The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release

NADPH oxidase (Nox) family proteins produce superoxide (O2.-) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2.- intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2.- dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2.- leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2.- even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2.- in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2.- leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2.-. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2.- release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2.- leakage. Although we identified Duox1 A-loop residues (His1071, His1072, and Gly1074) important for reducing O2.- release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2.- releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex. © 2015, American Society for Biochemistry and Molecular Biology Inc

Volumetric analysis of the aging auditory pathway using high resolution magnetic resonance histology

Numerous shown consequences of age-related hearing loss have been unveiled; however, the relationship of the cortical and subcortical structures of the auditory pathway with aging is not well known. Investigations into neural structure analysis remain sparse due to difficulties of doing so in animal models; however, recent technological advances have been able to achieve a resolution adequate to perform such studies even in the small mouse. We utilize 12 members of the BXD family of recombinant inbred mice and aged separate cohorts. Utilizing novel magnetic resonance histology imaging techniques, we imaged these mice and generated high spatial resolution three dimensional images which were then comprehensively labeled. We completed volumetric analysis of 12 separate regions of interest specific to the auditory pathway brainstem nuclei and cortical areas with focus on the effect of aging upon said structures. Our results showed significant interstrain variation in the age-related effect on structure volume supporting a genetic influence in this interaction. Through multivariable modeling, we observed heterogenous effects of aging between different structures. Six of the 12 regions of interests demonstrated a significant age-related effect. The auditory cortex and ventral cochlear nucleus were found to decrease in volume with age, while the medial division of the medial geniculate nucleus, lateral lemniscus and its nucleus, and the inferior colliculus increased in size with age. Additionally, no sex-based differences were noted, and we observed a negative relationship between auditory cortex volume and mouse weight. This study is one of the first to perform comprehensive magnetic resonance imaging and quantitative analysis in the mouse brain auditory pathway cytoarchitecture, offering both novel insights into the neuroanatomical basis of age-related changes in hearing as well as evidence toward a genetic influence in this interaction. High resonance magnetic resonance imaging provides a promising efficacious avenue in future mouse model hearing loss investigations