Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)-dependent protein kinase catalytic subunit (DNA-PKcs)-null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.     

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.     

Effects of PGF2α infusion on human cortisol biosynthesis

Intravenous administration of prostaglandin F_2α to normal volunteers induced a three-fold increase in urinary cortisol output. Dexamethasone administration significantly inhibited the cortisol response, indicating that prostaglandin does not directly stimulate adrenal cortisol biosynthesis. Prostaglandin infusion appears to increase cortisol biosynthesis through stimulation of pituitary ACTH release.     

2′-5′-Oligoadenylate synthetase is activated by a specific RNA sequence motif

2′-5′-Oligoadenylate synthetase plays a central role in the cellular innate antiviral response. Although activation of 2′-5′-oligoadenylate synthetase by double stranded RNA was discovered more than 30 years ago it is still unclear which sequence features are required by an RNA to activate the enzyme. A pool of chemically synthesized short double stranded RNAs of specific sequence was used to probe 2′-5′-oligoadenylate synthetase activation. It was found that activating double stranded RNAs contain the following motif: NNWWNNNNNNNNNWGN. Verification of this sequence motif in a pool of 102 small double stranded RNAs demonstrated a false positive prediction rate of 8% and a false negative prediction rate of 12%. The sequence motif identified provides mechanistic insight into the mechanism of 2′-5′-oligoadenylate synthetase activation by double stranded RNA and allows theoretical predictions whether a given RNA molecule has the capability to activate 2′-5′-oligoadenylate synthetase.     

Mothers' Perceptions of Their Adolescents' Weight Status: Are They Accurate?

Objective: To evaluate the accuracy of mothers’ perceptions of adolescents’ weight status. Research Methods and Procedures: Parent interviews and adolescent surveys (755) were conducted in an ethnically diverse sample from Project EAT (Eating among Teens). Results: Adolescent weight status was accurately assessed by 60% of mothers, underestimated by 35% of mothers, and overestimated by 5% of mothers. In multivariate analyses, mothers of female adolescents were about half as likely to underestimate their adolescent's weight status as mothers of male adolescents. Nonoverweight mothers were about half as likely as overweight mothers to underestimate their adolescent's weight status. Discussion: Most mothers are able to accurately assess their adolescent's weight status; however, adolescent gender and mothers’ weight status are related to accuracy. Mothers were more likely to underestimate their sons’ weights than their daughters’ weights, and overweight mothers were more likely to be inaccurate in their assessments than nonoverweight mothers. Findings suggest that the majority of parents do not need to be told that their children are overweight; instead, messages to parents of overweight teens should focus on how to provide support for healthy weight management.     

Physical Activity as a Predictor of Body Composition in American Indian Children

Objective: To examine physical activity in second grade American Indian children as a predictor of percentage body fat 3 years later. Research Methods and Procedures: Physical activity was assessed as average vector magnitude (AVM) counts from an accelerometer in 454 second grade children as part of the Pathways study. BMI was assessed, and skinfolds and bioelectrical impedance were used to estimate fat mass, fat‐free body mass, and percentage body fat in validated prediction equations. Associations were examined using mixed models regression controlling for baseline body composition. Results: In normal‐weight children, higher AVM counts were significantly associated with decreases in percentage body fat. Among overweight children, higher AVM counts were significantly associated with increases in BMI, fat mass, and fat‐free mass but not percentage body fat. Discussion: Higher physical activity levels in second grade were associated with lower levels of percentage body fat in fifth grade in normal‐weight but not in overweight children. BMI showed no association with physical activity among normal‐weight children, and increases in BMI were associated with increasing amounts of physical activity among overweight children. These findings emphasize the importance of valid body composition measures and may indicate important differences in associations between physical activity and adiposity in normal‐weight as compared with overweight children.     

Weight‐Bearing Physical Activity among Girls and Mothers: Relationships to Girls’ Weight Status

Objective: To assess weight‐bearing physical activity (WBPA) barriers, benefits, self‐efficacy, social influence, and behaviors [WBPA and physical activity (PA)] among girls and their mothers according to girls’ weight status (nonoverweight vs. overweight). Research Methods and Procedures: Participants were 9‐ to 11‐year‐old girls (n = 295) and their mothers who participated in the baseline assessment of a nutrition and PA intervention trial. Girls’ and mothers’ WBPA attitudes and mothers’ WBPA behaviors were self‐reported on questionnaires. Girls’ WBPA and total PA behaviors were self‐reported using a structured interview (Physical Activity Checklist Interview). Stature and weight were measured by standardized anthropometrics. Overweight status was based on BMI. Results: Compared with nonoverweight girls, overweight girls were significantly more likely to report barriers to WBPA participation and perceive social influence from family and friends to do more WBPA. They were also significantly less likely to report self‐efficacy regarding WBPA and to believe that they did enough WBPA. Compared with mothers of nonoverweight girls, mothers of overweight girls were significantly more likely to report that it is difficult to persuade their daughters to do more WBPA and significantly less likely to report that WBPA was fun for their daughters. Girls’ overweight status was not associated with girls’ reports of minutes spent per week in PA or WBPA. Discussion: The present study's findings of lower WBPA self‐efficacy, lack of enjoyment of WBPA, and higher perceived social influence to do WBPA among overweight girls suggest that efforts are needed to promote physical competencies and positive perceptions of PA among overweight girls.     

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.     

Influence of seed head-attacking biological control agents on spotted knapweed reproductive potential in western Montana over a 30-year period

Five insect biological control agents that attack flower heads of spotted knapweed, Centaurea stoebe L. subsp. micranthos (Gugler) Hayek, became established in western Montana between 1973 and 1992. In a controlled field experiment in 2006, seed-head insects reduced spotted knapweed seed production per seed head by 84.4%. The seed production at two sites in western Montana where these biological control agents were well established was 91.6-93.8% lower in 2004-2005 than 1974-1975, whereas the number of seed heads per square meter was 70.7% lower, and the reproductive potential (seeds/m(2)) was 95.9-99.0% lower. The average seed bank in 2005 at four sites containing robust spotted knapweed populations was 281 seeds/m(2) compared with 19 seeds/m(2) at four sites where knapweed density has declined. Seed bank densities were much higher at sites in central Montana (4,218 seeds/m(2)), where the insects have been established for a shorter period. Urophora affinis Frauenfeld was the most abundant species at eight study sites, infesting 66.7% of the seed heads, followed by a 47.3% infestation by Larinus minutus Gyllenhal and L. obtusus Gyllenhal. From 1974 to 1985, Urophora spp. apparently reduced the number of seeds per seed head by 34.5-46.9%; the addition of Larinus spp. further reduced seed numbers 84.2-90.5% by 2005. Path analysis indicated that both Larinus spp. and U. affinis contributed significantly to reduction of seed production over the 30-yr period. Spotted knapweed density may not decrease significantly until the seed bank falls below a critical threshold.     

An extremely diverse CD4 response to vaccinia virus in humans is revealed by proteome-wide T-cell profiling

CD4 T cells are required for the maintenance and recall of antiviral CD8 T cells and for antibody responses. Little is known concerning the overall architecture of the CD4 response to complex microbial pathogens. In a whole-proteome approach, 180 predicted open reading frames (ORFs) in the vaccinia virus genome were expressed and tested using responder cells from 20 blood samples from 11 vaccinees. Validation assays established the sensitivity and specificity of the system. Overall, CD4 responses were detected for 122 ORFs (68%). A mean of 39 ORFs were recognized per person (range, 13 to 63). The most frequently recognized ORFS were present in virions, including A3L and A10L (core proteins), WR148 (a fragmented homolog of an orthopoxvirus protein that forms inclusions in cells), H3L (a membrane protein), D13L (a membrane scaffold protein), and L4R (a nucleic acid binding protein). Serum immunoglobulin G profiling by proteome microarray detected responses to 45 (25%) of the ORFs and confirmed recent studies showing a diverse response directed to membrane and nonmembrane antigens. Our results provide the first empirical whole-proteome data set regarding the global CD4 response to full-length proteins in a complex virus and are consistent with the theory that abundant structural proteins are immunodominant.