The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity

The Escherichia coli Rep protein is a 3' to 5' SF1 DNA helicase required for replication of bacteriophage phiX174 in E. coli, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an approximately 130 degrees rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepDelta2B protein. This RepDelta2B protein can support phiX174 replication in a rep(-) E. coli strain, although the growth rate of E. coli containing the repDelta2B gene is approximately 1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepDelta2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.     

Validation of Peripheral Quantitative Computed Tomography–Derived Thigh Adipose Tissue Subcompartments in Young Girls Using a 3 T MRI Scanner

The ability to assess skeletal muscle adipose tissue is important given the negative clinical implications associated with greater fat infiltration of the muscle. Computed tomography and magnetic resonance imaging (MRI) are highly accurate for measuring appendicular soft tissue and muscle composition, but have limitations. Peripheral quantitative computed tomography (pQCT) is an alternative that investigators find valuable because of its low radiation, fast scan time, and comparatively lower costs. The present investigation sought to assess the accuracy of pQCT-derived estimates of total, subcutaneous, skeletal muscle, intermuscular, and calculated intramuscular adipose tissue areas, and muscle density in the midthigh of young girls using the gold standard, 3?T MRI, as the criterion. Cross-sectional data were analyzed for 26 healthy girls aged 9–12 years. Midthigh soft tissue composition was assessed by both pQCT and 3?T MRI. Mean tissue area for corresponding adipose compartments by pQCT and MRI was compared using t tests, regression analysis, and Bland-Altman plots. Muscle density was regressed on MRI skeletal muscle adipose tissue, intermuscular adipose tissue, and intramuscular adipose tissue, each expressed as a percentage of total muscle area. Correlations were high between MRI and pQCT for total adipose tissue (r²?=?0.98), subcutaneous adipose tissue (r²?=?0.95), skeletal muscle adipose tissue (r²?=?0.83), and intermuscular adipose tissue (r²?=?0.82), and pQCT muscle density correlated well with both MRI skeletal muscle adipose tissue (r²?=?0.70) and MRI intermuscular adipose tissue (r²?=?0.70). There was a slight, but statistically significant underestimation by pQCT for total and subcutaneous adipose tissue, whereas no significant difference was observed for skeletal muscle adipose tissue. Both pQCT-estimated intramuscular adipose tissue and muscle density were weakly correlated with MRI-intramuscular adipose tissue. We conclude that pQCT is a valid measurement technique for estimating all adipose subcompartments, except for intramuscular adipose tissue, for the midthigh region in young/adolescent girls.     

The association between aging-related monocyte transcriptional networks and comorbidity burden: the Multi-Ethnic Study of Atherosclerosis (MESA)

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