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  • 1.
    Adhikari, Deepak
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Flohr, Gilian
    Hogeschool Leiden, Zernikedreef 11,2333 CK Leiden, The Netherlands.
    Gorre, Nagaraju
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yang, Hairu
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Lan, Zijian
    University of Louisville Health Sciences Center, Louisville, Kentucky, USA.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles2009In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 15, no 12, p. 765-770Article in journal (Refereed)
    Abstract [en]

    To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor tuberous sclerosis complex 2 (Tsc2), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1-Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

  • 2.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Catch-Bond behavior of bacteria binding by slip bonds2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 5, p. 1331-1341Article in journal (Refereed)
    Abstract [en]

    It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.

  • 3.
    Blanco-Rivero, Amaya
    et al.
    Madrid, Spain.
    Shutova, Tatiana
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    José Román, María
    Madrid, Spain.
    Villarejo, Arsenio
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain.
    Martinez, Flor
    Madrid, Spain.
    Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, article id e49063Article in journal (Refereed)
    Abstract [en]

    Background: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions. Results/Conclusions: In the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. Significance: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

  • 4.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Höglund, Andreas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, David
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Jacobsson, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line2011In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 16, no 8, p. 783-794Article in journal (Refereed)
    Abstract [en]

    Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

  • 5. Chen, Xinyu
    et al.
    Yuan, Huwei
    Hu, Xiange
    Meng, Jingxiang
    Zhou, Xianqing
    Wang, Xiao-Ru
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. National Engineering Laboratory for Forest Tree Breeding, Key Laboratory of Genetic and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, People’s Republic of China.
    Li, Yue
    Variations in electrical impedance and phase angle among seedlings of Pinus densata and parental species in Pinus tabuliformis habitat environment2015In: Journal of Forestry Research, ISSN 1007-662X, E-ISSN 1993-0607, Vol. 26, no 3, p. 777-783Article in journal (Refereed)
    Abstract [en]

    Electrical impedance (EI) and phase angle (PHI) parameters in AC impedance spectroscopy are important electrical parameters in the study of medical pathology. However, little is known about their application in variation and genetic relationship studies of forest trees. In order to test whether impedance parameters could be used in genetic relationship analysis among conifer species, EI and PHI were measured in a seedling experiment test composed of Pinus tabuliformis, Pinus yunnanensis, and Pinus densata in a habitat of Pinus tabuliformis. The results showed that variations in both EI and PHI among species were significant in different electric frequencies, and the EI and PHI values measured in the two populations of P. densata were between the two parental species, P. yunnanensis and P. tabuliformis. These results show that these two impedance parameters could reflect the genetic relationship among pine species. This was the first time using the two AC impedance spectroscopy parameters to test the genetic relationship analysis between tree species, and would be a hopeful novel reference methodology for future studies in evolution and genetic variation of tree species.

  • 6.
    Cheregi, Otilia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Miranda, Hélder
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Inactivation of the Deg protease family in the cyanobacterium Synechocystis sp. PCC 6803 has impact on the outer cell layers2015In: Journal of Photochemistry and Photobiology. B: Biology, ISSN 1011-1344, E-ISSN 1873-2682, p. 383-394Article in journal (Refereed)
    Abstract [en]

    The serine type Deg/HtrA proteases are distributed in a wide range of organisms from Escherichia coli to humans. The cyanobacterium Synechocystis sp. PCC 6803 possesses three Deg protease orthologues: HtrA, HhoA and HhoB. Previously we compared Synechocystis 6803 wild type cells exposed to mild or severe stress conditions with a mutant lacking all three Deg proteases and demonstrated that stress had strong impact on the proteomes and metabolomes [1]. To identify the biochemical processes, which this protease family is involved in, here we compared Synechocystis sp. PCC 6803 wild type cells with a mutant lacking all three Deg proteases grown under normal growth conditions (30 °C and 40 μmol photons m−2 s−1). Deletion of the Deg proteases lead to the down-regulation of proteins related to the biosynthesis of outer cell layers (e.g. the GDP mannose 4,6-dehydratase) and affected protein secretion. During the late growth phase of the culture Deg proteases were found to be secreted to the extracellular medium of the Synechocystis sp. PCC 6803 wild type strain. While cyanobacterial Deg proteases seem to act mainly in the periplasmic space, deletion of the three proteases influences the proteome and metabolome of the whole cell. Impairments in the outer cell layers of the triple mutant might explain the higher sensitivity toward light and oxidative stress, which was observed earlier by Barker and coworkers [2].

  • 7.
    Christiansen, Alexander
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, no 7, p. 1689-1699Article in journal (Refereed)
    Abstract [en]

    Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.

  • 8. da Hora, G. C. A.
    et al.
    Archilha, N. L.
    Lopes, J. L. S.
    Mueller, D. M.
    Coutinho, K.
    Itri, R.
    Soares, T. A.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Departamento de Quı´mica Fundamental, Universidade Federal de Pernambuco, 50740-560 Cidade Universita´ria, Recife, Brazil.
    Membrane negative curvature induced by a hybrid peptide from pediocin PA-1 and plantaricin 149 as revealed by atomistic molecular dynamics simulations2016In: Soft Matter, ISSN 1744-683X, E-ISSN 1744-6848, Vol. 12, no 43, p. 8884-8898Article in journal (Refereed)
    Abstract [en]

    Antimicrobial peptides (AMPs) are cationic peptides that kill bacteria with a broad spectrum of action, low toxicity to mammalian cells and exceptionally low rates of bacterial resistance. These features have led to considerable efforts in developing AMPs as an alternative antibacterial therapy. In vitro studies have shown that AMPs interfere with membrane bilayer integrity via several possible mechanisms, which are not entirely understood. We have performed the synthesis, membrane lysis measurements, and biophysical characterization of a novel hybrid peptide. These measurements show that PA-Pln149 does not form nanopores, but instead promotes membrane rupture. It causes fast rupture of the bacterial model membrane (POPG-rich) at concentrations 100-fold lower than that required for the disruption of mammalian model membranes (POPC-rich). Atomistic molecular dynamics (MD) simulations were performed for single and multiple copies of PA-Pln149 in the presence of mixed and pure POPC/POPG bilayers to investigate the concentration-dependent membrane disruption by the hybrid peptide. These simulations reproduced the experimental trend and provided a potential mechanism of action for PA-Pln149. It shows that the PA-Pln149 does not form nanopores, but instead promotes membrane destabilization through peptide aggregation and induction of membrane negative curvature with the collapse of the lamellar arrangement. The sequence of events depicted for PA-Pln149 may offer insights into the mechanism of action of AMPs previously shown to induce negative deformation of membrane curvature and often associated with peptide translocation via non-bilayer intermediate structures.

  • 9.
    Dingeldein, Artur
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bax and oxidized phospholipids - a deadly complex: Apoptotic protein-lipid assemblies studied by MAS NMR spectroscopy2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mitochondria are renowned for their vital role as cellular powerhouses because they provide ATP via cellular respiration. Additionally, these organelles also play an important role in other physiological processes, such as apoptosis. Apoptosis, or regulated cell death, is an important mechanism that regulates, for example, tissue homeostasis and embryonic development. Malfunctioning apoptosis can cause severe diseases such as various types of cancer and neurodegenerative diseases. The significance of mitochondria for apoptosis is that mitochondria host a variety of apoptogenic factors, such as cytochrome c. The release of these factors after the formation of an apoptotic pore can be regarded as a point of no return in the onset of apoptosis as these factors trigger the activation of caspases and consequently nuclear fragmentation.

    The mitochondrial outer membrane (MOM) is essential for deciding the cell’s fate, since the MOM provides an interaction surface for the pro- and anti-apoptotic proteins of the Bcl-2 protein family. Further, oxidized phospholipids (OxPls) within the MOM that are generated under oxidative stress conditions (a potent pro-apoptotic stimulus) can directly affect the equilibrium between pro- and anti-apoptotic proteins at the MOM surface, hence influencing the formation of apoptotic pores.

    To characterize the impact of different OxPls on membrane dynamics and organization, several MOM-mimicking systems were studied by solid-state magic angle spinning nuclear magnetic resonance spectroscopy (MAS NMR). These main experiments were accompanied by fluorescence spectroscopy and differential scanning calorimetry (DSC) studies to investigate the impact of OxPls and their interactions with the pro-apoptotic Bax protein at both the macroscopic and molecular levels. By combining several orthogonal methods, we were able to obtain a detailed description of the changes in MOM-mimicking vesicles induced by several types of OxPls. Moreover, we demonstrated how these changes impact the interaction between liposomes and the pro-apoptotic Bax protein.

    By using DSC, we were able to determine not only the macroscopic effect of two OxPls – PazePC and PoxnoPC – but also a concentration threshold. Both OxPls disrupted the membrane order such that the melting behavior of the MOM-mimicking vesicles became less cooperative. A decrease in cooperativity was detectable for OxPl concentrations of up to 5 mol%, after which the cooperativity remained constant. The addition of Bax resulted in an observable ordering effect, as some of the membrane disorder was negated by Bax and the melting process became more cooperative again. The ordering effect of Bax was subsequently confirmed by 31P MAS NMR experiments and cross polarization (CP) buildup curves. Analysis of the buildup curves revealed that at the molecular level, Bax enabled more efficient CP transfer, which indicated rigidification of the membranes after Bax insertion. Furthermore, the 31P NMR experiments provided the first molecular evidence of the importance of cardiolipin as a membrane contact site for Bax.

    Despite having similar disordering effects when studied with DSC, PoxnoPC and PazePC exhibited opposing effects on the pore formation ability of Bax. In studies with fluorescent dye-based leakage assays, Bax was able to form long-lived, stable pores in PazePC-containing giant unilamellar vesicles (GUVs). However, the observed leakage mechanism in PoxnoPC-containing GUVs could no longer be explained by an all-or-none leakage mechanism due to the brevity of the formed pores, leading to partially leaked vesicles, indicating a graded leakage mechanism instead.

    To investigate the possible reasons for the different leakage activities and to obtain mechanistic insights, we conducted 13C MAS NMR experiments. These experiments enabled us to pinpoint specific carbon sites in the different MOM-mimicking systems and to study their dynamic profile as a function of temperature. Though the OxPl-containing multilamellar vesicles (MLVs), compared with non-oxidized systems, also showed drastic dynamic changes, the molecular differences between PazePC- and PoxnoPC-containing vesicles were not significant enough to constitute a structural reason for the opposing leakage activities.

    In addition to investigating membrane dynamics, we were able to establish a novel strategy for producing cytotoxic Bax protein. This novel expression and purification strategy increased the obtained yields by an order of magnitude. By deploying a double fusion approach, we were able to inhibit both termini of the protein from exhibiting their disruptive, yield-diminishing interactions with the host cell membranes.

    In conclusion, over the course of this thesis we were able to develop fast, yet powerful tools to investigate the dynamic changes of MOM-mimicking vesicles under the influence of OxPls and pro-apoptotic Bax. In the future, these tools could be used to characterize the underlying protein-lipid interactions that are responsible for the opposing leakage activities. Due to the development of a novel Bax production strategy, future research could shift to protein-focused studies with the primary goal of determining the structure of the apoptotic Bax pore.

  • 10.
    Dingeldein, Artur P G
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokorna, Sarka
    Hof, Martin
    Pedersen, Anders
    Karlsson, Göran
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    BCL-2 Family Proteins Effect on Mitochondrial-Mimicking Membrane Structure by Solid State NMR2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 251A-252AArticle in journal (Other academic)
    Abstract [en]

    Mitochondria are not only the cells' powerhouse, but also involved in their suicide via apoptosis. Key regulators of this pathway are members of the Bcl-2 protein family which interact with the outer mitochondrial membrane to modulate permeability and enable the release of apoptotic stimuli like cytochrome c. For a long time the mitochondrial membrane forming lipids have been seen as merely structural building units with proteins doing the actual work. This view changed in recent years, since lipids were shown to be also directly involved in apoptotic events e.g. under intracellular oxidative stress. Oxidized phospholipids (OxPls) generated under these stress conditions might trigger mitochondria-mediated apoptosis. Their presence in mitochondrial membranes can severely alter the properties of these membranes with yet unknown consequences regarding the formation of pores through membrane-mediated interplay with apoptotic Bax protein. We therefore devised a model system that embodies oxidative stress conditions by incorporating OxPls into mitochondria mimicking model membranes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) to study the impact of OxPls on apoptotic Bax-membrane interactions. To obtain molecular insight into hydrophobic fatty acid regions of membranes and their hydrophilic interface which is responsible for first protein-membrane contacts, we used differential scanning calorimetry (DSC) and solid state NMR spectroscopy. Upon incorporating OxPls with carboxyl (PoxnoPC) or aldehyde (PazePC) groups at their truncated sn-2-chains into our mitochondria model membranes, calorimetric and NMR measurements showed dramatic changes. 31P NMR experiments revealed major perturbation effects in these membranes; an effect which presumably elevates the membrane binding of apoptotic Bax to the charged membranes and its partial penetration, being a prerequisite for its final formation of pores which enable cytochrome c release from the mitochondrial interior. Currently structural studies of various Bax-lipid assemblies are ongoing.

  • 11.
    Dingeldein, Artur P. G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lidman, Martin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dynamical and Structural Alterations withing Lipid-Protein Assemblies Control Apoptotic Pore Formation - A Solid State NMR Study2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 59A-60AArticle in journal (Other academic)
  • 12.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Oxidatively stressed mitochondrial membranes: A molecular insight into their organization and function during apoptosisIn: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086Article in journal (Refereed)
    Abstract [en]

    Mitochondria are crucially involved in the removal of eukaryotic cells by theintrinsic pathway of programmed cell death (apoptosis). The mitochondrial outermembrane (MOM) is the platform where this pathway is regulated. Uponoxidative stress that triggers apoptotic action, the MOM undergoespermeabilization and releases cytochrome c, ultimately causing cell death. Notonly is this membrane perforation regulated by opposing members of the Bcl-2protein family meeting at the MOM but also is it regulated by the membrane itselfthat plays an active role. Upon oxidative damage, the membrane undergoessevere reorganization causing an imbalance towards cell death-causing apoptoticBcl-2 proteins. To understand the active role of MOM, we provided a detailedmolecular view of its structural and dynamic reorganization by solid-state13C MAS NMR (magic angle spinning nuclear magnetic resonance) accompaniedby calorimetric studies. By focusing on MOM-like vesicles doped with oxidizedlipid species, direct polarization 13C MAS NMR provided a quantitative overviewand identification of all lipid moieties across the membrane. 1H-13C crosspolarization and insensitive nuclei enhanced by polarization transfer MAS NMRgenerated a dynamic - mobile versus restricted – membrane profile. Evidently,oxidized phospholipids significantly perturb the structural membraneorganization and increase membrane dynamics. However, these perturbationsare not observed uniformly as the hydrophobic core is reflecting the melting ofthe lipid chains and the increase in lipid molecule disorder directly, whereas theinterface and headgroup region undergo complex changes in their dynamics,which are coupled to increased segmental disorder. These changes are potentiallycrucial in augmenting the pro-apoptotic action of proteins like Bax there.

  • 13.
    Domar, Ulla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Human intestinal alkaline phosphatase: tissue expression and serum levels1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human alkaline phosphatase (ALP) comprises four isozymes, viz liver/bone/ kidney or tissue unspecific (AP), intestinal (LAP), placental (PLAP) and germ cell or PLAP-like alkaline phosphatase, with their main expression in specific tissues as indicated by their names. The isozymes are coded by different genes, but they are closely related, with more than 50% amino acid sequence homologies. Their biological function is unclear. In certain malignant and benign diseases, serum elevations of one or more of the isozymes occur, which is of diagnostic importance. In this study, the special expression of the intestinal isozyme in human tissues and sera, in normal as well as in pathological conditions, has been investigated by use of isozyme specific monoclonal antibodies.

    Monoclonal antibodies against the AP, IAP and PLAP isozymes were prepared, and specific assays developed, based on these monoclonal antibodies and the catalytic activity of the isozymes. By use of these assays the basal levels of all three isozymes were examined in selected normal organs. The isozymes were found to be expressed in measurable amounts in all the examined organs.

    IAP was immunohistochemically localized to the epithelial cells of membranes lining the ducts and tubules of the kidney, liver, pancreas and small intestine.

    Normal human serum contained all three isozymes. The AP isozyme constituted about 90% of the total ALP activity, the IAP isozyme less than 10% and the PLAP isozyme about 1%. Considerable interindividual variations of the serum IAP activity were observed. The serum activities of the IAP isozyme were related to the individual ABO blood group and secretor status. Non-secretors had low levels of IAP activity amounting to about one tenth of the activity in sera from blood group B or 0 secretors, while blood group A secretors had serum IAP activities in the same order as non-secretors. High individual day to day variations were observed.

    Fat absorption caused serum IAP to increase significantly for all persons, but it was rapidly cleared from the blood. We found that the release of IAP into the blood was linked to lipid absorption, but removal from the blood was not linked to lipoprotein clearance.

    Certain tumors of the testis expressed elevated levels of all three ALP isozymes. The highest activitiy of LAP was observed in one yolk sac tumor, in agreement with the endodermal origin of this tumor. In seminoma tissue the AP and PLAP isozymes were significantly, and IAP moderately elevated.

    Cirrhosis of the liver caused significantly increased serum levels of IAP besides the AP isozyme. In inflammatory diseases of the small intestine, normal serum IAP activities were observed.

  • 14.
    Ehlers, Ina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    NMR studies of metabolites and xenobiotics: From time-points to long-term metabolic regulation2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 15.
    Eremenko, Ekaterina
    et al.
    Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Ben-Zvi, Anat
    National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Raveh, Dina
    Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, p. e58218-Article in journal (Refereed)
    Abstract [en]

    Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.

  • 16. Figueroa Karlström, Eduardo
    et al.
    Lundström, Ronnie
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Occupational Medicine.
    Stensson, Olle
    Hansson Mild, Kjell
    Therapeutic staff exposure to magnetic field pulses during TMS/rTMS treatments2006In: Bioelectromagnetics, ISSN 0197-8462, E-ISSN 1521-186X, Vol. 27, no 2, p. 156-158Article in journal (Refereed)
    Abstract [en]

    Transcranial magnetic stimulation or repetitive transcranial magnetic stimulation (TMS/rTMS) is currently being used in treatments of the central nervous system diseases, for instance, depressive states. The principles of localized magnetic stimulation are summarized and the risk and level of occupational field exposure of the therapeutic staff is analyzed with reference to lCNIRP guidelines for pulses below 100 kHz. Measurements and analysis of the occupational exposure to magnetic fields of the staff working with TMS/rTMS are presented.

  • 17.
    Florea, Cristina
    et al.
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Tanska, Petri
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Mononen, Mika
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Qu, Chengjuan
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lammi, Mikko
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). School of Public Health, Health Science Center of Xi’an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, Xi’an, China.
    Laasanen, Mikko
    School of Engineering and Technology, Savonia University of Applied Sciences, Kuopio, Finland.
    Korhonen, Rami
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland; Diagnostic Imaging Center, Kuopio University Hospital, Kuopio, Finland.
    A combined experimental atomic force microscopy-based nanoindentation and computational modeling approach to unravel the key contributors to the time-dependent mechanical behavior of single cells2017In: Biomechanics and Modeling in Mechanobiology, ISSN 1617-7959, E-ISSN 1617-7940, Vol. 16, no 1, p. 297-311, article id 27554263Article in journal (Refereed)
    Abstract [en]

    Cellular responses to mechanical stimuli are influenced by the mechanical properties of cells and the surrounding tissue matrix. Cells exhibit viscoelastic behavior in response to an applied stress. This has been attributed to fluid flow-dependent and flow-independent mechanisms. However, the particular mechanism that controls the local time-dependent behavior of cells is unknown. Here, a combined approach of experimental AFM nanoindentation with computational modeling is proposed, taking into account complex material behavior. Three constitutive models (porohyperelastic, viscohyperelastic, poroviscohyperelastic) in tandem with optimization algorithms were employed to capture the experimental stress relaxation data of chondrocytes at 5 % strain. The poroviscohyperelastic models with and without fluid flow allowed through the cell membrane provided excellent description of the experimental time-dependent cell responses (normalized mean squared error (NMSE) of 0.003 between the model and experiments). The viscohyperelastic model without fluid could not follow the entire experimental data that well (NMSE = 0.005), while the porohyperelastic model could not capture it at all (NMSE = 0.383). We also show by parametric analysis that the fluid flow has a small, but essential effect on the loading phase and short-term cell relaxation response, while the solid viscoelasticity controls the longer-term responses. We suggest that the local time-dependent cell mechanical response is determined by the combined effects of intrinsic viscoelasticity of the cytoskeleton and fluid flow redistribution in the cells, although the contribution of fluid flow is smaller when using a nanosized probe and moderate indentation rate. The present approach provides new insights into viscoelastic responses of chondrocytes, important for further understanding cell mechanobiological mechanisms in health and disease.

  • 18. Grønberg, Christina
    et al.
    Sitsel, Oleg
    Lindahl, Erik
    Gourdon, Pontus
    Andersson, Magnus
    Theoretical Physics and Swedish e-Science Research Center, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden.
    Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 11, p. 2417-2429Article in journal (Refereed)
    Abstract [en]

    Cu+-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu+-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu+ entry using molecular-dynamics simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu+ delivery. Mutational analyses and simulations in the presence and absence of Cu+ predict that the ion-entry path involves two ion-binding sites: one transient Met148-Cys382 site and one intramembranous site formed by trigonal coordination to Cys384, Asn689, and Met717. The results reconcile earlier biochemical and x-ray absorption data and provide a molecular understanding of ion entry in Cu+-transporting P-type ATPases.

  • 19.
    Hall, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lam, Xuan Tam
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The HhoA protease from Synechocystis sp. PCC 6803: novel insights into structure and activity regulation2017In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 198, no 3, p. 147-153Article in journal (Refereed)
    Abstract [en]

    Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.

  • 20.
    Hansson Mild, Kjell
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Biophysical aspects of permeation and diffusion of water in frog eggs1974Doctoral thesis, comprehensive summary (Other academic)
  • 21.
    Hansson Mild, Kjell
    et al.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Mattsson, Mats-Olof
    Örebro University, Sweden; Seibersdorf, Austria.
    ELF noise fields: a review2010In: Electromagnetic Biology and Medicine, ISSN 1536-8378, E-ISSN 1536-8386, Vol. 29, no 3, p. 72-97Article in journal (Refereed)
    Abstract [en]

    The debate as to whether low-level electromagnetic fields can affect biological systems and in the long term cause health effects has been going on for a long time. Yet the interaction of weak electromagnetic fields (EMF) with living cells, undoubtedly a most important phenomenon, is still not well understood. The exact mechanisms by which the effects are produced have not been identified. Furthermore, it is not possible to clearly define which aspects of an EMF exposure that constitute the "dose." One of the groups that contributed to solving this problem is the Bioelectromagnetics group at Catholic University of America (CUA), Washington, D. C. Their work has been devoted to investigating the physical parameters that are needed to obtain an effect of EMF exposure on biological systems, and also how to inhibit the effect. This is a review of their work on bioeffects caused by low-level EMF, their dependence on coherence time, constancy, spatial averaging, and also how the effects can be modified by an applied ELF noise magnetic field. The group has been using early chick embryos, and L929 and Daudi cells as their main experimental systems. The review also covers the work of other groups on low-level effects and the inhibition of the effects with an applied noise field. The group at CUA has shown that biological effects can be found after exposure to low-level ELF and RF electromagnetic fields, and when effects are observed, applying an ELF magnetic noise field inhibits the effects. Also, other research groups have tried to replicate the studies from the CUA group, or to apply EMF noise to suppress EMF-induced effects. Replications of the CUA effects have not always been successful. However, in all cases where the noise field has been applied to prevent an observed effect, it has been successful in eliminating the effect.

  • 22. Hoernke, M.
    et al.
    Mohan, J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kahra, Dana
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Westenhoff, S.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Schwieger, C.
    Determining membrane bound protein structures by infrared reflection-absorption spectroscopy2017In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S161-S161Article in journal (Other academic)
  • 23. Hoernke, Maria
    et al.
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mohan, Jagan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Westenhoff, Sebastian
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schwieger, Christian
    Structural Mechanism in a Membrane Remodelling ATP-ASE2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 578A-578AArticle in journal (Other academic)
  • 24. Hoernke, Maria
    et al.
    Mohan, Jagan
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kahra, Dana
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Westenhoff, Sebastian
    Schwieger, Christian
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 22, p. E4360-E4369Article in journal (Refereed)
    Abstract [en]

    The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

  • 25. Hoffmann, Andreas
    et al.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Institute of Physics, Martin Luther University Halle-Wittenberg, Halle, Germany.
    Oberwinkler, Tanja
    Siedler, Frank
    Socorro Cortina, Niña
    Balbach, Jochen
    Oesterhelt, Dieter
    Novel sulfated phosphoglycolipids from Natronomonas moolapensis2015In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 191, p. 8-15Article in journal (Refereed)
    Abstract [en]

    Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol. However, by NMR spectroscopy and enzymatic assays the existence of two isomeric molecules with different hexoses (1-(6-sulfo-D-glcp/galf-beta 1,2-glycero)phospho-2,3-diphytanylglycerol) could be shown. The structural origin from phosphatidylglycerol distinguishes these glycolipids within Archaea, because all other characterized haloarchaeal glycolipids consist of diphytanylglycerol directly linked to an oligoglycosyl moiety. Now the door is open to investigate the physical and functional consequences of these architectural differences of the head groups.

  • 26.
    Hubert, Madlen
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Caveolae dynamics is strongly influenced by the lipid composition of the plasma membrane2017In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, p. S121-S121Article in journal (Other academic)
  • 27.
    Hultin, Magnus
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Turnover of chylomicrons in the rat1995Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mechanisms involved in the clearance of chylomicrons and aspects of the interactions at the vascular endothelium were studied in the rat.

    The poly-anion heparin, known to release lipoprotein lipase (LPL) from the vascular endothelium, enhanced the clearance of chylomicrons. Five minutes after heparin injection, the clearance of chylomicron triglycerides and retinyl esters was markedly accelerated. The rapid initial clearance was followed by a slower clearance of heavily lipolyzed chylomicrons. In contrast, one hour after heparin the clearance of both triglycerides and retinyl esters was retarded. This decreased removal of chylomicrons coincided with a decrease in the heparin releasable LPL activity, indicating that the previous release to plasma by heparin had resulted in net loss of functional LPL in the tissues.

    The poly-cation protamine released hepatic lipase and some LPL from their binding sites to plasma. One hour after protamine, plasma triglyceride levels were increased, indicating that chylomicron removal was impeded. It has been speculated that protamine inactivates LPL in vivo, but this was not the case. Ten minutes after injection of protamine normal amounts of LPL could be released by heparin. Thus, the accumulation of plasma triglycerides was not due to a rapid inactivation of LPL by protamine.

    LPL has specificity for sn-1,3-ester bonds. To investigate if this specificity is important in vivo, a lipid emulsion containing medium-chain fatty acids (MCFA) in the sn-1,3-position and long-chain fatty acids (LCFA) in the sn-2-position was synthesized, as well as an emulsion containing MCFA-TG mixed with LCFA-TGs (MMM/LLL). In vitro experiments showed large differences in the hydrolysis of the emulsions, but in vivo there were only small differences in the metabolism.

    To further study if lipid emulsions are cleared by the same mechanisms as chylomicrons, an emulsion was made by the same formulation as Intralipid® with addition of 3H-triolein and ,4C-cholesteryl ester. As measured by the removal of cholesteryl esters, the emulsion was cleared at the same rate as was chylomicrons. The triglyceride label was, however, removed more slowly from the emulsion droplets than from chylomicrons. Together with the lower recirculation of labeled free fatty acids (FFA) in plasma, this suggests that there was less lipolysis of the emulsion. The current view that removal of lipid emulsions in vivo is mainly dependent on LPL-mediated hydrolysis might thus not be correct.

    To further analyze the metabolism of chylomicrons, a compartmental model was developed. In this process, the distribution volume for chylomicrons was shown to be larger than the blood volume, a model for the metabolism of FFA in the rat was validated, and the full tissue distribution of injected chylomicrons was determined. According to the model, about half of the triglyceride label was removed from the circulation together with the core label while for the emulsion this number was about 80 %. In fasted rats all labeled fatty acids appeared to mix with the plasma FFA pool, while in fed rats about one-fifth of the fatty acids did not mix with the FFA but was apparently channeled directly to tissue metabolism.

  • 28.
    Håkansson, Pär
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Westlund, Per-Olof
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lindahl, Erik
    Edholm, Olle
    A direct simulation of EPR slow-motion spectra of spin labelled phospholipids in liquid crystalline bilayers based on a molecular dynamics simulation of the lipid dynamics2001In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 3, no 23, p. 5311-5319Article in journal (Refereed)
    Abstract [en]

    EPR line shapes can be calculated from the stochastic Liouville equation assuming a stochastic model for the reorientation of the spin probe. Here we use instead and for the first time a detailed molecular dynamics (MD) simulation to generate the stochastic input to the Langevin form of the Liouville equation. A 0.1 μs MD simulation at T = 50°C of a small lipid bilayer formed by 64 dipalmitoylphosphatidylcholine (DPPC) molecules at the water content of 23 water molecules per lipid was used. In addition, a 10 ns simulation of a 16 times larger system consisting of 32 DPPC molecules with a nitroxide spin moiety attached at the sixth position of the sn2 chain and 992 ordinary DPPC molecules, was used to investigate the extent of the perturbation caused by the spin probe. Order parameters, reorientational dynamics and the EPR FID curve were calculated for spin probe molecules and ordinary DPPC molecules. The timescale of the electron spin relaxation for a spin-moiety attached at the sixth carbon position of a DPPC lipid molecule is 11.9 × 107 rad s−1 and for an unperturbed DPPC molecule it is 3.5 × 107 rad s−1.

  • 29.
    Hörnberg, Andreas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Eneqvist, Therese
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    The β-strand D of transthyretin trapped in two discrete conformations2004In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1700, no 1, p. 93-104Article in journal (Refereed)
    Abstract [en]

    Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.

  • 30.
    Ingemarson, Rolf
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Herpes simplex ribonucleotide reductase1989Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In all bacterial, plant and animal cells, as well as in many viruses, genetic information resides in DNA (deoxyribonucleic acid). Replication of DNA is essential for proliferation, and DNA-containing viruses (such as herpesviruses) must carry out this process within the mammalian cells they infect. The enzyme ribonucleotide reductase catalyzes the first unique step leading to the production of the four deoxy-ribonucleotides used to make DNA. Each deoxyribonucleotide is produced by reduction of the corresponding ribonucleotide. After infection of a mammalian cell with herpes simplex virus (HSV) a new ribonucleotide reductase activity appears, which is distinct from the mammalian enzyme activity. This is due to induction of a separate, virally-encoded ribonucleotide reductase. Two monoclonal antibodies were raised against HSV (type 1) ribonucleotide reductase, and were found to bind but not neutralize its enzyme activity. One antibody recognized a larger (140 kD) protein and the other a smaller (40 kD) protein, suggesting the HSV 1 ribonucleotide reductase had a heterodimeric composition similar to that found in many other organisms. The 140 kD protein was sequentially degraded to 110 kD, 93 kD and 81 kD proteins by a host (Vero) cell-specific serine protease. Of these different proteolytic products, at least the 93 kD residue was enzymatically active, suggesting that part of the 140 kD protein may have functions unrelated to ribonucleotide reduction. The 140 and 40 kD proteins bound tightly to each other in a complex of the a2ß2 type, as shown by analytical glycerol gradient centrifugation. An assay system for functional small and large subunits of HSV 1 ribonucleotide reductase was developed, using two temperaturesensitive mutant viruses, defective in either the large (tsl207) or small (tsl222) subunits. Active holoenzyme was reconstituted both in vitro, by mixing extracts from cells infected with either mutant, and in vivo by coinfection of cells with both mutants. The gene encoding the small subunit of HSV 1 ribonucleotide reductase was cloned into an expression plasmid under control of a tac promoter. The recombinant protein was purified to homogeneity from extracts of transfected E. coli, and was active when combined with large subunit, as provided by extracts of tsl222- infected hamster (BHK) cells. The protein contained a novel tyrosyl free radical that spectroscopically resembled, but was distinguishable from, the active-site free radical found in either the E. coli or mammalian small subunits of ribonucleotide reductase. The gene encoding the large subunit of HSV 1 ribonucleotide reductase was also expressed in E. coli, using similar techniques. The recombinant large subunit was immunoprecipitated from extracts of transfected bacteria, and showed weak activity when combined with small subunit, provided by extracts of tsl20-infected hamster (BHK) cells.

  • 31. Jo, Junghyo
    et al.
    Hörnblad, Andreas
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Kilimnik, German
    Department of Medicine, The University of Chicago, Chicago, IL, USA.
    Hara, Manami
    Department of Medicine, The University of Chicago, Chicago, IL, USA.
    Ahlgren, Ulf
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Periwal, Vipul
    Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
    The fractal spatial distribution of pancreatic islets in three dimensions: a self-avoiding growth model2013In: Physical Biology, ISSN 1478-3967, E-ISSN 1478-3975, Vol. 10, no 3, p. 036009-Article in journal (Refereed)
    Abstract [en]

    The islets of Langerhans, responsible for controlling blood glucose levels, are dispersed within the pancreas. A universal power law governing the fractal spatial distribution of islets in two-dimensional pancreatic sections has been reported. However, the fractal geometry in the actual three-dimensional pancreas volume, and the developmental process that gives rise to such a self-similar structure, has not been investigated. Here, we examined the three-dimensional spatial distribution of islets in intact mouse pancreata using optical projection tomography and found a power law with a fractal dimension of 2.1. Furthermore, based on two-dimensional pancreatic sections of human autopsies, we found that the distribution of human islets also follows a universal power law with a fractal dimension of 1.5 in adult pancreata, which agrees with the value previously reported in smaller mammalian pancreas sections. Finally, we developed a self-avoiding growth model for the development of the islet distribution and found that the fractal nature of the spatial islet distribution may be associated with the self-avoidance in the branching process of vascularization in the pancreas.

  • 32.
    Julkunen, Petro
    et al.
    Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland; Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland.
    Wilson, Wouter
    Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
    Jurvelin, Jukka
    Department of Physics, University of Kuopio, Kuopio, Finland; Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, Kuopio, Finland.
    Rieppo, Jarno
    Department of Biomedicine, Anatomy, University of Kuopio, Kuopio, FinlandDepartment of Biomedicine, Anatomy, University of Kuopio, Kuopio, Finland.
    Qu, Cheng-Juan
    Department of Biomedicine, Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Biosciences, Applied Biotechnology, University of Kuopio, Kuopio, Finland.
    Korhonen, Rami
    Department of Physics, University of Kuopio, Kuopio, Finland; Human Performance Laboratory, Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada.
    Stress-relaxation of human patellar articular cartilage in unconfined compression: prediction of mechanical response by tissue composition and structure.2008In: Journal of Biomechanics, ISSN 0021-9290, E-ISSN 1873-2380, Vol. 41, no 9, p. 1978-86, article id 18490021Article in journal (Refereed)
    Abstract [en]

    Mechanical properties of articular cartilage are controlled by tissue composition and structure. Cartilage function is sensitively altered during tissue degeneration, in osteoarthritis (OA). However, mechanical properties of the tissue cannot be determined non-invasively. In the present study, we evaluate the feasibility to predict, without mechanical testing, the stress-relaxation response of human articular cartilage under unconfined compression. This is carried out by combining microscopic and biochemical analyses with composition-based mathematical modeling. Cartilage samples from five cadaver patellae were mechanically tested under unconfined compression. Depth-dependent collagen content and fibril orientation, as well as proteoglycan and water content were derived by combining Fourier transform infrared imaging, biochemical analyses and polarized light microscopy. Finite element models were constructed for each sample in unconfined compression geometry. First, composition-based fibril-reinforced poroviscoelastic swelling models, including composition and structure obtained from microscopical and biochemical analyses were fitted to experimental stress-relaxation responses of three samples. Subsequently, optimized values of model constants, as well as compositional and structural parameters were implemented in the models of two additional samples to validate the optimization. Theoretical stress-relaxation curves agreed with the experimental tests (R=0.95-0.99). Using the optimized values of mechanical parameters, as well as composition and structure of additional samples, we were able to predict their mechanical behavior in unconfined compression, without mechanical testing (R=0.98). Our results suggest that specific information on tissue composition and structure might enable assessment of cartilage mechanics without mechanical testing.

  • 33.
    Kahra, Dana
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kovermann, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemistry Department, University of Konstanz, Konstanz, Germany.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox12016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 1, p. 95-102Article in journal (Refereed)
    Abstract [en]

    Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, K-D, of similar to 10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.

  • 34.
    Kamilla, Nørregaard
    et al.
    The Niels Bohr Institute; University of Copenhagen; Copenhagen, Denmark.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Sneppen, Kim
    The Niels Bohr Institute; University of Copenhagen; Copenhagen, Denmark.
    Nielsen, Peter Eigil
    Department of Cellular and Molecular Medicine; Faculty of Health and Sciences; Copenhagen, Denmark.
    Brown, Stanley
    The Niels Bohr Institute; University of Copenhagen; Copenhagen, Denmark.
    Oddershede, Lene
    The Niels Bohr Institute; University of Copenhagen; Copenhagen, Denmark.
    Effect of supercoiling on the λ switch2014In: Bacteriophage, ISSN 2159-7081, Vol. 4, no 1, p. e27517-1-e27517-5Article in journal (Refereed)
    Abstract [en]

    The lysogenic state of the λ switch is exceptionally stable, still, it is capable of responding to DNA-damage and rapidly enter the lytic state. We invented an assay where PNA mediated tethering of a plasmid allowed for single molecule investigations of the effect of supercoiling on the efficiency of the epigenetic λ switch. Compared with non-supercoiled DNA, the presence of supercoils enhances the CI-mediated DNA looping probability and renders the transition between the looped and unlooped states steeper, thus increasing the Hill coefficient. Interestingly, the transition occurs exactly at the CI concentration corresponding to the minimum number of CI molecules capable of maintaining the pRM-repressed state. Based on these results we propose that supercoiling maintains the pRM-repressible state as CI concentration decline during induction and thus prevent autoregulation of cI from interfering with induction.

  • 35.
    Koroidov, Sergey
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Water splitting in natural and artificial photosynthetic systems2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Photosynthesis is the unique biological process that converts carbon dioxide into organic compounds, for example sugars, using the energy of sunlight. Thereby solar energy is converted into chemical energy. Nearly all life depends on this reaction, either directly, or indirectly as the ultimate source of their food. Oxygenic photosynthesis occurs in plants, algae and cyanobacteria. This process created the present level of oxygen in the atmosphere, which allowed the formation of higher life, since respiration allows extracting up to 15-times more energy from organic matter than anaerobic fermentation. Oxygenic photosynthesis uses as substrate for the ubiquitous water. The light-induced oxidation of water to molecular oxygen (O2), catalyzed by the Mn4CaO5 cluster associated with the photosystem II (PS II) complex, is thus one of the most important and wide spread chemical processes occurring in the biosphere. Understanding the mechanism of water-oxidation by the Mn4CaO5 cluster is one of today’s great challenges in science. It is believed that one can extract basic principles of catalyst design from the natural system that than can be applied to artificial systems. Such systems can be used in the future for the generation of fuel from sunlight.

    In this thesis the light-induced production of molecular oxygen and carbon dioxide (CO2) by PSII was observed by membrane-inlet mass spectrometry. By analyzing this observation is shown that CO2 not only is the substrate in photosynthesis for the production of sugars, but that it also regulates the efficiency of the initial steps of the electron transport chain of oxygenic photosynthesis by acting, in form of HCO3-, as acceptor for protons produced during water-splitting. This finding concludes the 50-years old search for the function of CO2/HCO3 in photosynthetic water oxidation.

    For understanding the mechanism of water oxidation it is crucial to resolve the structures of all oxidation states, including transient once, of the Mn4CaO5 cluster. With this application in mind a new illumination setup was developed and characterized that allowed to bring the Mn4CaO5 cluster of PSII microcrystals into known oxidation states while they flow through a narrow capillary. The optimized illumination conditions were employed at the X-ray free electron laser at the Linac Coherent Light Source (LCLS) to obtain simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) at room temperature. This two methods probe the overall protein structure and the electronic structure of the Mn4CaO5 cluster, respectively. Data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. This approach opens new directions for studying structural changes during the catalytic cycle of the Mn4CaO5 cluster, and for resolving the mechanism of O-O bond formation.

    In two other projects the mechanism of molecular oxygen formation by artificial water oxidation catalysts containing inexpensive and abundant elements were studied. Oxygen evolution catalyzed by calcium manganese and manganese only oxides was studied in 18O-enriched water. It was concluded that molecular oxygen is formed by entirely different pathways depending on what chemical oxidant was used.  Only strong non-oxygen donating oxidants were found to support ‘true’ water-oxidation. For cobalt oxides a study was designed to understand the mechanistic details of how the O-O bond forms. The data demonstrate that O-O bond formation occurs by direct coupling between two terminal water-derived ligands. Moreover, by detailed theoretical modelling of the data the number of cobalt atoms per catalytic site was derived.

  • 36.
    Koroidov, Sergey
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shevela, Dmitriy
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Shutova, Tatyana
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Messinger, Johannes
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 11, no 17, p. 6299-6304Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria, algae and plants oxidize water to the O2 we breathe, and consume CO2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO2/HCO3 in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO3 acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water-oxidation out into the chloroplast's lumen, resulting in a light-driven production of O2 and CO2. Depletion of HCO3 from the media leads, in the absence of added buffers, to a reversible down-regulation of O2 production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis, and conclude the more than 50-y-long quest for the function of CO2/ HCO3 in photosynthetic water oxidation.

  • 37. Koukalova, Alena
    et al.
    Amaro, Mariana
    Aydogan, Gokcan
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Williamson, Philip T. F.
    Mikhalyov, Ilya
    Hof, Martin
    Sachl, Radek
    Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5460Article in journal (Refereed)
    Abstract [en]

    It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)-and cholesterol (Chol)-driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Forster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solidstate wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

  • 38.
    Kovermann, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rogne, Per
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Protein dynamics and function from solution state NMR spectroscopy2016In: Quarterly reviews of biophysics (Print), ISSN 0033-5835, E-ISSN 1469-8994, Vol. 49, article id e6Article, review/survey (Refereed)
    Abstract [en]

    It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

  • 39.
    Lammi, Mikko
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Sironen, Reijo
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Elo, Mika
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Oksala, Niku
    Department of Physiology, University of Kuopio, Kuopio, Finland; Department of Surgery, Kuopio University Hospital, Kuopio, Finland.
    Kaarniranta, Kai
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Karjalainen, Hannu
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Responses of mammalian cells to mechanical forces2001In: Recent Research Developments in Biophysics and Biochemistry / [ed] Pandalai, S. G., Trivandrum, India: Research Signpost , 2001, Vol. 1, p. 77-89Chapter in book (Other academic)
    Abstract [en]

    All cells and tissues of our body are continuously subject to various mechanical stresses. These forces include, e.g., compression, shear stress, hydrostatic pressure and osmotic pressure. The range of forces vary from few pascals to several megapascals in magnitude. In many cases, mechanical forces are required for the tissues to maintain their normal functional structure and composition. However, excessive forces in the end may lead to adverse responses. In this paper, we review the data available from many different tissues in order to compare the signaling mechanisms involved in cellular mechanotransduction, and how the cells respond to forces that are too strenuous for them to withstand. The possible stress reactions caused by excessive forces are also discussed.

  • 40.
    Lee, Eun
    et al.
    Sungkyunkwan University.
    Holme, Petter
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Network characteristics of individual pigments in cyanobacterial photosystem II core complexes2013In: Journal of the Korean Physical Society, ISSN 0374-4884, E-ISSN 1976-8524, Vol. 63, no 11, p. 2255-2261Article in journal (Refereed)
    Abstract [en]

    Part of the excitation energy transfer (EET) characteristics of the photosystem II (PSII) comes from the interconnection between pigments. To understand the correlation between the EET and the pigments’ interaction structure, we construct a network from the EET rates which are related to both the distance between the pigments (chlorophylls and pheophytins) and their spatial orientations. Especially, we investigate how well the PS II core complex’s EET functionality can be explained by using only the network topology in Thermosynechococcus vulcanus 1.9 °A. Starting from the Förster theory, we construct a network of EET pathways. For an analysis of the network structure, we calculate common network-structural measures like betweenness centrality, eigenvector centrality and weighted clustering. These measures can reflect the role of individual pigments in the EET network. In our work, we found that some well-known properties were reproduced by the network analysis of the simplified network, which means that the topology of the network encodes functionally relevant information. For example, from the network structural analysis, we can infer that most of the chlorophyll molecules (clorophylls) in the pigment-protein complex CP47 have heightened probability to transfer energy compared with other chlorophylls. We also see that the active branch chlorophylls in the reaction center are characterized by a high eigenvector centrality, a high betweenness centrality and a low weighted clustering coefficient. This is indicative of functionally important vertices.

  • 41. Lendel, Christofer
    et al.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mayzel, Maxim
    Andersson, C. Evalena
    Karlsson, Göran
    Härd, Torleif
    Combined Solution- and Magic Angle Spinning NMR Reveals Regions of Distinct Dynamics in Amyloid β Protofibrils2016In: ChemistrySelect, ISSN 2365-6549, Vol. 1, no 18, p. 5850-5853Article in journal (Refereed)
    Abstract [en]

    Solid-state magic angle spinning (MAS) NMR has emerged as an important tool for investigations of protein aggregates and amyloid fibrils, which are not accessible for solution NMR experiments. We recently presented a structural model for amyloid β (Aβ) protofibrils based on MAS-NMR data. The absence of resonances for the N-terminus of Aβ in this dataset suggested that it might be disordered and more dynamic than the structural core. We here provide evidence for a distinct dynamic regime in the N-terminal part of the peptide and show that the structural characteristics of this region can be elucidated using 13C-detected solution NMR. The results shed more light on the structural properties of pre-fibrillar Aβ species and demonstrate the potential of combining MAS and solution NMR experiments for the characterization of structure and dynamics of complex protein assemblies.

  • 42.
    Lindahl, Viveca
    et al.
    KTH, Fysik.
    Gourdon, Pontus
    Department of Biomedical Sciences, University of Copenhagen, Denmark.
    Andersson, Magnus
    KTH, Fysik.
    Hess, Berk
    KTH, Fysik.
    Permeability and ammonia selectivity in aquaporin TIP2;1: linking structure to functionIn: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322Article in journal (Refereed)
    Abstract [en]

    Aquaporin TIP2;1 is a protein channel that is permeable to both water and ammonia. Thestructural origin of ammonia selectivity remains obscure, but experiments have revealed that adouble mutation renders it impermeable to ammonia without affecting water permeability. Here,we aim to reproduce and explain these observations by performing an extensive mutationalstudy using microsecond long molecular dynamics simulations, applying two popular force fields.We calculate permeabilities and free energy profiles along the channel axis, for ammonia andwater. For one force field, the permeability of the double mutant decreases by a factor of 2.5 forwater and a factor of 4 for ammonia, thus increasing the selectivity for water. We attribute thiseffect to decreased entropy of water in the pore, due to the observed increase in pore–waterinteractions and narrower pore. Additionally, we observe spontaneous opening and closing ofthe pore on the cytosolic side, which suggests a gating mechanism for the pore. Our resultsshow that sampling methods and simulation times are sufficient to delineate even subtle effectsof mutations on structure and function and to capture important long-timescale events, butalso underline the importance of improving models further.

  • 43. Lugmaier, Robert A
    et al.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Kuehner, Ferdinand
    Benoit, Martin
    Dynamic restacking of Escherichia coli P-pili2008In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 37, no 2, p. 111-120Article in journal (Refereed)
    Abstract [en]

    P-pili of uropathogenic Escherichia coli mediate the attachment to epithelial cells in the human urinary tract and kidney and therefore play an important role in infection. A better understanding of this mechanism could help to prevent bacteria from spreading but also provides interesting insights into molecular mechanics for future nanotech applications. The helical rod design of P-pili provides an efficient design to withstand hydrodynamic shear forces. The adhesive PapG unit at the distal end of the P-pilus forms a specific bond with the glycolipid Galabiose. This bond has a potential width Delta x = 0.7 (+/-) 0.15 nm and a dissociation rate K-Off = 8.0 center dot 10(-4) +/- 5.0 center dot 10(-4) s(-1). It with-stands a force of similar to 49 pN under physiological conditions. Additionally, we analyzed the behavior of unstacking and restacking of the P-pilus with dynamic force spectroscopy at velocities between 200 and 7,000 nm/s. Up to a critical extension of 66% of the totally stretched P-pilus, un/restacking was found to be fully reversible at velocities up to 200 nm/s. If the P-pilus is stretched beyond this critical extension a characteristic hysteresis appears upon restacking. This hysteresis originates from a nucleation process comparable to a first-order phase transition in an under-cooled liquid. Analysis of the measurement data suggests that 20 PapA monomers are involved in the formation of a nucleation kernel.

  • 44. Lycksell, P O
    et al.
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Bengtsson-Olivecrona, G
    Johansson, L B
    Wijmenga, S S
    Wernic, D
    Gräslund, A
    Sequence specific 1H-NMR assignments and secondary structure of a carboxy-terminal functional fragment of apolipoprotein CII.1992In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 205, no 1Article in journal (Refereed)
    Abstract [en]

    The structural properties of a synthetic fragment of human apolipoprotein CII (apoCII) has been studied by circular dichroism and proton nuclear magnetic resonance. The fragment corresponds to the carboxy-terminal 30 amino acid residues and retains the ability of apoCII to activate lipoprotein lipase. Like native apoCII, the fragment has a tendency to self-associate in pure aqueous solution. Addition of 1,1,1,3,3,3-hexafluoro-2-isopropanol to aqueous solvent dissolves the aggregates and leads to an increase in the alpha-helical content of the peptide, probably by stabilizing transient helical structures. The resonances in the 1H-NMR spectrum of the fragment in 35% (CF3)2CHOH were assigned through standard procedures from nuclear Overhauser enhancement spectroscopy, correlated spectroscopy and total correlated spectroscopy experiments. The NMR data indicates the formation of a stable alpha helix spanning Ile66-Gly77. Another alpha helical turn may be formed between Lys55 and Ala59 and possibly span even further towards the carboxyl terminus. These structural elements are different from those previously predicted for this part of the sequence of apoCII.

  • 45.
    Malisauskas, Mantas
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Weise, Christoph
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yanamandra, Kiran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lability landscape and protease resistance of human insulin amyloid: a new insight into its molecular properties2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, no 1, p. 60-74Article in journal (Refereed)
    Abstract [en]

    Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as AFM, thioflavin-T fluorescence, circular dichroism and gas phase electrophoretic mobility macromolecule analysis we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pHs and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding the inflection point of the growth phase; (ii) an abrupt cut-off between growth and disaggregation at pH 8-10; (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time AFM and thioflavin-T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca. 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.

  • 46.
    Messinger, Johannes
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lubitz, Wolfgang
    Shen, Jian-Ren
    Photosynthesis: from natural to artificial2014In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 16, no 24, p. 11810-11811Article in journal (Other academic)
  • 47. Metsälä, Markus
    et al.
    Schmidt, Florian M.
    University of Helsinki.
    Skytta, Mirva
    Vaittinen, Olavi
    Halonen, Lauri
    Acetylene in breath: background levels and real-time elimination kinetics after smoking2010In: Journal of Breath Research, ISSN 1752-7155, E-ISSN 1752-7163, Vol. 4, no 4, article id 046003Article in journal (Refereed)
    Abstract [en]

    We have measured the acetylene concentration in the exhaled breath of 40 volunteers (31 non-smokers, nine smokers) using near-infrared cavity ring-down spectroscopy. The acetylene levels were found to be the same as in ambient air for non-smokers, whereas elevated levels were observed for smokers. Real-time measurements with sub-second time resolution have been applied to measure the elimination kinetics of acetylene in breath after exposure to tobacco smoke. Three exponential time constants can be distinguished from the data and these can be used to define the residence times for different compartments, according to the multi-compartment model of the human body.

  • 48.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 3, p. 694-704Article in journal (Refereed)
    Abstract [en]

    Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

  • 49.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding effects on two homologs of ribosomal protein S16: protein-dependent structural changes and local interactions2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 2, p. 401-410Article in journal (Refereed)
    Abstract [en]

    Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16T(herme)) and mesophilic (S161homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic Meso, protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Forster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16 Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16me, allowing measurements of three intraprotein distances. All S16meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two 516-rhermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.

  • 50. Moles, Angela T
    et al.
    Peco, Begoña
    Wallis, Ian R
    Foley, William J
    Poore, Alistair G B
    Seabloom, Eric W
    Vesk, Peter A
    Bisigato, Alejandro J
    Cella-Pizarro, Lucrecia
    Clark, Connie J
    Cohen, Philippe S
    Cornwell, William K
    Edwards, Will
    Ejrnæs, Rasmus
    Gonzales-Ojeda, Therany
    Graae, Bente J
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Biology, NTNU, Trondheim, Norway.
    Hay, Gregory
    Lumbwe, Fainess C
    Magaña-Rodríguez, Benjamín
    Moore, Ben D
    Peri, Pablo L
    Poulsen, John R
    Stegen, James C
    Veldtman, Ruan
    Zeipel, Hugovon
    Andrew, Nigel R
    Boulter, Sarah L
    Borer, Elizabeth T
    Cornelissen, Johannes H C
    Farji-Brener, Alejandro G
    DeGabriel, Jane L
    Jurado, Enrique
    Kyhn, Line A
    Low, Bill
    Mulder, Christa P H
    Reardon-Smith, Kathryn
    Rodríguez-Velázquez, Jorge
    De Fortier, An
    Zheng, Zheng
    Blendinger, Pedro G
    Enquist, Brian J
    Facelli, Jose M
    Knight, Tiffany
    Majer, Jonathan D
    Martínez-Ramos, Miguel
    McQuillan, Peter
    Hui, Francis K C
    Correlations between physical and chemical defences in plants: tradeoffs, syndromes, or just many different ways to skin a herbivorous cat?2013In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 198, no 1, p. 252-263Article in journal (Refereed)
    Abstract [en]

    Most plant species have a range of traits that deter herbivores. However, understanding of how different defences are related to one another is surprisingly weak. Many authors argue that defence traits trade off against one another, while others argue that they form coordinated defence syndromes. We collected a dataset of unprecedented taxonomic and geographic scope (261 species spanning 80 families, from 75 sites across the globe) to investigate relationships among four chemical and six physical defences. Five of the 45 pairwise correlations between defence traits were significant and three of these were tradeoffs. The relationship between species' overall chemical and physical defence levels was marginally nonsignificant (P=0.08), and remained nonsignificant after accounting for phylogeny, growth form and abundance. Neither categorical principal component analysis (PCA) nor hierarchical cluster analysis supported the idea that species displayed defence syndromes. Our results do not support arguments for tradeoffs or for coordinated defence syndromes. Rather, plants display a range of combinations of defence traits. We suggest this lack of consistent defence syndromes may be adaptive, resulting from selective pressure to deploy a different combination of defences to coexisting species.

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