The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA–damage site.</p

The Novel Calpain Inhibitor SJA6017 Improves Functional Outcome after Delayed Administration in a Mouse Model of Diffuse Brain Injury

A principal mechanism of calcium-mediated neuronal injury is the activation of neutral proteases known as calpains. Proteolytic substrates for calpain include receptor and cytoskeletal proteins, signal transduction enzymes and transcription factors. Recently, calpain inhibitors have been shown to provide benefit in rat models of focal head injury and focal cerebral ischemia. The present study sought to investigate, in experiment 1, the time course of calpain-mediated cytoskeletal injury in a mouse model of diffuse head injury by measuring the 150- and 145-kDa α-spectrin breakdown products (SBDP). Secondly, in experiment 2, we examined the effect of early (20 min postinjury) administration of the novel calpain inhibitor SJA6017 on functional outcome measured 24 h following injury and its effect on posttraumatic α-spectrin degradation. Lastly, in experiment 3, we examined the effect of delayed (4 or 6 h postinjury) administration of SJA6017 on 24-h postinjury functional outcome. In experiment 1, isoflurane-anesthetized male CF-1 mice (18-22 g) were subjected to a 750 g-cm weight drop-induced injury and were sacrificed for SBDP analysis at postinjury times of 30 min, and 1, 2, 6, 24 and 48 h (plus sham). In experiments 2 and 3, mice were injured as described, and delivered a single tail vein injection of either SJA6017 (0.3, 1, or 3 mg/kg) or vehicle (administered immediately, 4 or 6 h postinjury [3 mg/kg]). Functional outcome was evaluated in both studies , and, in experiment 2, 24-h postinjury assessment of SBDPs was determined. Following injury, the level of SBDP 145 was significantly different from sham at 24 and 48 h in cortical and at 24 h in the hippocampal tissues and at 48 h in the striatum. Immediate postinjury administration of SJA6017 resulted in a dose-related improvement in 24-h functional outcome (p < 0.05 at 3 mg/kg). Significance was maintained after a 4-h delay of the 3 mg/kg, but was lost after a 6-h delay. Despite improvement in functional outcome at 24 h, SJA6017 did not reduce spectrin breakdown in cortical or hippocampal tissues. These results support a role for calpain-mediated neuronal injury and the potential for a practical therapeutic window for calpain inhibition following traumatic brain injury. However, measurements of regional spectrin degradation may not be the most sensitive marker for determining the effects of calpain inhibition.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63231/1/089771501317095269.pd

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems

ALCHEMI Finds a “Shocking” Carbon Footprint in the Starburst Galaxy NGC 253

The centers of starburst galaxies may be characterized by a specific gas and ice chemistry due to their gas dynamics and the presence of various ice desorption mechanisms. This may result in a peculiar observable composition. We analyse the abundances of CO2, a reliable tracer of ice chemistry, from data collected as part of the Atacama Large Millimeter/submillimeter Array large program ALCHEMI, a wide-frequency spectral scan toward the starburst galaxy NGC 253 with an angular resolution of 1.″6. We constrain the CO2 abundances in the gas phase using its protonated form HOCO+. The distribution of HOCO+ is similar to that of methanol, which suggests that HOCO+ is indeed produced from the protonation of CO2 sublimated from ice. The HOCO+ fractional abundances are found to be (1-2) 7 10−9 at the outer part of the central molecular zone (CMZ), while they are lower (∼10−10) near the kinematic center. This peak fractional abundance at the outer CMZ is comparable to that in the Milky Way CMZ, and orders of magnitude higher than that in Galactic disk, star-forming regions. From the range of HOCO+/CO2 ratios suggested from chemical models, the gas-phase CO2 fractional abundance is estimated to be (1-20) 7 10−7 at the outer CMZ, and orders of magnitude lower near the center. We estimate the CO2 ice fractional abundances at the outer CMZ to be (2-5) 7 10−6 from the literature. A comparison between the ice and gas CO2 abundances suggests an efficient sublimation mechanism. This sublimation is attributed to large-scale shocks at the orbital intersections of the bar and CMZ