DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

Mek2 is dispensable for mouse growth and development

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.     

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

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Highlights? DCC and netrin-1 are enriched at synapses in the adult mouse forebrain ? DCC is enriched in the PSD and regulates dendritic spine morphology ? LTP induction and memory formation require DCC expression by neurons ? DCC activation of Src is required for NMDAR-dependent LTP in adult CNS     

Growth Monitoring: A Survey of Current Practices of Primary Care Paediatricians in Europe

Objective

We aimed to study current practices in growth monitoring by European primary care paediatricians and to explore their perceived needs in this field.

Methods

We developed a cross-sectional, anonymous on-line survey and contacted primary care paediatricians listed in national directories in the 18 European countries with a confederation of primary care paediatricians. Paediatricians participated in the survey between April and September 2011.

Results

Of the 1,198 paediatricians from 11 European countries (response rate 13%) who participated, 29% used the 2006 World Health Organization Multicentre Growth Reference Study growth charts, 69% used national growth charts; 61% used software to draw growth charts and 79% did not use a formal algorithm to detect abnormal growth on growth charts. Among the 21% of paediatricians who used algorithms, many used non-algorithmic simple thresholds for height and weight and none used the algorithms published in the international literature. In all, 69% of paediatricians declared that a validated algorithm to monitor growth would be useful in daily practice. We found important between-country variations.

Conclusion

The varied growth-monitoring practices declared by primary care paediatricians reveals the need for standardization and evidence-based algorithms to define abnormal growth and the development of software that would use such algorithms.     

Altered expression of flowering class B and class C genes in the Appendix tobacco mutant

 In the tobacco (Nicotiana tabacum) Appendix mutant, anthers are tipped by a miniature style and stigma. The outgrowth appears on the anther when it is already differentiating and follows the developmental timing of the central carpel. The Appendix mutation thus represents a late homeotic transformation suggesting that the APPENDIX (APX) gene either could be a misregulated organ identity gene or could be involved in regulating the expression of such genes. RFLP analysis with two class B (TM6 and NTGLO) and a class C (NAG) probes revealed that the Appendix phenotype is not caused by a mutation in one of these genes. However, in situ hybridization showed important changes in the expression of NTGLO and NAG in the mutant when compared with wild-type tobacco. Surprisingly, although no phenotypic alteration other than the style and stigma outgrowth is observed in the Appendix mutant, changes in class B and class C gene expession were not restricted to the anther tip cells from which the outgrowth originates. As expected, NAG was expressed in the Appendix outgrowth but it was also overexpressed in the normal third and fourth whorl organs at the time the outgrowth, as well as the central styles and stigmas, differentiated. Overexpression of a class C gene is probably responsible for the Appendix phenotype. In normal and mutant flowers, NTGLO was expressed in the second, third and fourth whorls up to the time of carpel fusion. Expression of this class B gene then ceased in the fourth whorl organs but was reactivated at later stages only in the styles and stigmas as well as in the outgrowths of the mutant. It thus seems that the function of the APX gene is either to regulate the late expression of organ identity genes or to control cell proliferation in such a way that, in the mutant, some cells are in a state where they respond in an unusual way to developmental signals. Received: 17 October 1997 / Revision accepted: 24 March 1998