An imperfect G2M checkpoint contributes to chromosome instability following irradiation of S and G2 phase cells

DNA double strand break (DSB) repair and checkpoint control represent two major mechanisms that function to reduce chromosomal instability following ionising irradiation (IR). Ataxia telangiectasia (A-T) cells have long been known to have defective checkpoint responses. Recent studies have shown that they also have a DSB repair defect following IR raising the issue of how ATM’s repair and checkpoint functions interplay to maintain chromosomal stability. A-T and Artemis cells manifest an identical and epistatic repair defect throughout the cell cycle demonstrating that ATM’s major repair defect following IR represents Artemis-dependent end-processing. Artemis cells show efficient G2/M checkpoint induction and a prolonged arrest relative to normal cells. Following irradiation of G2 cells, this checkpoint is dependent on ATM and A-T cells fail to show checkpoint arrest. In contrast, cells irradiated during S phase initiate a G2/M checkpoint which is independent of ATM and, significantly, both Artemis and A-T cells show a prolonged arrest at the G2/M checkpoint likely reflecting their repair defect. Strikingly, the G2/M checkpoint is released before the completion of repair when approximately 10-20 DSBs remain both for S phase and G2 phase irradiated cells. This defined sensitivity level of the G2/M checkpoint explains the prolonged arrest in repair-deficient relative to normal cells and provides a conceptual framework for the co-operative phenotype between checkpoint and repair functions in maintaining chromosomal stability

Chromosome breakage after G2 checkpoint release

DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATM's G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and approximately 20 gammaH2AX foci remain

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ∼15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ

The SREBF-1 locus is associated with type 2 diabetes and plasma adiponectin levels in a middle-aged Austrian population

Funding Information: This study was supported by grants from the Oesterrei-chische Nationalbank (Project No. 10678 and 10932), the Medizinische Forschungsgesellschaft Salzburg and a grant from the Land Salzburg. Copyright: Copyright 2011 Elsevier B.V., All rights reserved.Context: The sterol regulatory element-binding protein-1c (SREBP-1c) is a transcription factor involved in the regulation of lipid and glucose metabolism and has been implicated in the pathophysiology of type 2 diabetes mellitus (T2DM). Objective: We aimed to confirm associations of the SREBF-1 gene with T2DM in an Austrian population and to study possible associations with diabetes-related quantitative traits. Design, settings and participants: We genotyped a diabetic cohort (n=446) along with a control group (n=1524) for a common C/G variation that is located in exon 18c (rs2297508) and has been associated with obesity and T2DM in French populations. Main outcome measures: Body mass index (BMI), indices of insulin sensitivity and β-cell function, plasma adiponectin, T2DM and single-nucleotide polymorphism rs2297508. Results: Genotype distributions associated with rs2297508 differed by T2DM status (P=0.0045), but not by BMI. The variant G allele was associated with a modest, but significant, increase in the prevalence of T2DM after adjustment for age, sex and BMI (G/G: odds ratios (OR) (95% confidence intervals)=1.45 (0.99-2.11) and G/C: OR=1.37 (1.04-1.81)). In a cross-sectional population of non-diabetic subjects, associations of rs2297508 genotypes with plasma adiponectin levels adjusted for age, sex and BMI (P=0.0017) were observed in that the risk G/G genotype displayed the lowest adiponectin levels. Conclusions: We observed associations of rs2297508 with T2DM prevalence and plasma adiponectin. SREBP-1c has been implicated in the regulation of adiponectin gene expression. Our results therefore raise the possibility that sequence variations at the SREBF-1 gene locus might contribute to T2DM risk, at least in part, by altering circulating adiponectin levels.publishersversionPeer reviewe

High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection". The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation

Automated Generation of User Guidance by Combining Computation and Deduction

Herewith, a fairly old concept is published for the first time and named "Lucas Interpretation". This has been implemented in a prototype, which has been proved useful in educational practice and has gained academic relevance with an emerging generation of educational mathematics assistants (EMA) based on Computer Theorem Proving (CTP). Automated Theorem Proving (ATP), i.e. deduction, is the most reliable technology used to check user input. However ATP is inherently weak in automatically generating solutions for arbitrary problems in applied mathematics. This weakness is crucial for EMAs: when ATP checks user input as incorrect and the learner gets stuck then the system should be able to suggest possible next steps. The key idea of Lucas Interpretation is to compute the steps of a calculation following a program written in a novel CTP-based programming language, i.e. computation provides the next steps. User guidance is generated by combining deduction and computation: the latter is performed by a specific language interpreter, which works like a debugger and hands over control to the learner at breakpoints, i.e. tactics generating the steps of calculation. The interpreter also builds up logical contexts providing ATP with the data required for checking user input, thus combining computation and deduction. The paper describes the concepts underlying Lucas Interpretation so that open questions can adequately be addressed, and prerequisites for further work are provided.Comment: In Proceedings THedu'11, arXiv:1202.453

The Mitochondrial T16189C Polymorphism Is Associated with Coronary Artery Disease in Middle European Populations

BACKGROUND: The pivotal role of mitochondria in energy production and free radical generation suggests that the mitochondrial genome could have an important influence on the expression of multifactorial age related diseases. Substitution of T to C at nucleotide position 16189 in the hypervariable D-loop of the control region (CR) of mitochondrial DNA (mtDNA) has attracted research interest because of its suspected association with various multifactorial diseases. The aim of the present study was to compare the frequency of this polymorphism in the CR of mtDNA in patients with coronary artery disease (CAD, n = 482) and type 2 diabetes mellitus (T2DM, n = 505) from two study centers, with healthy individuals (n = 1481) of Middle European descent in Austria. METHODOLOGY AND PRINCIPAL FINDINGS: CR polymorphisms and the nine major European haplogroups were identified by DNA sequencing and primer extension analysis, respectively. Frequencies and Odds Ratios for the association between cases and controls were calculated. Compared to healthy controls, the prevalence of T16189C was significantly higher in patients with CAD (11.8% vs 21.6%), as well as in patients with T2DM (11.8% vs 19.4%). The association of CAD, but not the one of T2DM, with T16189C remained highly significant after correction for age, sex and body mass index (BMI) and was independent of the two study centers. CONCLUSIONS AND SIGNIFICANCE: Our results show for the first time a significant association of T16189C with CAD in a Middle European population. As reported in other studies, in patients with T2DM an association with T16189C in individuals of European decent remains questionable

The T-381C SNP in BNP gene may be modestly associated with type 2 diabetes: an updated meta-analysis in 49 279 subjects

A recent study reported an association between the brain natriuretic peptide (BNP) promoter T-381C polymorphism (rs198389) and protection against type 2 diabetes (T2D). As replication in several studies is mandatory to confirm genetic results, we analyzed the T-381C polymorphism in seven independent case-control cohorts and in 291 T2D-enriched pedigrees totalling 39 557 subjects of European origin. A meta-analysis of the seven case-control studies (n = 39 040) showed a nominal protective effect [odds ratio (OR) = 0.86 (0.79-0.94), P = 0.0006] of the CC genotype on T2D risk, consistent with the previous study. By combining all available data (n = 49 279), we further confirmed a modest contribution of the BNP T-381C polymorphism for protection against T2D [OR = 0.86 (0.80-0.92), P = 1.4 × 10−5]. Potential confounders such as gender, age, obesity status or family history were tested in 4335 T2D and 4179 normoglycemic subjects and they had no influence on T2D risk. This study provides further evidence of a modest contribution of the BNP T-381C polymorphism in protection against T2D and illustrates the difficulty of unambiguously proving modest-sized associations even with large sample size