| 1. |
- Althage, Magnus, et al.
(författare)
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Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain.
- 2004
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002 .- 0005-2728. ; 1659:1, s. 73-82
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Tidskriftsartikel (refereegranskat)abstract
- Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha and a beta subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane alpha-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 A), and visualized by SDS-PAGE. In the alpha(2)beta(2) tetramer, alphabeta cross-links were obtained with the alphaG476C-betaS2C, alphaG476C-betaT54C and alphaG476C-betaS183C double mutants. Significant alphaalpha cross-links were obtained with the alphaG476C single mutant in the loop connecting helix 3 and 4, whereas betabeta cross-links were obtained with the betaS2C, betaT54C and betaS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the alpha and beta subunits in the dimer is lined along an axis formed by helices 3 and 4 from the alpha subunit and helices 6, 7 and 8 from the beta subunit. In addition, helices 2 and 4 in the alpha subunit together with helices 6 and 12 in the beta subunit interact with their counterparts in the alpha(2)beta(2) tetramer. Each beta subunit in the alpha(2)beta(2) tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.
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| 2. |
- Goransson, Gunnel, et al.
(författare)
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Ship-Generated Waves and Induced Turbidity in the Gota alv River in Sweden
- 2014
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Ingår i: Journal of Waterway, Port, Coastal and Ocean Engineering. - 0733-950X. ; 140:3
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Tidskriftsartikel (refereegranskat)abstract
- Ship-generated waves were investigated in the Gota alv river, which is a major waterway on the Swedish west coast between the sea and Lake Vanern. Ships with a typical size of 85x15x5 m (lengthxwidthxdraft) travel at speeds between 5 and 10 knots, generating waves that cause sediment transport and erosion along the river bed and banks. Field measurements of the wave properties and turbidity were carried out during 17 ship passages, and comparisons were made with the most commonly used formulas for predicting ship waves. The formula proposed by the Permanent International Association of Navigation Congresses yielded the overall best agreement for the divergent (secondary) waves, whereas the drawdown (primary wave) could best be estimated from the vessel sinkage. The maximum recorded turbidity was mainly a function of the drawdown, and it could be well predicted from the parameterized bed shear stress. In conclusion, ship waves often induce bed and bank erosion in restricted waterways and, although simplistic formulas involve significant uncertainties, they are still useful tools for predictions. However, more studies are needed to determine the influence of a limited river cross section on the wave generation and the relationship between ship waves and sediment transport.
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| 3. |
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| 4. |
- Karlsson, J, et al.
(författare)
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Roles of Individual Amino Acids in Helix 14 of the Membrane Domain of Proton-Translocating Transhydrogenase from Escherichia coli As Deduced from Cysteine Mutagenesis
- 2003
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42, s. 6575-6581
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Tidskriftsartikel (refereegranskat)abstract
- Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues Leu240 to Ser260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of Gly245, Gly249, and Gly252, also to leucines. In addition to the residues forming the helix, residues Asn238 and Asp239 were also mutated. Except for I242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines Gly245, Gly249, and Gly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of Gly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines Ser250, Ser251, and Ser256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.
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| 5. |
- Löfgren, Lars, et al.
(författare)
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Patient-Centric Quantitative Microsampling for Accurate Determination of Urine Albumin to Creatinine Ratio (UACR) in a Clinical Setting
- 2024
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Ingår i: The Journal of Applied Laboratory Medicine. - : Oxford University Press (OUP). - 2576-9456 .- 2475-7241. ; 9:2, s. 329-341
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Tidskriftsartikel (refereegranskat)abstract
- Background: Developing and implementing new patient-centric strategies for drug trials lowers the barrier to participation for some patients by reducing the need to travel to research sites. In early chronic kidney disease (CKD) trials, albuminuria is the key measure for determining treatment effect prior to pivotal kidney outcome trials. Methods: To facilitate albuminuria sample collection outside of a clinical research site, we developed 2 quantitative microsampling methods to determine the urinary albumin to creatinine ratio (UACR). Readout was performed by LC-MS/MS. Results: For the Mitra device the within-batch precision (CV%) was 2.8% to 4.6% and the between-batch precision was 5.3% to 6.1%. Corresponding data for the Capitainer device were 4.0% to 8.6% and 6.7% to 9.0%, respectively. The storage stability at room temperature for 3 weeks was 98% to 103% for both devices. The recovery for the Mitra and Capitainer devices was 104% (SD 7.0%) and 95 (SD 7.4%), respectively. The inter-assay comparison of UACR assessment generated results that were indistinguishable regardless of microsampling technique. The accuracy based on LC-MS/MS vs analysis of neat urine using a clinical chemistry analyzer was assessed in a clinical setting, resulting in 102 ± 8.0% for the Mitra device and 95 ± 10.0% for the Capitainer device. Conclusions: Both UACR microsampling measurements exhibit excellent accuracy and precision compared to a clinical chemistry analyzer using neat urine. We applied our patient-centric sampling strategy to subjects with heart failure in a clinical setting. Precise UACR measurements using quantitative microsampling at home would be beneficial in clinical drug development for kidney therapies.
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| 6. |
- Nguyen, Duong T., et al.
(författare)
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Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.
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| 7. |
- Pedersen, Anders, 1976, et al.
(författare)
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Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme
- 2003
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Ingår i: Biochim.Biophys. Acta (Bioenergetics). ; 1604, s. 55-59
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Tidskriftsartikel (refereegranskat)abstract
- Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called “occluded” intermediary state of the reaction cycle of the intact enzyme. Despite a Kd in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP+ is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2′-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD+ by NADH, indicating that 3-acetylpyridine-NAD+ and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2′-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424–Arg425–Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.
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