The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.

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More than just strand breaks: the recognition of structural DNA discontinuities by DNA-dependent protein kinase catalytic subunit Dip, R; Naegeli, H Dip, R; Naegeli, H (2005). More than just strand breaks: the recognition of structural DNA discontinuities by DNA-dependent protein kinase catalytic subunit. FASEB Journal , 19 (7) More than just strand breaks: the recognition of structural DNA discontinuities by DNA-dependent protein kinase catalytic subunit AbstractThe DNA-dependent protein kinase (DNA-PK) is a trimeric factor originally identified as an enzyme that becomes activated upon incubation with DNA. Genetic defects in either the catalytic subunit (DNA-PK(CS)) or the two Ku components of DNA-PK result in immunodeficiency, radiosensitivity, and premature aging. This combined phenotype is generally attributed to the requirement for DNA-PK in the repair of DNA double strand breaks during various biological processes. However, recent studies revealed that DNA-PK(CS), a member of the growing family of phosphatidylinositol 3-kinases, participates in signal transduction cascades related to apoptotic cell death, telomere maintenance and other pathways of genome surveillance. These manifold functions of DNA-PK(CS) have been associated with an increasing number of protein interaction partners and phosphorylation targets. Here we review the DNA binding properties of DNA-PK(CS) and highlight its ability to interact with an astounding diversity of nucleic acid substrates. This survey indicates that the large catalytic subunit of DNA-PK functions as a sensor of not only broken DNA molecules, but of a wider spectrum of aberrant, unusual, or specialized structures that interrupt the standard double helical conformation of DNA. ABSTRACTThe DNA-dependent protein kinase (DNA-PK) is a trimeric factor originally identified as an enzyme that becomes activated upon incubation with DNA. Genetic defects in either the catalytic subunit (DNA-PK CS ) or the two Ku components of DNA-PK result in immunodeficiency, radiosensitivity, and premature aging. This combined phenotype is generally attributed to the requirement for DNA-PK in the repair of DNA double strand breaks during various biological processes. However, recent studies revealed that DNA-PK CS , a member of the growing family of phosphatidylinositol 3-kinases, participates in signal transduction cascades related to apoptotic cell death, telomere maintenance and other pathways of genome surveillance. These manifold functions of DNA-PK CS have been associated with an increasing number of protein interaction partners and phosphorylation targets. Here we review the DNA binding properties of DNA-PK CS and highlight its ability to interact with an astounding diversity of nucleic acid substrates. This survey indicates that the large catalytic subunit of DNA-PK functions as a sensor of not only broken DNA molecules, but of a wider spectrum of aberrant, unusual, or specialized structures that interrupt the standard double helical conformation of DNA.-Dip, R., Naegeli, H. More than j...

BackgroundA randomized, unmasked, multicenter study was conducted to evaluate the rate of pruritus reduction and improvement in clinical scoring by cyclosporine A (5 mg/kg orally, once daily for 28 days) either alone (n = 25 dogs) or with concurrent prednisolone (1 mg/kg once daily for 7 days, followed by alternate dosing for 14 days; n = 23 dogs) for the treatment of atopic dermatitis in dogs. Dogs were included in the study after exclusion of other causes of pruritic dermatitis, and were assessed by dermatologists on days 0, 14 ± 1 and 28 ± 2. Assessments included: general physical examination, CADESI-03 lesion scoring, overall clinical response, evaluation of adverse events (AEs), body weight and clinical pathology (hematology, clinical chemistry and urinalysis). Owner assessments, including pruritus (visual analogue scale, VAS) and overall assessment of response were conducted every 3–4 days, either during visits to the clinic or at home. Owners reported AEs to the investigator throughout the study.ResultsBy day 28 ± 2 both treatment groups resulted in a significant improvement of the atopic dermatitis. Both investigators and owners agreed that concurrent therapy resulted in a quicker improvement of the dogs ‘overall’ skin condition and of pruritus (significant reduction of pruritus by day 3–4, 72.8% improvement by day 14 ± 1), when compared to cyclosporine A alone (significant reduction of pruritus by day 7–8, 24.7% improvement by day 14 ± 1). CADESI-03 scores significantly improved in both groups by day 14 ± 1 onwards, and there were no significant differences in the scores between treatment groups at any time points. A total of 56 AEs (cyclosporine A alone = 34; concurrent therapy = 22) were reported in 33 dogs. No dogs died or stopped treatment due to an AE. The most commonly reported AEs in the cyclosporine A group were associated with the digestive tract, whilst systemic disorders were reported more frequently observed following concurrent therapy. Evaluation of body weight change and clinical pathology indices showed no overall clinically significant abnormalities.ConclusionsIn dogs with atopic dermatitis, a short initiating course of prednisolone expedited the efficacy of cyclosporine A in resolving pruritus and associated clinical signs. The observed adverse events were consistent with those expected for the individual veterinary medicinal products.

The red fox (Vulpes vulpes) is a representative of the canid family with wide distribution in the Northern Hemisphere and Australia. The increasing utilization of urbanized habitats by red foxes prompted us to test whether this species may be used to monitor the presence of anthropogenic pollutants in cities or suburbs. For that purpose, we compared the concentrations of heavy metals (Cd, Pb, Cu, Zn) in foxes from urban, suburban, and rural areas within the municipality of Zürich (Switzerland). The kidney and liver of suburban and rural foxes contained the highest Cd concentrations, whereas urban foxes contained the highest Pb levels. In the kidney of suburban foxes, Cd concentrations increased from a median value of 0.73 mg/kg in juvenile animals to 1.82 mg/kg in adults. Similarly, the liver of suburban foxes contained increasing Cd levels from a median of 0.21 mg/kg in juvenile animals to 0.94 mg/kg in adults. An age-dependent storage of Cd was also found in foxes from the rural surroundings, but no such accumulation occurred in urban foxes from the city center, where even adult animals contained very low Cd levels. Conversely, foxes from the urban center were characterized by elevated Pb concentrations during the first 2 years of life, but this transient Pb accumulation was absent in suburban or rural animals. The liver of juvenile foxes contained a median Pb concentration of 0.99 mg/kg in the city compared to only 0.47 and 0.37 mg/kg in the suburban and rural area, respectively. Thus, we found that animals from separate environmental compartments contain different patterns of tissue residues, implying that red foxes may serve as a bioindicator species to detect certain toxic hazards in urbanized habitats.

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Nuclear orphan receptors 4A (NR4A) are early responsive genes that belong to the superfamily of hormone receptors and comprise NR4A1, NR4A2 and NR4A3. They have been associated to transcriptional activation of multiple genes involved in inflammation, apoptosis and cell cycle control. Here, we establish a link between NR4As and adenosine, a paradoxical inflammatory molecule that can contribute to persistence of inflammation or mediate inflammatory shutdown. Transcriptomics screening of the human mast cell-line HMC-1 revealed a sharp induction of transcriptionally active NR4A2 and NR4A3 by the adenosine analogue NECA. The concomitant treatment of NECA and the adenosine receptor A 2A (A 2A AR) selective antagonist SCH-58261 exaggerated this effect, suggesting that upregulation of these factors in mast cells is mediated by other AR subtypes (A 2B and A 3 ) and that A 2A AR activation counteracts NR4A2 and NR4A3 induction. In agreement with this, A 2A ARsilencing amplified NR4A induction by NECA. Interestingly, a similar A 2A AR modulatory effect was observed on ERK1/2 phosphorylation because A 2A AR blockage exacerbated NECA-mediated phosphorylation of ERK1/2. In addition, PKC or MEK1/2 inhibition prevented ERK1/2 phosphorylation and antagonized AR-mediated induction of NR4A2 and NR4A3, suggesting the involvement of these kinases in AR to NR4A signaling. Finally, we observed that selective A 2A AR activation with CGS-21680 blocked PMA-induced ERK1/2 phosphorylation and modulated the overexpression of functional nuclear orphan receptors 4A. Taken together, these results establish a novel PKC/ERK/nuclear orphan receptors 4A axis for adenosinergic signaling in mast cells, which can be modulated by A 2A AR activation, not only in the context of adenosine but of other mast cell activating stimuli as well.