Kristina Wilhelm
Umeå University
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Publication
Featured researches published by Kristina Wilhelm.
FEBS Journal | 2009
Kristina Wilhelm; Adas Darinskas; Wim Noppe; Elke Duchardt; K. Hun Mok; Vladana Vukojević; Jürgen Schleucher; Ludmilla A. Morozova-Roche
Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self‐assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid–liquid interface within an ion‐exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein α‐lactalbumin, known as humanα‐lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4–30 lysozyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8‐anilinonaphthalene‐1‐sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin‐T dye, shows a spherical morphology, assembles into ring‐shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well‐populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.
European Journal of Neurology | 2007
Kristina Wilhelm; Kiran Yanamandra; M. A. Gruden; Vladimir Zamotin; Mantas Malisauskas; Vida Casaite; Adas Darinskas; Lars Forsgren; Ludmilla A. Morozova-Roche
Peripheral immune responses can be sensitive indicators of disease pathology. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of 26 Parkinsons disease (PD) patients compared with controls by using ELISA. We found a statistically significant increase of the autoimmune responses to both antigens in PD patients compared with controls with a mean increase of 70% and 50% in the autoimmune reactions towards insulin and S100B, respectively. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in PD patients, indicating that an amyloid‐specific conformational epitope is not involved in immune recognition of this amyloid type, while sequential epitope of native insulin is hidden within the amyloid structures. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.
Langmuir | 2010
Vladana Vukojević; Alice M. Bowen; Kristina Wilhelm; Yu Ming; Zhang Ce; Jürgen Schleucher; P. J. Hore; Lars Terenius; Ludmilla A. Morozova-Roche
Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying PC12 cell death caused by ELOA oligomers. ELOA, a lipoprotein complex formed by equine lysozyme (EL) and oleic acid (OA), induces cell death in all tested cell lines, but the actual mechanism of its action is not known. We have used methods with single-molecule sensitivity, fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and confocal laser scanning microscopy (CLSM) imaging by avalanche photodiodes (APD), so-called APD imaging, to study ELOA interactions with the plasma membrane in live PC12 cells. We detected ELOA accumulation in the cell surroundings, observed ELOA interactions with the plasma membrane, and local changes in plasma membrane lipid dynamics in the vicinity of ELOA complexes. These interactions resulted in plasma membrane rupture, followed by rapid influx and distribution of ELOA inside the already dead cell. In order to probe the ELOA-plasma membrane interaction sites at the molecular and atomic levels, the ELOA complexes were further studied by photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, nuclear magnetic resonance (NMR) and atomic force microscopy (AFM). We observed a novel mechanism of oligomer toxicity-cell death induced by continuous disturbance of the plasma membrane, eventually causing permanent plasma membrane damage and identified the sites in ELOA that are potentially involved in the interactions with the plasma membrane.
ChemBioChem | 2014
Jørn Døvling Kaspersen; Jannik Nedergaard Pedersen; Jon Gade Hansted; Søren Bang Nielsen; Srinivasan Sakthivel; Kristina Wilhelm; Ekaterina L. Nemashkalova; Sergei E. Permyakov; Eugene A. Permyakov; Cristiano L. P. Oliveira; Ludmilla A. Morozova-Roche; Daniel E. Otzen; Jan Skov Pedersen
The cytotoxic complex formed between α‐lactalbumin and oleic acid (OA) has inspired many studies on protein–fatty acid complexes, but structural insight remains sparse. After having used small‐angle X‐ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein–oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core–shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off‐loading of the OA into cell membranes.
American Journal of Physiology-lung Cellular and Molecular Physiology | 2012
Esra Roan; Kristina Wilhelm; Alex Bada; Patrudu S. Makena; Vijay K. Gorantla; Scott E. Sinclair; Christopher M. Waters
Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.
American Journal of Physiology-gastrointestinal and Liver Physiology | 2014
Geetha Samak; Ruchika Gangwar; Lynn M. Crosby; Leena P. Desai; Kristina Wilhelm; Christopher M. Waters; Radhakrishna Rao
The intestinal epithelium is subjected to various types of mechanical stress. In this study, we investigated the impact of cyclic stretch on tight junction and adherens junction integrity in Caco-2 cell monolayers. Stretch for 2 h resulted in a dramatic modulation of tight junction protein distribution from a linear organization into wavy structure. Continuation of cyclic stretch for 6 h led to redistribution of tight junction proteins from the intercellular junctions into the intracellular compartment. Disruption of tight junctions was associated with redistribution of adherens junction proteins, E-cadherin and β-catenin, and dissociation of the actin cytoskeleton at the actomyosin belt. Stretch activates JNK2, c-Src, and myosin light-chain kinase (MLCK). Inhibition of JNK, Src kinase or MLCK activity and knockdown of JNK2 or c-Src attenuated stretch-induced disruption of tight junctions, adherens junctions, and actin cytoskeleton. Paracellular permeability measured by a novel method demonstrated that cyclic stretch increases paracellular permeability by a JNK, Src kinase, and MLCK-dependent mechanism. Stretch increased tyrosine phosphorylation of occludin, ZO-1, E-cadherin, and β-catenin. Inhibition of JNK or Src kinase attenuated stretch-induced occludin phosphorylation. Immunofluorescence localization indicated that phospho-MLC colocalizes with the vesicle-like actin structure at the actomyosin belt in stretched cells. On the other hand, phospho-c-Src colocalizes with the actin at the apical region of cells. This study demonstrates that cyclic stretch disrupts tight junctions and adherens junctions by a JNK2, c-Src, and MLCK-dependent mechanism.
FEBS Journal | 2014
Kristina Wilhelm; Esra Roan; Manik C. Ghosh; Kaushik Parthasarathi; Christopher M. Waters
Patients with acute lung injury are administered high concentrations of oxygen during mechanical ventilation, and while both hyperoxia and mechanical ventilation are necessary, each can independently cause additional injury. However, the precise mechanisms that lead to injury are not well understood. We hypothesized that alveolar epithelial cells may be more susceptible to injury caused by mechanical ventilation because hyperoxia causes cells to be stiffer due to increased filamentous actin (f‐actin) formation via the GTPase RhoA and its effecter Rho kinase (ROCK). We examined cytoskeletal structures in cultured murine lung alveolar epithelial cells (MLE‐12) under normoxic and hyperoxic (48 h) conditions. We also measured cell elasticity (E) using an atomic force microscope in the indenter mode. Hyperoxia caused increased f‐actin stress fibers and bundle formation, an increase in g‐ and f‐actin, an increase in nuclear area and a decrease in nuclear height, and cells became stiffer (higher E). Treatment with an inhibitor (Y‐27632) of ROCK significantly decreased E and prevented the cytoskeletal changes, while it did not influence the nuclear height and area. Pre‐exposure of cells to hyperoxia promoted detachment when cells were subsequently stretched cyclically, but the ROCK inhibitor prevented this effect. Hyperoxia caused thickening of vinculin focal adhesion plaques, and inhibition of ROCK reduced the formation of distinct focal adhesion plaques. Phosphorylation of focal adhesion kinase was significantly reduced by both hyperoxia and treatment with Y‐27632. Hyperoxia caused increased cell stiffness and promoted cell detachment during stretch. These effects were ameliorated by inhibition of ROCK.
PLOS ONE | 2013
Emily A. Clementi; Kristina Wilhelm; Juergen Schleucher; Ludmilla A. Morozova-Roche; Anders P. Hakansson
HAMLET and ELOA are complexes consisting of oleic acid and two homologous, yet functionally different, proteins with cytotoxic activities against mammalian cells, with HAMLET showing higher tumor cells specificity, possibly due to the difference in propensity for oleic acid binding, as HAMLET binds 5-8 oleic acid molecules per protein molecule and ELOA binds 11-48 oleic acids. HAMLET has been shown to possess bactericidal activity against a number of bacterial species, particularly those with a respiratory tropism, with Streptococcus pneumoniae displaying the greatest degree of sensitivity. We show here that ELOA also displays bactericidal activity against pneumococci, which at lower concentrations shows mechanistic similarities to HAMLET’s bactericidal activity. ELOA binds to S. pneumoniae and causes perturbations of the plasma membrane, including depolarization and subsequent rupture, and activates an influx of calcium into the cells. Selective inhibition of calcium channels and sodium/calcium exchange activity significantly diminished ELOA’s bactericidal activity, similar to what we have observed with HAMLET. Finally, ELOA-induced death was also accompanied by DNA fragmentation into high molecular weight fragments – an apoptosis-like morphological phenotype that is seen during HAMLET-induced death. Thus, in contrast to different mechanisms of eukaryote cell death induced by ELOA and HAMLET, these complexes are characterized by rather similar activities towards bacteria. Although the majority of these events could be mimicked using oleic acid alone, the concentrations of oleic acid required were significantly higher than those present in the ELOA complex, and for some assays, the results were not identical between oleic acid alone and the ELOA complex. This indicates that the lipid, as a common denominator in both complexes, is an important component for the complexes’ bactericidal activities, while the proteins are required both to solubilize and/or present the lipid at the bacterial membrane and likely to confer other and separate functions during the bacterial death.
Journal of Visualized Experiments | 2015
Gabriel Rapalo; Josh D. Herwig; Robert Hewitt; Kristina Wilhelm; Christopher M. Waters; Esra Roan
There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.
Cellular & Molecular Biology Letters | 2007
Adas Darinskas; Renata Gasparaviciute; Mantas Malisauskas; Kristina Wilhelm; Jurij A. Kozhevnikov; Evaldas Liutkevicius; Audrone Pilinkiene; Ludmilla A. Morozova-Roche
We have shown the fetal liver cell engraftments into multiple tissues of adult healthy mice, achieved without suppressing the animals’ immune systems. Fetal cells from the livers of male C57Bl/6J Black lineage mice at day 13 to 15 of gestation were injected intravenously into female adult CC57W/MY White mice. The grafting was evaluated by Y-chromosome-specific PCR, cytometric analysis of fluorescently stained donor cells, and histological analysis. All the methods consistently showed the presence of multiple engraftments randomly distributed through the various organs of the recipients. After 60 days, the grafts still constituted 0.1 to 2.75% of the tissues. The grafted cells did not change their appearance in any of the organs except the brain, where they became enlarged. Inflammatory reactions were not detected in any of the histological preparations. The frequency of engraftments was higher in the liver, indicating that similarity between the donor and recipient cells facilitates engraftment. The high inherent plasticity of fetal liver cells underlies their ability to integrate into healthy recipient organs, which can be governed by environmental conditions and connections with neighboring cells rather than by the initial cellular developmental programs. The fact that fetal liver cells can be grafted into multiple tissues of healthy animals indicates that they can be used to replace the natural loss of cells in adult organisms.