Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb

Low Prevalence of Lactase Persistence in Bronze Age Europe Indicates Ongoing Strong Selection over the Last 3,000 Years

Lactase persistence (LP), the continued expression of lactase into adulthood, is the most strongly selected single gene trait over the last 10,000 years in multiple human populations. It has been posited that the primary allele causing LP among Eurasians, rs4988235-A [1], only rose to appreciable frequencies during the Bronze and Iron Ages [2, 3], long after humans started consuming milk from domesticated animals. This rapid rise has been attributed to an influx of people from the Pontic-Caspian steppe that began around 5,000 years ago [4, 5]. We investigate the spatiotemporal spread of LP through an analysis of 14 warriors from the Tollense Bronze Age battlefield in northern Germany (∼3,200 before present, BP), the oldest large-scale conflict site north of the Alps. Genetic data indicate that these individuals represent a single unstructured Central/Northern European population. We complemented these data with genotypes of 18 individuals from the Bronze Age site Mokrin in Serbia (∼4,100 to ∼3,700 BP) and 37 individuals from Eastern Europe and the Pontic-Caspian Steppe region, predating both Bronze Age sites (∼5,980 to ∼3,980 BP). We infer low LP in all three regions, i.e., in northern Germany and South-eastern and Eastern Europe, suggesting that the surge of rs4988235 in Central and Northern Europe was unlikely caused by Steppe expansions. We estimate a selection coefficient of 0.06 and conclude that the selection was ongoing in various parts of Europe over the last 3,000 years

In vitro and in vivo Metabolism of a Potent Inhibitor of Soluble Epoxide Hydrolase, 1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea

1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) is a potent soluble epoxide hydrolase (sEH) inhibitor that is used extensively in research for modulating inflammation and protecting against hypertension, neuropathic pain, and neurodegeneration. Despite its wide use in various animal disease models, the metabolism of TPPU has not been well-studied. A broader understanding of its metabolism is critical for determining contributions of metabolites to the overall safety and effectiveness of TPPU. Herein, we describe the identification of TPPU metabolites using LC-MS/MS strategies. Four metabolites of TPPU (M1–M4) were identified from rat urine by a sensitive and specific LC-MS/MS method with double precursor ion scans. Their structures were further supported by LC-MS/MS comparison with synthesized standards. Metabolites M1 and M2 were formed from hydroxylation on a propionyl group of TPPU; M3 was formed by amide hydrolysis of the 1-propionylpiperdinyl group on TPPU; and M4 was formed by further oxidation of the hydroxylated metabolite M2. Interestingly, the predicted α-keto amide metabolite and 4-(trifluoromethoxy)aniline (metabolite from urea cleavage) were not detected by the LC-MRM-MS method. This indicates that if formed, the two potential metabolites represent <0.01% of TPPU metabolism. Species differences in the formation of these four identified metabolites was assessed using liver S9 fractions from dog, monkey, rat, mouse, and human. M1, M2, and M3 were generated in liver S9 fractions from all species, and higher amounts of M3 were generated in monkey S9 fractions compared to other species. In addition, rat and human S9 metabolism showed the highest species similarity based on the quantities of each metabolite. The presence of all four metabolites were confirmed in vivo in rats over 72-h post single oral dose of TPPU. Urine and feces were major routes for TPPU excretion. M1, M4 and parent drug were detected as major substances, and M2 and M3 were minor substances. In blood, M1 accounted for ~9.6% of the total TPPU-related exposure, while metabolites M2, M3, and M4 accounted for <0.4%. All four metabolites were potent inhibitors of human sEH but were less potent than the parent TPPU. In conclusion, TPPU is metabolized via oxidation and amide hydrolysis without apparent breakdown of the urea. The aniline metabolites were not observed either in vitro or in vivo. Our findings increase the confidence in the ability to translate preclinical PK of TPPU in rats to humans and facilitates the potential clinical development of TPPU and other sEH inhibitors

Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts

The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines. The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non-homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5–7. The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 Gy, ranging from 0.03 to 0.28. These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs. We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated. In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines. Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines. Obviously, in this respect, other proteins/pathways appear to be more relevant. © 1999 Cancer Research Campaig

Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.</p> <p>Methods</p> <p>Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.</p> <p>Results</p> <p>MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.</p> <p>Conclusions</p> <p>The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC <it>in vivo</it>. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.</p

Induction and processing of the radiation-induced gamma-H2AX signal and Its link to the underlying pattern of DSB: A combined experimental and modelling study

We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular

Measurement of the kinetics of DNA double Strand break repair in ehrlich ascites tumour cells using the unwinding method.

Two main components of DNA strand break repair have been found using the unwinding method. The first has a time constant (t37) of some minutes and the second, much slower component, a time constant of several hours. The time constant for the slower component of repair was found to vary with the conditions of incubation and to depend on the quality of the radiation. The t37 values obtained for slow repair under various conditions after X-irradiation and after alpha-irradiation were found to be close to those for repair of double strand breaks as measured by velocity sedimentation. Values for initial breaks, obtained by extrapolation of slow repair data back to time zero, were also close to those obtained for double strand breaks. We therefore propose that the unwinding method can be a useful technique for monitoring the repair of double strand breaks

Irreparable DNA double-strand breaks induced in eukaryotic cells by sparsely or densely ionizing radiation and their importance for cell killing.

In yeast cells, DNA double-strand breaks (dsb) are induced linearly with dose both by sparsely ionizing 30 MeV electrons and by densely ionizing 3.5 MeV &alpha; particles. The RBE value for initial dsb is 2.6. Most dsb produced by either type of radiation can be repaired when cells are kept at 30&deg;C under non-growth conditions. Repair of dsb is completed after 72 hr of this liquid holding treatment. Dsb that remain unrepaired after this period are dsb that are irreparable under non-growth conditions. These irreparable dsb follow a quadratic function of dose of sparsely or densely ionizing radiation. It was found that the RBE and OER values were similar for both the survival of a mutant that cannot repair dsb and for dsb induction. Moreover, 1-2 dsb per cell per lethal event were measured for both sparsely and densely ionizing radiation under oxic or anoxic conditions. These results suggest that the dsb is the lethal lesion for a yeast cell deficient is dsb repair irrespective of the induction of dsb by sparsely or densely ionizing radiation and irrespective of oxic or anoxic conditions

The influence of oxygen on the survival and yield of DNA double-Strand breaks in irradiated yeast cells.

Survival and induction of DNA double-strand breaks were studied in cells of Saccharomyces cerevisiae irradiated under oxic or anoxic conditions with 30 MeV electrons. A linear relationship between DNA double-strand breakage and dose was found in both cases. The o.e.r.-value for colony forming ability was found to be 1&middot;9 &plusmn; 0&middot;2, whereas the o.e.r.-value for DNA double-strand breakage was 3&middot;0 &plusmn; 0&middot;1. These results are not inconsistent with the idea that DNA double-strand breaks are involved in killing of yeast cells. The frequency of induction of DNA double-strand breaks was found to be 0&middot;74 &times; 10-11 double-strand breaks per g/mol per Gy when cells were irradiated under oxygen and 0&middot;24 &times; 10-11 double-strand breaks per g/mol per Gy under nitrogen