| 1. |
- Akkuratov, Evgeny E.
(författare)
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The Biophysics of Na+,K+-ATPase in neuronal health and disease
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Na+,K+-ATPase is one of the most important proteins in the mammalian cell. It creates sodium and potassium gradients which are fundamental for the membrane potential and sodium-dependent secondary active transport. It has a second role in the cell as a receptor that by binding chemicals from the cardiotonic steroids family, the most knowledgeable of them is ouabain, triggers various signaling pathways in the cell which regulate gene activation, proliferation, apoptosis, etc. It has been shown that several severe neurological diseases are associated with mutations in the Na+,K+-ATPase encoding genes. Although Na+,K+-ATPase was discovered already in 1957 by the Danish scientist Jens Skou, the knowledge about the function of this enzyme is still not complete. In the studies included in the thesis, we have learned more about the function of Na+,K+-ATPase in different aspects of health and disease. In study I we showed a mechanism of ouabain-dependent regulation of the NMDA receptor, one of the most important receptors in the nervous system, via binding with Na+,K+-ATPase. This allows us to look at the Na+,K+-ATPase as regulator via protein-protein interaction. In study II we investigated a different aspect of Na+,K+-ATPase functioning – to look at how binding of ouabain to Na+,K+-ATPase activates a number of signaling cascades by looking at the phosphoproteome status of the cells. This allows us to see the whole picture of ouabain-mediated cascades and further characterize them. In study III we focused on the role of Na+,K+-ATPase in severe epileptic encephalopathy caused by a mutation in the ATP1A1 gene. We performed a molecular and cellular study to describe how mutations affects protein structure and function and found that this mutation converts the ion pump to a nonspecific leak channel. In study IV we performed a translational study of the most common mutation for rapid-onset dystonia-parkinsonism. We studied how this mutation affects the nervous system on the protein-, cellular-, and organism level and found that the complete absence of ultraslow afterhyperpolarization (usAHP) could explain gait disturbances found in patients. In the on-going study we showed that Na+,K+-ATPase can oligomerize and that this effect is triggered by ouabain binding to the Na+,K+-ATPase. In this study, we utilized a novel fluorescence labelling approach and used biophysical techniques with single molecule sensitivity to track Na+,K+-ATPase interactions. In summary, we applied biophysical and molecular methods to study different aspects of the function of Na+,K+-ATPase, and gained insights that could be helpful not only for answering fundamental questions about Na+,K+-ATPase but also to find a treatment for patients with diseases associated with mutations in this protein.
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| 2. |
- Andersson, Magnus, et al.
(författare)
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Transport Pathway in Cu+ P-Type ATPases
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 106:2, s. 427A-427A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 3. |
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| 4. |
- García Pérez, Ana Elia, et al.
(författare)
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Oxygen abundances in metal-poor subgiants as determined from [OI], OI and OH lines
- 2006
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Ingår i: Astronomy and Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 451:2, s. 621-642
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Tidskriftsartikel (refereegranskat)abstract
- The debate on the oxygen abundances of metal-poor stars has its origin in contradictory results obtained using different abundance indicators. To achieve a better understanding of the problem we have acquired high quality spectra with the Ultraviolet and Visual Echelle Spectrograph at VLT, with a signal-to-noise of the order of 100 in the near ultraviolet and 500 in the optical and near infrared wavelength range. Three different oxygen abundance indicators, OH ultraviolet lines around 310.0 nm, the [O I] line at 630.03 nm and the O I lines at 777.1-5 nm were observed in the spectra of 13 metal-poor subgiants with . Oxygen abundances were obtained from the analysis of these indicators which was carried out assuming local thermodynamic equilibrium and plane-parallel model atmospheres. Abundances derived from O I were corrected for departures from local thermodynamic equilibrium. Stellar parameters were computed using -vs.-color calibrations based on the infrared flux method and Balmer line profiles, Hipparcos parallaxes and lines. [O/Fe] values derived from the forbidden line at 630.03 nm are consistent with an oxygen/iron ratio that varies linearly with [Fe/H] as . Values based on the O I triplet are on average (s.d.) higher than the values based on the forbidden line while the agreement between OH ultraviolet lines and the forbidden line is much better with a mean difference of the order of (s.d.). In general, our results follow the same trend as previously published results with the exception of the ones based on OH ultraviolet lines. In that case our results lie below the values which gave rise to the oxygen abundance debate for metal-poor stars.
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| 5. |
- Han, Huijong, et al.
(författare)
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The XBI BioLab for life science experiments at the European XFEL
- 2021
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Ingår i: Journal of applied crystallography. - 0021-8898 .- 1600-5767. ; 54, s. 7-21
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Tidskriftsartikel (refereegranskat)abstract
- The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.
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| 6. |
- Hjorth-Jensen, Samuel John, et al.
(författare)
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Prospects for membrane protein crystals in NMX
- 2020
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Ingår i: Neutron Crystallography in Structural Biology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 634, s. 47-68
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Bokkapitel (refereegranskat)abstract
- Adding hydrogen atoms and protonation states to structures of membrane proteins requires successful implementation of neutron macromolecular crystallography (NMX). This information would significantly increase our fundamental understanding of the transport processes membrane proteins undertake. To grow the large crystals needed for NMX studies requires significant amounts of stable protein, but once that challenge is overcome there is no intrinsic property of membrane proteins preventing the growth of large crystals per se. The calcium-transporting P-type ATPase (SERCA) has been thoroughly characterized biochemically and structurally over decades. We have extended our crystallization efforts to assess the feasibility of growing SERCA crystals for NMX—exploring microdialysis and capillary counterdiffusion crystallization techniques as alternatives to the traditional vapor diffusion crystallization experiment. Both methods possess crystallization dynamics favorable for maximizing crystal size and we used them to facilitate the growth of large crystals, validating these approaches for membrane protein crystallization for NMX.
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| 7. |
- Jonsell, Karin, et al.
(författare)
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Chemical abundances in 43 metal-poor stars
- 2005
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Ingår i: Astronomy & Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 440:1, s. 321-343
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Tidskriftsartikel (refereegranskat)abstract
- We have derived abundances of O, Na, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Fe, Ni, and Ba for 43 metal-poor field stars in the solar neighbourhood, most of them subgiants or turn-off-point stars with iron abundances [Fe/H] ranging from -0.4 to -3.0. About half of this sample has not been spectroscopically analysed in detail before. Effective temperatures were estimated from uvby photometry, and surface gravities primarily from Hipparcos parallaxes. The analysis is differential relative to the Sun, and was carried out with plane-parallel MARCS models. Various sources of error are discussed and found to contribute a total error of about 0.1-0.2 dex for most elements, while relative abundances, such as [Ca/Fe], are most probably more accurate. For the oxygen abundances, determined in an NLTE analysis of the 7774 Å triplet lines, the errors may be somewhat larger. We made a detailed comparison with similar studies and traced the reasons for the, in most cases, relatively small differences. Among the results we find that [O/Fe] possibly increases beyond [Fe/H] = -1.0, though considerably less so than in results obtained by others from abundances based on OH lines. We did not trace any tendency toward strong overionization of iron, and find the excesses, relative to Fe and the Sun, of the α elements Mg, Si, and Ca to be smaller than those of O. We discuss some indications that also the abundances of different α elements relative to Fe vary and the possibility that some of the scatter around the trends in abundances relative to iron may be real. This may support the idea that the formation of Halo stars occurred in smaller systems with different star formation rates. We verify the finding by Gratton et al. (2003b, A&A, 406, 131) that stars that do not participate in the rotation of the galactic disk show a lower mean and larger spread in [ α/Fe] than stars participating in the general rotation. The latter stars also seem to show some correlation between [ α/Fe] and rotation speed. We trace some stars with peculiar abundances, among these two Ba stars, HD 17072 and HD 196944, the second already known to be rich in s elements. Finally we advocate that a spectroscopic study of a larger sample of halo stars with well-defined selection criteria is very important, in order to add to the very considerable efforts that various groups have already made.
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| 8. |
- Kirscht, Andreas, et al.
(författare)
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Crystal Structure of an Ammonia-Permeable Aquaporin
- 2016
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Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1545-7885. ; 14:3
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.
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| 9. |
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| 10. |
- Mattle, Daniel, et al.
(författare)
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A sulfur-based transport pathway in Cu+-ATPases
- 2015
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Ingår i: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 16:6, s. 40-728
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Tidskriftsartikel (refereegranskat)abstract
- Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB -type Cu(+)-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu(+) across cellular membranes. Crystal structures of a copper-free Cu(+)-ATPase are available, but the mechanism of Cu(+) recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu(+)-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu(+) is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway.
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