably accurate for MCA and ICA aneurysms, but not for ACA or VBA aneurysms.

The Use of a Vaginal Conductivity Probe to Influence Calf Sex Ratio via Altered Insemination Time

One hundred eighty-nine mixed breed beef heifers from 13 consignors enrolled in the MACEP heifer development project were utilized in this study. Heifers were synchronized by feeding 0.5 mg melengestrol acetate (MGA) per head per day for 14 days followed by an injection of prostaglandin F2a (PGF2a; 25 mg Lutalyse®) 17 days after the last MGA feeding. Each heifer was fitted with a Heatwatch® transmitter on the morning of PGF2a administration to facilitate detection of estrus. Vaginal conductivity measurements were taken using an Ovatec® probe every 12 hours for 96 hours beginning at the time of PGF2a injection. Heifers randomly assigned to produce a female calf were inseminated near the onset of estrus (as indicated by probe values of £ 55 on the decline). Heifers randomly assigned to produce a male calf were inseminated approximately 24 hours after the onset of estrus (as indicated by probe values of ³ 60 on the incline). All heifers not inseminated by 96 hours after PGF2a were mass inseminated in an attempt to impregnate as many heifers as possible. Heifers that were diagnosed as pregnant as a result of the artificial insemination were subjected to ultrasonography for fetal sex determination. Only 70 of the 189 heifers (37.0%) exhibited estrus according to Heatwatch® and incidence of estrus was influenced by heifer average daily gain, reproductive tract score, and disposition score. Heifers receiving a disposition score of 3 (78.7) had a higher (P<.05) probe reading at AI than those receiving a disposition score of 1 or 2 (70.8 and 72.5, respectively). Heifers with probe readings at insemination of 80 - 84 and > 84 had lower (P<.05) pregnancy rates to AI (13.6 and 0.0%, respectively) than heifers with probe readings in the ranges of < 60, 60 - 64, 65 - 69, 70 - 74, and 75 - 79 (35.7, 40.9, 31.4, 35.3, and 26.9% respectively). Heifers that were bred when probe values were increasing had a lower (P<.05) percentage of male fetuses (34.4%) than those bred during a period of decreasing probe values (69.2% male fetuses). These results demonstrate that a vaginal conductivity probe may be a useful tool to determine an insemination time that could potentially alter calf sex ratio.