Spectroscopic and in vitro Investigations of Fe2+/α-Ketoglutarate-Dependent Enzymes Involved in Nucleic Acid Repair and Modification

The activation of molecular oxygen for the highly selective functionalization and repair of DNA and RNA nucleobases is achieved by α-ketoglutarate (α-KG)/iron-dependent dioxygenases. Of special interest are the human homologues AlkBH of Escherichia coli EcAlkB and ten-eleven translocation (TET) enzymes. These enzymes are involved in demethylation or dealkylation of DNA and RNA, although additional physiological functions are continuously being found. Given their importance, studying enzyme-substrate interactions, turnover and kinetic parameters is pivotal for the understanding of the mode of action of these enzymes. Diverse analytical methods, including X-ray crystallography, UV/Vis absorption, electron paramagnetic resonance (EPR), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy have been employed to study the changes in the active site and the overall enzyme structure upon substrate, cofactor, and inhibitor addition. Several methods are now available to assess the activity of these enzymes. By discussing limitations and possibilities of these techniques for EcAlkB, AlkBH and TET we aim to give a comprehensive synopsis from a bioinorganic point-of-view, addressing researchers from different disciplines working in the highly interdisciplinary and rapidly evolving field of epigenetic processes and DNA/RNA repair and modification.     

Evolution of Martensite Tetragonality in High-Carbon Steels Revealed by In Situ High-Energy X-Ray Diffraction

Metallurgical and Materials Transactions A - The martensitic transformation was studied by in situ and ex situ experiments in two high-carbon, 0.54 and 0.74 wt pct C, steels applying three...     

Bestimmung von Gamma-Hydroxybuttersäure und Gamma- Hydroxybuttersäure- Metaboliten im Haar und deren forensische Bedeutung

Gamma-Hydroxybutyrate (GHB) has been subject of scientific research due to its endogenous occurrence in humans and at the same time sedating effect after exogenous uptake, whether intentional or not. Of particular interest in this context is the distinction between possible endogenous GHB-concentrations and those that were in hair after a (single) exogenous uptake. In the present study, methods of extraction and quantification by LC-MS/MS for GHB and its metabolites glycolic acid, 2,4-dihydroxybutanoic acid (2,4-DHB), 3,4-dihydroxybutanoic acid (3,4-DHB) and 4-(sulfooxy)butanoic acid (GHB-sulf) were established. After validation of those methods, hair samples of persons without known exogenous GHB uptake were analysed to define an endogenous reference range for each substance. Subsequently hair samples were analysed after single or regular uptake of GHB. GHB-concentrations of 928 hair samples from persons without known uptake of GHB were between < 0.1 and 6.3 ng/mg. Only 29 samples showed concentrations higher than 3.0 ng/mg. Interestingly, hair samples that were stored for two to eight years showed significantly higher GHB-concentrations between 1.5 and 130 ng/mg, with 48 of 52 analysed segments showing concentrations above 3.0 ng/mg. Thus, storing seems to have a greater effect on the observed GHB-concentration than the quantity of GHB taken. In accordance with that, of 136 analysed hair samples of persons with a daily uptake of 3 to 9 g GHB, only 25 of the measured concentrations were above 3.0 ng/mg and only three outside of the prior defined endogenous reference range. The association between the quantity of exogenous GHB-uptake and the observed GHB-concentration in hair was found to be very weak. In agreement with those results, no raise in GHB-concentration was observed in hair samples after a single uptake of GHB. All concentrations were in the lower range of the prior defined endogenous reference range. Regarding the four analysed GHB-metabolites, the following endogenous reference ranges (including outliers) were defined: glycolic acid 0.39 - 11 (24) ng/mg, GHB-sulf < 0.40 - 1.7 (12) ng/mg, 3,4-DHB < 0.10 - 0.63 (4.7) ng/mg and 2,4-DHB < 0.20 - 0.45 (0.65) ng/mg. In hair samples from persons with regular uptake of GHB, at least one metabolite was found to be elevated (i.e., above the prior defined endogenous reference range) in the average of each hair strand, in contrast to the observed GHB-concentrations. In four of eleven hair strands even all four metabolites were above their endogenous reference range. Nevertheless, no statistically significant association between the quantity of GHB-uptake and the observed concentration in hair was found. Hair strands examined after a single GHB-uptake partially showed elevated concentrations of GHB-metabolites. The elevated segments, however, were not in each case those one would expect assuming the average hair growth rate of 1 cm/month. In the present study, it was shown that the exogenous uptake of GHB does not necessarily result in an elevated GHB-concentration in hair. Thus, the sole quantification of GHB will most likely not allow a clear decision whether or not GHB has been taken up exogenously. Thus, the here developed analytical methods do not yield legal certainty. However, in combination with the detection of one or more elevated GHB-metabolite concentrations, a better evaluation of this issue seems possible.     

Spirooxindol-1,3-oxazine Alkaloids: Highly Potent and Selective Antitumor Agents Evolved from Iterative Structure Optimization

Spirooxindole-1,3-oxazines are a small and structurally unique class of spirooxindole alkaloids. To date, only four of these compounds have been isolated from natural sources, and their biological properties remained unknown thus far. Dioxyreserpine is a synthetic spirooxindole-1,3-oxazine, that can readily be prepared from the Rauvolfia alkaloid (–)-reserpine by catalytic photooxygenation. While dioxyreserpine itself was now identified as a moderately effective antitumoral agent, structurally modified analogs of it emerged as a new class of highly potent and selective growth inhibitors of various human cancers, including pancreatic cancers. Systematic structural optimization ultimately led to an inhibitor displaying low-micromolar IC₅₀-values against six cancer cell lines as well as selective apoptosis induction in vitro.     

Grafting PEG and alkyl comb polymers onto bleached wood pulp fibres

Comb polymers were prepared by reacting a poly(ethylene-alt-maleic anhydride) with alkyl amines or poly(ethylene glycol) (PEG) amines. The resulting polymers were used to modify bleached softwood kraft pulp fibre surfaces by catalyst-free grafting in a process suitable for pulp mill implementation. Pulp fibres were impregnated with a polymer solution and cured above 100°C. High grafting yields were obtained despite having up to 88% of the anhydride groups consumed by amine derivatization. Grafting yields were more than 90% when the polymer dosage was <13 g/kg (dry polymer/dry fibre) for alkyl derivatives and < 38 g/kg for PEG derivatives. We propose that the upper dosage limit for efficient grafting reflects the need for direct contact between cellulose and every polymer chain for ester linkage formation. For a given polymer dosage, the cured pulp sheets had a maximum wet tensile index, TI_max, when either curing time or temperature was increased. Both the alkyl and PEG derivatives fit the power law for the wet TI_max~βΓ_ru^0.54–0.62 where β values were the estimated conversion of succinic acid moieties to anhydrides when the pulp sheets were cured, and Γ_ru is the dimensionless polymer content that is numerically equal to the amount of applied polymer in mmol repeat units/g dry fibre. However, high polymer dosages give experimental TI_max values that fall below the power law, irrespective of curing intensity, because the pulp sheets contain unfixed polymer chains that lubricate fibre/fibre joints, lowering wet strength.     

Dispersal of brown trout (Salmo trutta L.) fry in a low gradient stream - implications for egg stocking practices

Stocking of eggs is a common strategy to support declining or reintroduce extirpated salmonid populations. Data on how juveniles disperse from stocking points is crucial to be able to design efficient stocking programs. Detailed information of dispersal is limited for many salmonids, for example, brown trout. In this study, dispersal distance was measured at the end of the first growing season in a low gradient (0.7%) stream in Sweden where the trout population had been depleted. Eggs from 17 separate sets of parents were stocked as eyed eggs in March. During the following fall fry were sampled throughout the stream. The majority of the fry dispersed downstream and remained within a distance of 200 m from the stocking point with no difference between sizes of fry and the presence of a competing cohort or not. There was no dissimilarity in dispersal distances across offspring originating from different parents indicating absence of genetic influence. Our results suggest that, in streams similar to our study site, stocking points should be separated by approximately 330 m in order to avoid overlap in habitat use of fry from different stocking points and that the presence of competing cohorts, fry size and within population variability in dispersal can be neglected.