| 1. |
- Walker, Anthony P., et al.
(author)
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Integrating the evidence for a terrestrial carbon sink caused by increasing atmospheric CO2
- 2021
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In: New Phytologist. - : John Wiley & Sons. - 0028-646X .- 1469-8137. ; 229:5, s. 2413-2445
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Journal article (peer-reviewed)abstract
- Atmospheric carbon dioxide concentration ([CO2]) is increasing, which increases leaf‐scale photosynthesis and intrinsic water‐use efficiency. These direct responses have the potential to increase plant growth, vegetation biomass, and soil organic matter; transferring carbon from the atmosphere into terrestrial ecosystems (a carbon sink). A substantial global terrestrial carbon sink would slow the rate of [CO2] increase and thus climate change. However, ecosystem CO2 responses are complex or confounded by concurrent changes in multiple agents of global change and evidence for a [CO2]‐driven terrestrial carbon sink can appear contradictory. Here we synthesize theory and broad, multidisciplinary evidence for the effects of increasing [CO2] (iCO2) on the global terrestrial carbon sink. Evidence suggests a substantial increase in global photosynthesis since pre‐industrial times. Established theory, supported by experiments, indicates that iCO2 is likely responsible for about half of the increase. Global carbon budgeting, atmospheric data, and forest inventories indicate a historical carbon sink, and these apparent iCO2 responses are high in comparison to experiments and predictions from theory. Plant mortality and soil carbon iCO2 responses are highly uncertain. In conclusion, a range of evidence supports a positive terrestrial carbon sink in response to iCO2, albeit with uncertain magnitude and strong suggestion of a role for additional agents of global change.
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| 2. |
- Aguilar, Ximena, 1978-, et al.
(author)
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Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors
- 2014
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:5, s. e98575-
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Journal article (peer-reviewed)abstract
- Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.
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| 3. |
- Augusti, Angela, et al.
(author)
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Deriving correlated climate and physiological signals from deuterium isotopomers in tree rings
- 2008
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In: Chemical Geology. - : Elsevier BV. - 0009-2541 .- 1872-6836. ; 252:1-2, s. 1-8
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Journal article (peer-reviewed)abstract
- he deuterium (D) abundance of tree-ring cellulose archives past climatic conditions, but previous attempts to access this archive have led to conflicting results. Based on an overview of D fractionation mechanisms in plants, we explain why past measurements of D abundance yield unreliable paleo signals. Our data show that variation in D abundance among the C–H groups (isotopomer variation) of tree-ring cellulose is generally greater than variation in D abundance due to climatic influences. A comparison of the D isotopomer abundances of soluble sugars of annual plants and of trees, and of tree-ring cellulose shows that an “isotopomer pattern” of the C3 photosynthetic pathway is transmitted from soluble sugars to tree-ring cellulose. Differences in this pattern between oaks and conifers appear to be related to differences in metabolism. Furthermore, the patterns are modified by hydrogen isotope exchange between C–H groups and source water during cellulose synthesis. Based on these results, we propose a strategy to simultaneously reconstruct climate signals and signals related to tree physiology from D isotopomers of tree rings. Combination of climate signals and physiological signals may allow the detection of century-time-scale adaptations of trees to past environmental change, and help to forecast future adaptations.
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| 4. |
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| 5. |
- Augusti, Angela, et al.
(author)
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Hydrogen exchange during cellulose synthesis distinguishes climatic and biochemical isotope fractionations in tree rings.
- 2006
-
In: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 172:3, s. 490-499
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Journal article (peer-reviewed)abstract
- • The abundance of the hydrogen isotope deuterium (D) in tree rings is an attractive record of climate; however, use of this record has proved difficult so far, presumably because climatic and physiological influences on D abundance are difficult to distinguish.• Using D labelling, we created a D gradient in trees. Leaf soluble sugars of relatively low D abundance entered cellulose synthesis in stems containing strongly D-labelled water. We used nuclear magnetic resonance (NMR) spectroscopy to quantify D in the C-H groups of leaf glucose and of tree-ring cellulose.• Ratios of D abundances of individual C-H groups of leaf glucose depended only weakly on leaf D labelling, indicating that the D abundance pattern was determined by physiological influences. The D abundance pattern of tree-ring cellulose revealed C-H groups that exchanged strongly (C(2)-H) or weakly (C(6)-H2) with water during cellulose synthesis.• We propose that strongly exchanging C-H groups of tree-ring cellulose adopt a climate signal stemming from the D abundance of source water. C-H groups that exchange weakly retain their D abundance established in leaf glucose, which reflects physiological influences. Combining both types of groups may allow simultaneous reconstruction of climate and physiology from tree rings.
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| 6. |
- Augusti, Angela, 1968-
(author)
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Monitoring climate and plant physiology using deuterium isotopomers of carbohydrates
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Climate is changing and it is certain that this change is due to human activities. Atmospheric greenhouse gases have been rising in an unprecedented way during the last two centuries, although the land biosphere has dampened their increase by absorbing CO2 emitted by anthropogenic activities. However, it is unclear if this will continue in the future. This uncertainty makes it difficult to predict future climate changes and to determine how much greenhouse gas emissions must be reduced to protect climate. To understand the future role of plants in limiting the atmospheric CO2 level, the effect of increasing CO2 on plant photosynthesis and productivity has been studied. However, studies on trees showed contradictory results, which depended on the duration of the experiment. This revealed that an initial strong CO2 fertilization may be a transient response that disappears after a few years. Because climate changes over centuries, we must explore the response of vegetation to increasing CO2 on this time scale. Studying tree rings is a good alternative to impractical decade-long experiments, because trees have experienced the CO2 increase during the last 200 years and may already have responded to it. This thesis shows that the intramolecular distribution of the stable hydrogen isotope deuterium (deuterium isotopomer distribution, DID) of tree rings is a reliable tool to study long-term plant-climate adaptations. The premise for this is that the deuterium abundance in tree rings depends on environmental as well as physiological factors. Using newly developed methodology for DID measurements, the influences of both factors can be separated. Applied to tree rings, separating both factors opens a strategy for simultaneous reconstruction of climate and of physiological responses. The results presented show that DIDs are influenced by kinetic isotope effects of enzymes, allowing studies of metabolic regulation. We show that the abundances of specific D isotopomers in tree-ring cellulose indeed allow identifying environmental and physiological factors. For example, the D2 isotopomer is mostly influenced by environment, its abundance should allow better reconstruction of past temperature. On the other hand, the abundance ratio of two isotopomers (D6R and D6S) depends on atmospheric CO2, and might serve as a measure of the efficiency of photosynthesis (ratio of photorespiration to assimilation). The presence of this dependence in all species tested and in tree-ring cellulose allows studying adaptations of plants to increasing CO2 on long time scales, using tree-ring series or other remnant plant material.
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| 7. |
- Augusti, Angela, et al.
(author)
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The ins and outs of stable isotopes in plants.
- 2007
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In: New Phytologist. - Oxford : Blackwell Scientific Publications Ltd. - 0028-646X .- 1469-8137. ; 174:3, s. 473-475
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Journal article (peer-reviewed)
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| 8. |
- Baskaran, Preetisri, et al.
(author)
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Nitrogen dynamics of decomposing Scots pine needle litter depends on colonizing fungal species
- 2019
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In: FEMS Microbiology Ecology. - : Oxford University Press. - 0168-6496 .- 1574-6941. ; 95:6
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Journal article (peer-reviewed)abstract
- In boreal ecosystems plant production is often limited by low availability of nitrogen. Nitrogen retention in below-ground organic pools plays an important role in restricting recirculation to plants and thereby hampers forest production. Saprotrophic fungi are commonly assigned to different decomposer strategies, but how these relate to nitrogen cycling remains to be understood. Decomposition of Scots pine needle litter was studied in axenic microcosms with the ligninolytic litter decomposing basidiomycete Gymnopus androsaceus or the stress tolerant ascomycete Chalara longipes. Changes in chemical composition were followed by 13C CP/MAS NMR spectroscopy and nitrogen dynamics was assessed by the addition of a 15N tracer. Decomposition by C. longipes resulted in nitrogen retention in non-hydrolysable organic matter, enriched in aromatic and alkylic compounds, whereas the ligninolytic G. androsaceus was able to access this pool, counteracting nitrogen retention. Our observations suggest that differences in decomposing strategies between fungal species play an important role in regulating nitrogen retention and release during litter decomposition, implying that fungal community composition may impact nitrogen cycling at the ecosystem level.
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| 9. |
- Betson, Tatiana, 1975-
(author)
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Deuterium isotopomers as a tool in environmental research
- 2006
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Doctoral thesis (other academic/artistic)abstract
- This thesis describes the development and the use of quantitative deuterium Nuclear Magnetic Resonance spectroscopy (NMR) as a tool in two areas of environmental research: the study of long term climate-plant interactions and the source tracking of persistent organic pollutant. Long-term interactions between plants and climate will influence climate change during this century and beyond, but cannot be studied in manipulative experiments. We propose that long tree rings series can serve as records for tracking such interactions during past centuries. The abundance of the stable hydrogen isotope deuterium (D) is influenced by physical and biochemical isotope fractionations. Because the overlapping effects of these fractionations are not understood, studies of the D abundance of tree rings led to conflicting results. We hypothesized that both types of fractionations can be separated if the D abundance of individual C-H groups of metabolites can be measured, that is if individual D isotopomers are quantified. The first paper describes a technique for quantification of D isotopomers in tree-ring cellulose by NMR. The technique showed that the D isotopomers distribution (DID) was non-random. Therefore, the abundance of each isotopomer potentially contains individual information which suggests an explanation for the conflicting results obtained by measuring the overall D abundance (dD). In the second paper, this technique was used to study hydrogen isotope exchange during cellulose synthesis in tree rings. This revealed that some C-H positions exchange strongly with xylem water, while others do not. This means that the exchanging C-H positions should acquire the D abundance of source water, which is determined by physical fractionations, while non-exchanging C-H positions of tree-ring cellulose should retain biochemical fractionations from the leaf level. Therefore, the abundance of the corresponding D isotopomers should contain information about climate and physiology. When analysing tree-ring series, the DIDs should reflect information about temperature, transpiration and regulation of photosynthesis. In the third paper, we showed that CO2 concentration during photosynthesis determines a specific abundance ratio of D isotopomers. This dependence was found in metabolites of annual plants, and in tree-ring cellulose. This result shows that D isotopomers of tree-ring series may be used to detect long-term CO2 fertilisation effects. This information is essential to forecast adaptations of plants to increasing CO2 concentrations on time scales of centuries. In the fourth paper, the source of persistent organic pollutants in the environment was tracked using DID measurements. The dD values of two compounds of related structures were not enough to show indisputably that they did not originate from the same source. However, the DIDs of the common part between the two compounds proved that they did not originate from the same source. These results underline the superior discriminatory power of DIDs, compared to dD measurements. The versatility of DID measurements makes them a precious tool in addressing questions that can not be answered by dD measurements.
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| 10. |
- Betson, Tatiana R, et al.
(author)
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Quantification of deuterium isotopomers of tree-ring cellulose using nuclear magnetic resonance.
- 2006
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:24, s. 8406-8411
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Journal article (peer-reviewed)abstract
- Stable isotopes in tree rings are important tools for reconstruction of past climate. Deuterium (D) is of particular interest since it may contain climate signals and report on tree physiology. Measurements of the D/H ratio of tree-ring cellulose have proven difficult to interpret, presumably because the D/H ratio of the whole molecule blends the abundances of the seven D isotopomers of cellulose. Here we present a method to measure the abundance of the D isotopomers of tree-ring cellulose by nuclear magnetic resonance spectroscopy (NMR). The method transforms tree-ring cellulose into a glucose derivative that gives highly resolved, quantifiable deuterium NMR spectra. General guidelines for measurement of D isotopomers by NMR are described. The transformation was optimized for yield and did not alter the original D isotopomer abundances, thus, conserving the original signals recorded in wood cellulose. In the tree-ring samples tested, the abundances of D isotopomers varied by approximately ±10% (2% standard error). This large variability can only be caused by biochemistry processes and shows that more information is present in D isotopomer abundances, compared to the D/H ratio. Therefore, measurements of the D isotopomer distribution of tree rings may be used to obtain information on long-term adaptations to environmental changes and past climate change.
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| 11. |
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| 12. |
- Chaintreau, Alain, et al.
(author)
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Site-specific C-13 content by quantitative isotopic C-13 nuclear magnetic resonance spectrometry : a pilot inter-laboratory study
- 2013
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In: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 788, s. 108-113
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Journal article (peer-reviewed)abstract
- Isotopic C-13 NMR spectrometry, which is able to measure intra-molecular C-13 composition, is of emerging demand because of the new information provided by the C-13 site-specific content of a given molecule. A systematic evaluation of instrumental behaviour is of importance to envisage isotopic C-13 NMR as a routine tool. This paper describes the first collaborative study of intra-molecular C-13 composition by NMR. The main goals of the ring test were to establish intra-and inter-variability of the spectrometer response. Eight instruments with different configuration were retained for the exercise on the basis of a qualification test. Reproducibility at the natural abundance of isotopic C-13 NMR was then assessed on vanillin from three different origins associated with specific delta C-13(i) profiles. The standard deviation was, on average, between 0.9 and 1.2 parts per thousand for intra-variability. The highest standard deviation for inter-variability was 2.1%. This is significantly higher than the internal precision but could be considered good in respect of a first ring test on a new analytical method. The standard deviation of delta C-13(i) in vanillin was not homogeneous over the eight carbons, with no trend either for the carbon position or for the configuration of the spectrometer. However, since the repeatability for each instrument was satisfactory, correction factors for each carbon in vanillin could be calculated to harmonize the results.(C) 2013 Elsevier B.V. All rights reserved.
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| 13. |
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| 14. |
- Cormier, Marc-André, et al.
(author)
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2H-fractionations during the biosynthesis of carbohydrates and lipids imprint a metabolic signal on the δ2H values of plant organic compounds
- 2018
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In: New Phytologist. - : Wiley-Blackwell. - 0028-646X .- 1469-8137. ; 218:2, s. 479-491
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Journal article (peer-reviewed)abstract
- Hydrogen (H) isotope ratio (δ2H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2H variability observed in plant compounds not being fully elucidated.We present a conceptual biochemical model based on empirical H isotope data that we generated in two complementary experiments that clarifies a large part of the unexplained variability in the δ2H values of plant organic compounds.The experiments demonstrate that information recorded in the δ2H values of plant organic compounds goes beyond hydrological signals and can also contain important information on the carbon and energy metabolism of plants. Our model explains where 2H‐fractionations occur in the biosynthesis of plant organic compounds and how these 2H‐fractionations are tightly coupled to a plant's carbon and energy metabolism. Our model also provides a mechanistic basis to introduce H isotopes in plant organic compounds as a new metabolic proxy for the carbon and energy metabolism of plants and ecosystems.Such a new metabolic proxy has the potential to be applied in a broad range of disciplines, including plant and ecosystem physiology, biogeochemistry and palaeoecology.
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| 15. |
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| 16. |
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| 17. |
- Drotz, Stina Harrysson, et al.
(author)
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Effects of soil organic matter composition on unfrozen water content and heterotrophic CO2 production of frozen soils
- 2010
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In: Geochimica et Cosmochimica Acta. - : Elsevier Ltd. - 0016-7037 .- 1872-9533. ; 74:8, s. 2281-90
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Journal article (peer-reviewed)abstract
- Several recent studies have highlighted the importance of soil organic matter (SOM) mineralization at high latitudes during winter for ecosystem carbon (C) balances, and the ability of the soil to retain unfrozen water at sub-zero temperatures has been shown to be a major determinant of C mineralization rates. Further, SOM is believed to strongly influence the liquid water contents in frozen surface layers of boreal forest soils and tundra, but the mechanisms and specific factors involved are currently unknown. Here we evaluate the effects of the chemical composition of SUM on the amount of unfrozen water, the pore size equivalents in which unfrozen water can exist, and the microbial heterotrophic activity at sub-zero temperatures in boreal forest soils. To do this, we have characterized the chemical composition of SUM in forest soil samples (surface O-horizons) using solid state CP-MAS (cross polarization magic angle spinning) NMR spectroscopy. The acquired information was then used to elucidate the extent to which different fractions of SUM can explain the observed variations in unfrozen water content, pore size equivalents, and biogenic CO2 production rates in the examined soil samples under frozen conditions (-4 degrees C). The data evaluation was done by the use of principal component analysis (PCA) and projections to latent structures by means of partial least square (PLS). We conclude that aromatic, O-aromatic, methoxy/N-alkyl and alkyl C are the major SOM components affecting frozen boreal forest soil's ability to retain unfrozen water and sustain heterotrophic activity (95% confidence level). Our results reveal that solid carbohydrates have a significant negative impact (95% confidence level) on CO2 production in frozen boreal spruce forest soils, in contrast to the positive effects of carbohydrate polymers during unfrozen conditions. We conclude that the hierarchy of environmental factors controlling SOM mineralization changes as soils freeze. The effect of SUM composition on pore size distribution and unfrozen water content has a superior influence on SUM mineralization and hence on heterotrophic CO2 production of frozen soils. (C) 2010 Elsevier Ltd. All rights reserved.
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| 18. |
- Duchardt, Elke, et al.
(author)
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Cytosine ribose flexibility in DNA : a combined NMR 13C spin relaxation and molecular dynamics simulation study.
- 2008
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In: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 36:12, s. 4211-9
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Journal article (peer-reviewed)abstract
- Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.
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| 19. |
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| 20. |
- Ehlers, Ina, 1984-, et al.
(author)
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Elucidating Turnover Pathways of Bioactive Small Molecules by Isotopomer Analysis : The Persistent Organic Pollutant DDT
- 2014
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In: PLOS ONE. - : PLoS ONE. - 1932-6203. ; 9:10, s. e110648-
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Journal article (peer-reviewed)abstract
- The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight againstmalaria, although DDT and related compounds pose toxicological hazards. Technical DDT contains the dichloro congenerDDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene) as by-product, but DDD is also formed by reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis. In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess DDT turnover in the environment. Because intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of smallbioactive molecules in chemistry, physiology and environmental science.
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| 21. |
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| 22. |
- Ehlers, Ina, 1984-, et al.
(author)
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Limited suppression of photorespiration by 20th century atmospheric CO2 increase in trees worldwide
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Other publication (other academic/artistic)abstract
- Forests are a key component of the global carbon and hydrological cycle and forest responses to environmental drivers create important feedbacks to these cycles. Photosynthetic efficiency of most forest tree species is strongly limited by photorespiration, a side reaction using O2 instead of CO2 as substrate, leading to a carbon loss for the plant. Photorespiration occurs in all trees and is reduced under elevated CO2 concentrations and increased under elevated temperature. Because the CO2 concentration of the atmosphere has increased in past decades, long-lived trees may have benefited from reduced photorespiration, but the temperature increase would have been a compensating detriment; but direct quantification of long-term changes in metabolic fluxes is lacking. Realistic forecasting of responses of trees and forests to future CO2 and temperature demands quantifying the reduction of photorespiration. In twelve tree species from five continents, we observe that photorespiration has been reduced by the CO2 increase during the past century, but for most the reduction is smaller than predicted from plant responses in CO2 alone. Comparison with data from a combined CO2 and temperature manipulation experiment shows that the reduced response can be explained by increases in leaf temperatures, which might result directly from increased air temperatures or indirectly from reduced transpirative cooling. These data suggest that global warming has already inhibited plant fertilization by increasing CO2, and that biomass increases may have been smaller than deduced from measurements of the heavy carbon isotope 13C. Observation of this centennial metabolic shift in tree physiology worldwide provides new insights into forest-climate feedbacks and can be used to improve coupled climate-vegetation models.
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| 23. |
- Ehlers, Ina, 1984-
(author)
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NMR studies of metabolites and xenobiotics : From time-points to long-term metabolic regulation
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science.The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs.Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches.The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects.The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.
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| 24. |
- Ehlers, Ina, 1984-, et al.
(author)
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Quantification of a metabolic shift towards photosynthesisin C3 plants driven by 20th-century CO2 rise
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Other publication (other academic/artistic)abstract
- Terrestrial vegetation currently absorbs approximately a third of the annual anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However,terrestrial net primary production is highly sensitive to atmospheric [CO2] and associated climatic changes. In C3-plants, which dominate terrestrial vegetation, netphotosynthesis depends on the ratio between gross photosynthesis and photorespiration, which strongly depends on [CO2]. However, our knowledge of feedbacks betweenterrestrial biomes and increasing atmospheric [CO2] is nearly entirely based on atmospheric inversion models and manipulation experiments, which do not reveal physiological mechanisms or are limited in duration and to step increases in [CO2]. By applying novel NMR (Nuclear Magnetic Resonance) spectroscopy methodology we examine isotopomer ratios of plant carbohydrates to probe shifts in the photosynthesis/photorespiration ratio in C3 plants over more than a century. Using herbarium samples of natural vascular plant species, crops and a Sphagnum species, we detect a consistent 35% increase in the 2photosynthesis/photorespiration ratio in responseto the ~100 ppm CO2 increase between approximately 1900 and 2013, with no evidencefor feedback regulation by the plants. Our data provide direct quantitative information on the “CO2 fertilization effect” over century time scales, thus addressing a major uncertainty in Earth system models, enabling improved predictions of the future [CO2] sink strength of terrestrial ecosystems. Further, relating the detected metabolic shift in crop plants to historic yield trends indicates that only a fraction of the increased net photosynthesis has translated into increased yield. Our results also demonstrate that archives of plant material contain metabolic information embedded in their isotopomer ratios covering centuries, bridging a fundamental gap between experimental plant science and paleoenvironmental studies.
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| 25. |
- Erhagen, Björn, et al.
(author)
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Temperature response of litter and soil organic matter decomposition is determined by chemical composition of organic material
- 2013
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In: Global Change Biology. - : Wiley-Blackwell. - 1354-1013 .- 1365-2486. ; 19:12, s. 3858-3871
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Journal article (peer-reviewed)abstract
- The global soil carbon pool is approximately three times larger than the contemporary atmospheric pool, therefore even minor changes to its integrity may have major implications for atmospheric CO2 concentrations. While theory predicts that the chemical composition of organic matter should constitute a master control on the temperature response of its decomposition, this relationship has not yet been fully demonstrated. We used laboratory incubations of forest soil organic matter (SOM) and fresh litter material together with NMR spectroscopy to make this connection between organic chemical composition and temperature sensitivity of decomposition. Temperature response of decomposition in both fresh litter and SOM was directly related to the chemical composition of the constituent organic matter, explaining 90% and 70% of the variance in Q10 in litter and SOM respectively. The Q10 of litter decreased with increasing proportions of aromatic and O-aromatic compounds, and increased with increased contents of alkyl- and O-alkyl carbons. In contrast, in SOM, decomposition was affected only by carbonyl compounds. To reveal why a certain group of organic chemical compounds affected the temperature sensitivity of organic matter decomposition in litter and SOM, a more detailed characterisation of the (13) C aromatic region using Heteronuclear Single Quantum Coherence (HSQC) was conducted. The results revealed considerable differences in the aromatic region between litter and SOM. This suggests that the correlation between chemical composition of organic matter and the temperature response of decomposition differed between litter and SOM. The temperature response of soil decomposition processes can thus be described by the chemical composition of its constituent organic matter, this paves the way for improved ecosystem modelling of biosphere feedbacks under a changing climate.
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| 26. |
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| 27. |
- Flodell, Sara, et al.
(author)
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Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.
- 2006
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 34:16, s. 4449-4457
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Journal article (peer-reviewed)abstract
- Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.
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| 28. |
- Flodell, Sara, et al.
(author)
-
Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs.
- 2002
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In: Journal of Biomolecular Structure and Dynamics. - 0739-1102 .- 1538-0254. ; 19:4, s. 627-636
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Journal article (other academic/artistic)abstract
- Hepatitis B virus (HBV) HBV is DNA virus with a unique replication strategy, which involves reverse transcription of its pregenomic RNA. Essential for this reverse transcription are the 5'- and 3'-ends of its pregenomic RNA (5'-RT-RNA and 3'-RT-RNA, respectively) which form conserved bulged stem-loop structures. The 5'-RT-RNA consists of a 67 nucleotide bulged stem-loop structure, epsilon, which constitutes the signal for encapsidation of the pregenomic RNA and subsequent reverse transcription. The reverse transcriptase (RT) initially binds to the completely conserved apical loop of epsilon and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. Structural studies of epsilon can provide important parameters required for the design of RNA targeted anti- viral drugs directed against Hepatitis B virus. NMR studies of large RNA systems (> ca. 50 nucleotides) require novel approaches, e.g., different labeling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing (13)C/(15)N/(2)H labeled nucleotides has been developed based on converting specifically labeled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway and nucleotide and salvage pathways. These NTPs give a large freedom in designing different labeling patterns in in vitro synthesized RNAs under study for NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides). Here this new technique is applied for structural studies on 27, 36 and 61 nucleotides long RNA fragments, mimicking different regions of epsilon.
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| 29. |
- Flodell, Sara, et al.
(author)
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The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges.
- 2002
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 30:21, s. 4803-4811
-
Journal article (peer-reviewed)abstract
- Reverse transcription of hepatitis B virus (HBV) pregenomic RNA is essential for virus replication. In the first step of this process, HBV reverse transcriptase binds to the highly conserved encapsidation signal, epsilon (epsilon), situated near the 5' end of the pregenome. epsilon has been predicted to form a bulged stem-loop with the apical stem capped by a hexa- loop. After the initial binding to this apical stem- loop, the reverse transcriptase synthesizes a 4 nt primer using the bulge as a template. Here we present mutational and structural data from NMR on the apical stem-loop of epsilon. Application of new isotope-labeling techniques (13C/15N/2H-U-labeling) allowed resolution of many resonance overlaps and an extensive structural data set could be derived. The NMR data show that, instead of the predicted hexa-loop, the apical stem is capped by a stable UGU tri-loop closed by a C-G base pair, followed by a bulged out C. The apical stem contains therefore two unpaired pyrimidines (C1882 and U1889), rather than one as was predicted, spaced by 6 nt. C1882, the 3' neighbour to the G of the loop-closing C-G base pair, is completely bulged out, while U1889 is at least partially intercalated into the stem. Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, reveals a high degree of conservation of epsilon. In particular, the residues essential for this fold are either totally conserved or show rare non-disruptive mutations. These data strongly indicate that this fold is essential for recognition by the reverse transcriptase.
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| 30. |
- Greule, Markus, et al.
(author)
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Improved rapid authentication of vanillin using δ13C and δ2H values
- 2010
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In: European Food Research and Technology. - : Springer Science and Business Media LLC. - 1438-2377 .- 1438-2385. ; 231:6, s. 933-941
-
Journal article (peer-reviewed)abstract
- Vanilla still remains one of the most important and widely used flavours in the food industry and is also extensively employed by fragrance and pharmaceutical manufacturing companies. Natural vanilla flavour, extracted from the pods of the tropic orchid vanilla, is considerably more expensive than synthetic vanillin. The disparity of prices between natural vanillin and that derived from other sources has given rise to many cases of fraudulent adulteration, and for more than 30 years, strenuous efforts have been made to authenticate sources of vanillin. Stable isotope analysis is one of the most powerful analytical tools to distinguish between natural vanillin and that originating from other sources. Recently, a rapid and precise method for analysis of both δ13C and δ2H values of plant methoxyl groups has been published. Here, we report an application of the method for the control of authenticity of vanillin. Carbon and hydrogen stable isotope values of the vanillin molecule and vanillin methoxyl groups of vanillin samples of different origins including authentic and synthetic samples were measured. The results clearly show that use of this approach provides a rapid and reliable authenticity assessment of vanillin. The technique used for these studies is robust and rapid, involves minimum sample preparation and requires only a small amount of vanillin sample, usually 1 mg for stable carbon and 4 mg for stable hydrogen analysis.
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| 31. |
- Haddad, Lenny, et al.
(author)
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Small molecules dominate organic phosphorus in NaOH-EDTA extracts of soils as determined by 31P NMR
- 2024
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In: Science of the Total Environment. - : Elsevier. - 0048-9697 .- 1879-1026. ; 931
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Journal article (peer-reviewed)abstract
- Understanding the composition of organic phosphorus (P) in soils is relevant to various disciplines, from agricultural sciences to ecology. Despite past efforts, the precise nature of soil organic P remains an enigma, especially that of the orthophosphate monoesters, which dominate 31P NMR spectra of NaOH-EDTA extracts of soils worldwide. The monoester region often exhibits an unidentified, broad background believed to represent high molecular weight (MW) P. We investigated this monoester background using 1D 31P NMR and 2D 1H[sbnd]31P NMR, as well as 31P transverse relaxation (T2) measurements to calculate its intrinsic linewidth and relate it to MW. Analyzing seven soils from different ecosystems, we observed linewidths of 0.5 to 3 Hz for resolved monoester signals and the background, indicating that it consists of many, possibly >100, sharp signals associated with small (<1.5 kDa) organic P molecules. This result was further supported by 2D 1H[sbnd]31P NMR spectra revealing signals not resolved in the 1D spectra. Our findings align with 31P NMR studies detecting background signals in soil-free samples and modern evidence that alkali-soluble soil organic matter consists of self-assemblies of small organic compounds mimicking large molecules.
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| 32. |
- Harrysson Drotz, Stina, et al.
(author)
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Both catabolic and anabolic heterotrophic microbial activity proceed in frozen soils
- 2010
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:49, s. 21046-21051
-
Journal article (peer-reviewed)abstract
- A large proportion of the global soil carbon pool is stored in soils of high-latitude ecosystems in which microbial processes and production of greenhouse gases proceed during the winter months. It has been suggested that microorganisms have limited ability to sequester substrates at temperatures around and below 0 °C and that a metabolic shift to dominance of catabolic processes occurs around these temperatures. However, there are contrary indications that anabolic processes can proceed, because microbial growth has been observed at far lower temperatures. Therefore, we investigated the utilization of the microbial substrate under unfrozen and frozen conditions in a boreal forest soil across a temperature range from -9 °C to +9 °C, by using gas chromatography-isotopic ratio mass spectrometry and (13)C magic-angle spinning NMR spectroscopy to determine microbial turnover and incorporation of (13)C-labeled glucose. Our results conclusively demonstrate that the soil microorganisms maintain both catabolic (CO(2) production) and anabolic (biomass synthesis) processes under frozen conditions and that no significant differences in carbon allocation from [(13)C]glucose into [(13)C]CO(2) and cell organic (13)C-compounds occurred between +9 °C and -4 °C. The only significant metabolic changes detected were increased fluidity of the cell membranes synthesized at frozen conditions and increased production of glycerol in the frozen samples. The finding that the processes in frozen soil are similar to those in unfrozen soil has important implications for our general understanding and conceptualization of soil carbon dynamics in high-latitude ecosystems.
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| 33. |
- Harrysson Drotz, Stina, et al.
(author)
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Contributions of matric and osmotic potentials to the unfrozen water content of frozen soils
- 2009
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In: Geoderma. - : Elsevier B.V.. - 0016-7061 .- 1872-6259. ; 148:3-4, s. 392-8
-
Journal article (peer-reviewed)abstract
- Recent reports show that biogeochemical processes continue when the soil is frozen, but are limited by water availability. However, there is little knowledge about the interactive effects of soil and environmental variables on amounts of unfrozen water in frozen soils. The aims of this study were to determine the contributions of matric and osmotic potentials to the unfrozen water content of frozen soil. We determined the effects of matric and osmotic potential on unfrozen water contents of frozen mineral soil fractions (ranging from coarse sand to fine silt) at − 7 °C, and estimated the contributions of these potentials to liquid water contents in samples from organic surface layers of boreal soils frozen at − 4 °C. In the mineral soil fractions the unfrozen water contents appeared to be governed solely by the osmotic potential, but in the humus layers of the sampled boreal soils both the osmotic and matric potentials control unfrozen water content, with osmotic potential contributing 20 to 69% of the total water potential. We also determined pore size equivalents, where unfrozen water resides at − 4 °C, and found a strong correlation between these equivalents and microbial CO2 production. The larger the pores in which the unfrozen water is found the larger the microbial activity that can be sustained. The osmotic potential may therefore be a key determinant of unfrozen water and carbon dynamics in frozen soil.
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| 34. |
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| 35. |
- Hägglund, Gunnar, et al.
(author)
-
1915-Pos 195 Pt Nmr: Interactions Of The Cancer Drug Cis-platin With Membranes And Mgst1, A Integral Membrane Detoxification Protein
- 2008
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In: Biophysical Journal. ; , s. 1915-
-
Conference paper (other academic/artistic)abstract
- The microsomal Glutathione-transferase (MGST1) is an integral membrane protein, which catalyses the conjugation of glutathione (tripeptide GSH) with xenobiotics; a process essential for cells to remove and detoxify e. g. carcinogens. While this glutathione system plays an essential role in healthy cell survival, glutathione has been shown to have a pivotal role in the development of acquired drug resistance. It prevents successful chemotherapies against a range of cancer types, therapies often based on cisplatin based drugs. These Pt compounds are initially quite effective, they become non-effective e.g. during the treatment of prostate cancer (very common 10000 new cases/a in Sweden) which progresses into a non-curable form during therapy. To understand the molecular mechanism behind the activity of Pt drugs and their inhibition by the human defense system, we use an solid state NMR approach (complemented by liquid NMR) to elucidate for cis-platin (diamino-dichloroplatinat II): conversion of cis-platin complex into an diamino-diaqua-complex, essential for its membrane passage into the cell interior. lipid membrane - drug interactions: binding to cell membrane surface, solubility and membrane transport. Pt drug binding to MGST1 enzyme, followed by glutathione conjugation into more water soluble compounds.
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| 36. |
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| 37. |
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| 38. |
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| 39. |
- Kaffarnik, Stefanie, et al.
(author)
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Two-Dimensional P-31,H-1 NMR Spectroscopic Profiling of Phospholipids in Cheese and Fish
- 2013
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In: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 61:29, s. 7061-7069
-
Journal article (peer-reviewed)abstract
- Phospholipids (PLs) comprise an important lipid class in food because of their technological use as emulsifiers and their nutritional value. This study used one-dimensional P-31 NMR and two-dimensional (2D) P-31,H-1 COSY NMR spectroscopy for the determination of the PL composition of cheese and fish after liquid liquid enrichment. This extraction step enabled the identification of 10 PLs in cheese and 9 PLs in fish by 2D P-31,H-1 NMR. Variations in the P-31 shifts indicated differences in the fatty acids attached to the individual PLs. The total PL content in cheese fat and fish oil ranged from 0.3 to 0.4% and from 5 to 12%, respectively. Phosphatidylcholine was the most prominent PL in both matrices (up to 6596). Minor PLs (limit of detection = 4 nmol, i.e. 500 mu L of an 8 mu M solution) were identified in forms of phosphatidic acid, lysophosphatidic acid, and phosphatidylglycerol. Specific cross couplings and H-1 fine structures in the 2D P-31,H-1 NMR spectra proved to be valuable for the assignment and verification of known and uncommon PLs in the samples.
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| 40. |
- Larsson, Göran, et al.
(author)
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A high-resolution HCANH experiment with enhanced sensitivity via multiple quantum line narrowing
- 1999
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In: Journal of Biomolecular NMR. - 0925-2738 .- 1573-5001. ; 14:2, s. 169-174
-
Journal article (peer-reviewed)abstract
- We report a 3D constant-time HCANH experiment (CTSL-HCANH) that uses the slower relaxation of multiple-quantum coherence to increase sensitivity and provides high C(α) resolution. In this experiment the H(α) of the (H(α), C(α)) multiple quanta are selectively spin locked, so that H(α) chemical shift evolution and (1) H-(1)H J-dephasing become ineffective during the relatively long delay needed for C(α) to N coherence transfer. As compared to an HCANH experiment that uses C(α) single-quantum coherence, an average enhancement of 20% was observed on calmodulin in complex with the binding domain of the transcription factor SEF2-1. Compared to CBCANH the signal intensity is approximately twice as good. The favorable relaxation properties of multiple quanta, together with the outstanding C(α) resolution, make the experiment a very good complement to CBCANH and CBCA(CO)NH for sequential assignment of larger proteins for which deuteration is not yet necessary.
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| 41. |
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| 42. |
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| 43. |
- Larsson, Göran, et al.
(author)
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Backbone dynamics of a symmetric calmodulin dimer in complex with the calmodulin-binding domain of the basic-helix-loop-helix transcription factor SEF2-1/E2-2 : a highly dynamic complex.
- 2005
-
In: Biophys J. - 0006-3495. ; 89:2, s. 1214-26
-
Journal article (peer-reviewed)abstract
- Calmodulin (CaM) interacts specifically as a dimer with some dimeric basic-Helix-Loop-Helix (bHLH) transcription factors via a novel high affinity binding mode. Here we report a study of the backbone dynamics by (15)N-spin relaxation on the CaM dimer in complex with a dimeric peptide that mimics the CaM binding region of the bHLH transcription factor SEF2-1. The relaxation data were measured at multiple magnetic fields, and analyzed in a model-free manner using in-house written software designed to detect nanosecond internal motion. Besides picosecond motions, all residues also experience internal motion with an effective correlation time of approximately 2.5 ns with squared order parameter (S(2)) of approximately 0.75. Hydrodynamic calculations suggest that this can be attributed to motions of the N- and C-terminal domains of the CaM dimer in the complex. Moreover, residues with significant exchange broadening are found. They are clustered in the CaM:SEF2-1mp binding interface, the CaM:CaM dimer interface, and in the flexible helix connecting the CaM N- and C-terminal domains, and have similar exchange times (approximately 50 micros), suggesting a cooperative mechanism probably caused by protein:protein interactions. The dynamic features presented here support the conclusion that the conformationally heterogeneous bHLH mimicking peptide trapped inside the CaM dimer exchanges between different binding sites on both nanosecond and microsecond timescales. Nature has thus found a way to specifically recognize a relatively ill-fitting target. This novel mode of target-specific binding, which neither belongs to lock-and-key nor induced-fit binding, is characterized by dimerization and continuous exchange between multiple flexible binding alternatives.
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| 44. |
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| 45. |
- Nielsen, Søren B, et al.
(author)
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The interaction of equine lysozyme : oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism
- 2010
-
In: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 398:2, s. 351-361
-
Journal article (peer-reviewed)abstract
- The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOA) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appears to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells (HAMLET). Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation (QCM-D) data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and "soft" lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with BSA, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA which shifts towards free OA as ELOA is progressively diluted indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA toward a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein. Abbreviations QCM-D, Quartz crystal microbalance with dissipation; CD, Circular dichroism; EL, equine lysozyme; ELOA, EL complex with oleic acid; OA, oleic acid, CSLM, Confocal scanning laser microscopy, Df, dissipation-frequency.
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| 46. |
- Olofsson, Annelie, et al.
(author)
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Biochemical and functional characterization of Helicobacter pylori vesicles
- 2010
-
In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:6, s. 1539-1555
-
Journal article (peer-reviewed)abstract
- Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigor of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and NMR. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.
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| 47. |
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| 48. |
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| 49. |
- Petzold, Katja, 1981-, et al.
(author)
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Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns.
- 2007
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 35:20, s. 6854-6861
-
Journal article (peer-reviewed)abstract
- The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function.
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| 50. |
- Petzold, Katja, 1981-
(author)
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NMR studies of host-pathogen interactions
- 2009
-
Doctoral thesis (other academic/artistic)abstract
- This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.
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