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  • 101.
    Bergman, Hilde-Marlene
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lindfors, Lina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Palm, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Lanekoff, Ingela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Metabolite aberrations at early onset of diabetes detected in rat kidney using mass spectrometry imaging2019In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 13, p. 2809-2816Article in journal (Refereed)
    Abstract [en]

    Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes.

  • 102.
    Bergman, Hilde-Marléne
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Applications of nanospray desorption electrospray ionization mass spectrome: Analysis of lipids and metabolites in brain tissue sections and single cells2016Licentiate thesis, comprehensive summary (Other academic)
    List of papers
    1. Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization
    Open this publication in new window or tab >>Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization
    2016 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 12, p. 3686-3695Article in journal (Refereed) Published
    Abstract [en]

    Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease.

    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-281314 (URN)10.1039/c5an02620b (DOI)000378942900021 ()26859000 (PubMedID)
    Funder
    Swedish Research Council, VR 621-2013-4231Swedish Foundation for Strategic Research , SSF ICA-6
    Available from: 2016-03-22 Created: 2016-03-22 Last updated: 2018-11-29
    2. Detection of endogenous lipids and metabolites in single cells using nano-DESI
    Open this publication in new window or tab >>Detection of endogenous lipids and metabolites in single cells using nano-DESI
    (English)Manuscript (preprint) (Other academic)
    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-291338 (URN)
    Available from: 2016-05-01 Created: 2016-05-01 Last updated: 2018-11-29
  • 103.
    Bergman, Hilde-Marléne
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Applications of nanospray desorption electrospray ionization mass spectrometry: In situ lipid and metabolite analysis from cells to tissue2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ambient mass spectrometry (MS) has proved to be an important addition to the bioanalytical toolbox. These methods perform analyte sampling and ionization under atmospheric pressure, and require very little sample preparation other than the sampling process in front of the machine. Nanospray desorption electrospray ionization (nano-DESI) is an ambient MS technique developed in 2010 that utilizes localized liquid extraction for surface sampling. The aim of this thesis was to explore the possibilities of this technique, and identify areas in which nano-DESI MS could further contribute to the community of MS-based surface analysis.

    One such area was found to be mass spectrometry imaging (MSI) of small-molecule neurotransmitters. By the use of deuterated standards of acetylcholine, γ-aminobutyric acid and glutamate, the respective endogenous compounds were successfully imaged in coronal sections of rat brain. The use of internal standards was shown to be essential to compensatee for matrix effects in different regions of the brain. In a second imaging study, nano-DESI MSI was used to compare the chemical profiles of diabetic rat kidney tissue and control. Analysis was performed on kidney two weeks after diabetic onset, before any pathohistological changes relating to diabetic nephropathy can be seen in a microscope. In our study, it was shown that a large number of chemical species related to energy metabolism were detected with altered signal intensity in diabetic kidney tissue.

    To push the limits of nano-DESI analysis, its use for single-cell analysis was evaluated. By placing buccal epithelial cells in contact with the nano-DESI probe, it was possible to identify 46 endogenous compounds and detect differences between cells from three human donors. In addition, it was shown that molecules from single cells on a surface could be detected by scanning the surface with the nano-DESI probe, which opens up for development of an automated analysis with higher throughput.

    The last study in this thesis was concerned with method development rather than application, as it presented a setup for pneumatically assisted nano-DESI. Evaluation showed that the setup provided improved sensitivity in the analysis of small metabolites, and provided the possibility of using pure water as nano-DESI solvent.

    List of papers
    1. Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization
    Open this publication in new window or tab >>Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization
    2016 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 12, p. 3686-3695Article in journal (Refereed) Published
    Abstract [en]

    Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease.

    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-281314 (URN)10.1039/c5an02620b (DOI)000378942900021 ()26859000 (PubMedID)
    Funder
    Swedish Research Council, VR 621-2013-4231Swedish Foundation for Strategic Research , SSF ICA-6
    Available from: 2016-03-22 Created: 2016-03-22 Last updated: 2018-11-29
    2. Metabolite aberrations at early onset of diabetes detected in rat kidney using mass spectrometry imaging
    Open this publication in new window or tab >>Metabolite aberrations at early onset of diabetes detected in rat kidney using mass spectrometry imaging
    Show others...
    2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 13, p. 2809-2816Article in journal (Refereed) Published
    Abstract [en]

    Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes.

    National Category
    Analytical Chemistry
    Research subject
    Chemistry with specialization in Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-347672 (URN)10.1007/s00216-019-01721-5 (DOI)000468133600008 ()30895347 (PubMedID)
    Funder
    Swedish Foundation for Strategic Research Swedish Research CouncilSwedish Diabetes AssociationAstraZeneca
    Note

    Title in dissertation list of papers: Metabolite aberrations at early onset of diabetes detected in rat kidney using mass spectrometry imaging

    Available from: 2018-04-05 Created: 2018-04-05 Last updated: 2019-06-19Bibliographically approved
    3. Profiling and quantifying endogenous molecules in single cells using nano-DESI MS
    Open this publication in new window or tab >>Profiling and quantifying endogenous molecules in single cells using nano-DESI MS
    2017 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 142, no 19, p. 3639-3647Article in journal (Refereed) Published
    Abstract [en]

    Molecular profiling of single cells has the potential to significantly advance our understanding of cell function and cellular processes of importance to health and disease. In particular, small molecules with rapid turn-over rates can reveal activated metabolic pathways resulting from an altered chemical environment or cellular events such as differentiation. Consequently, techniques for quantitative metabolite detection acquired in a higher throughput manner are needed to characterize the biological variability between seemingly homogenous cells. Here, we show that nanospray desorption electrospray ionization (nano-DESI) mass spectrometry ( MS) enables sensitive molecular profiling and quantification of endogenous species in single cells in a higher throughput manner. Specifically, we show a large number of detected amino acids and phospholipids, including plasmalogens, readily detected from single cheek cells. Further, by incorporating a phosphatidylcholine ( PC) internal standard into the nano-DESI solvent, we determined the total amount of PC in one cell to be 1.2 pmoles. Finally, we describe a higher throughput approach where molecules in single cells are automatically profiled. These developments in single cell analysis provide a basis for future studies to understand cellular processes related to drug effects, cell differentiation and altered chemical microenvironments.

    Place, publisher, year, edition, pages
    ROYAL SOC CHEMISTRY, 2017
    National Category
    Analytical Chemistry
    Research subject
    Chemistry with specialization in Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-336430 (URN)10.1039/c7an00885f (DOI)000411703800013 ()28835951 (PubMedID)
    Funder
    Swedish Research Council, VR 621-2013-4231Swedish Foundation for Strategic Research , SSF ICA-6
    Available from: 2017-12-15 Created: 2017-12-15 Last updated: 2018-11-29
    4. A pneumatically assisted nanospray desorption electrospray ionization source for increased solvent versatility and enhanced metabolite detection from tissue
    Open this publication in new window or tab >>A pneumatically assisted nanospray desorption electrospray ionization source for increased solvent versatility and enhanced metabolite detection from tissue
    2017 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 142, no 18, p. 3424-3431Article in journal (Refereed) Published
    Abstract [en]

    Nanospray desorption electrospray ionization (nano-DESI) has been established as a powerful technique for mass spectrometry imaging (MSI) of biomolecules from tissue samples. The direct liquid extraction of analytes from a surface at ambient pressure negates the need for significant sample preparation or matrix application. Although many recent studies have applied nano-DESI to new and exciting applications, there has not been much work in the development and improvement of the nano-DESI source. Here, we incorporate a nebulizer to replace the self-aspirating secondary capillary in the conventional nano-DESI setup, and characterize the device by use of rat kidney tissue sections. We find that the pneumatically assisted nano-DESI device offers improved sensitivity for metabolite species by 1-3 orders of magnitude through more complete desolvation and reduced ionization suppression. Further, the pneumatically assisted nano-DESI device reduces the dependence on probe-to-surface distance and enables sampling and imaging using pure water as the nano-DESI solvent. This provides exclusive detection and imaging of many highly polar endogenous species. Overall, the developed pneumatically assisted nano-DESI device provides more versatile solvent selection and an increased sensitivity for metabolites, which generates ion images of higher contrast - allowing for more intricate studies of metabolite distribution.

    Place, publisher, year, edition, pages
    ROYAL SOC CHEMISTRY, 2017
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-334756 (URN)10.1039/c7an00901a (DOI)000409919200016 ()28828451 (PubMedID)
    Funder
    Swedish Foundation for Strategic Research , SSF ICA-6Swedish Research Council, VR 621-2013-4231
    Available from: 2017-11-27 Created: 2017-11-27 Last updated: 2018-11-29
  • 104. Bergman, Hilde-Marléne
    et al.
    Andersson, Ingela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Detection of endogenous lipids and metabolites in single cells using nano-DESIManuscript (preprint) (Other academic)
  • 105.
    Bergman, Hilde-Marléne
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lundin, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lanekoff, Ingela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization2016In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 12, p. 3686-3695Article in journal (Refereed)
    Abstract [en]

    Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease.

  • 106.
    Bergman, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ingvar Eidhammer, Harald Barsnes, Geir Egil Eide, and Lennart Martens:: Computational and statistical methods for protein quantification by mass spectrometry2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 6, p. 1575-1576Article, book review (Refereed)
  • 107.
    Bergman, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Recent developments in proteomic methods and disease biomarkers2014In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, p. 3836-3851Article in journal (Refereed)
    Abstract [en]

    Proteomic methodologies for identification and analysis of biomarkers have gained more attention during recent years and have evolved rapidly. Identification and detection of disease biomarkers are important to foresee outbreaks of certain diseases thereby avoiding surgery and other invasive and expensive medical treatments for patients. Thus, more research into discovering new biomarkers and new methods for faster and more accurate detection is needed. It is often difficult to detect and measure biomarkers because of their low concentrations and the complexity of their respective matrices. Therefore it is hard to find and validate methods for accurate screening methods suitable for clinical use. The most recent developments during the last three years and also some historical considerations of proteomic methodologies for identification and validation of disease biomarkers are presented in this review.

  • 108.
    Bergman, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Shevchenko, Denys
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Approaches for the analysis of low molecular weight compounds with laser desorption/ionization techniques and mass spectrometry2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 1, p. 49-61Article, review/survey (Refereed)
    Abstract [en]

    This review summarizes various approaches for the analysis of low molecular weight (LMW) compounds by different laser desorption/ionization mass spectrometry techniques (LDI-MS). It is common to use an agent to assist the ionization, and small molecules are normally difficult to analyze by, e.g., matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the common matrices available today, because the latter are generally small organic compounds themselves. This often results in severe suppression of analyte peaks, or interference of the matrix and analyte signals in the low mass region. However, intrinsic properties of several LDI techniques such as high sensitivity, low sample consumption, high tolerance towards salts and solid particles, and rapid analysis have stimulated scientists to develop methods to circumvent matrix-related issues in the analysis of LMW molecules. Recent developments within this field as well as historical considerations and future prospects are presented in this review.

  • 109.
    Bergman, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Thapper, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Styring, Stenbjorn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Shevchenko, Denys
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Quantitative determination of the Ru(bpy)(3)(2+) cation in photochemical reactions by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry2014In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, no 21, p. 8513-8518Article in journal (Refereed)
    Abstract [en]

    The coordination compound of Ru(II) with three 2,2'-bipyridine ligands possesses a potent photosensitization capacity for electron- and energy-transfer processes. In combination with salts of peroxydisulfate acid as sacrificial electron acceptors, Ru(bpy)(3)(2+) is widely used for photocatalytic oxidative transformations in organic synthesis and water splitting. The drawback of this system is that bipyridine degrades under the resulting strongly oxidative conditions, the concentration of Ru(bpy)(3)(2+) diminishes, and the photocatalytic reaction eventually stops. A commonly employed assay for the determination of Ru(bpy)(3)(2+), UV-Vis spectroscopy, has low selectivity and does not distinguish between the intact complex and its decayed forms. Here, we report a matrix assisted laser desorption/ionisation mass spectrometric method for the quantitative analysis of Ru(bpy)(3)(2+) in photochemical reaction mixtures. The developed method was successfully used for the determination of intact Ru(bpy)(3)(2+) during the course of the water photooxidation reaction. The significant difference between the results of MALDI MS and UV-Vis analyses was observed.

  • 110.
    Bergman, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Estrada, Sergio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Hall, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Rahman, Rashidur
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Blomgren, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Svedberg, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Thibblin, Alf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
    Wångsell, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Synthesis and Labelling of a Piperazine-Based Library of 11C-Labeled Ligands for Imaging of the Vesicular Acetylcholine Transporter2014In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 57, no 8, p. 525-532Article in journal (Refereed)
    Abstract [en]

    The cholinergic system is involved in neurodegenerative diseases, and visualization of cholinergic innervations with positron emission tomography (PET) would be a useful tool in understanding these diseases. A ligand for the vesicular acetylcholine transporter (VAChT), acknowledged as a marker for cholinergic neurons, could serve as such a PET tracer. The aim was to find a VAChT PET tracer using a library concept to create a small but diverse library of labeled compounds. From the same precursor and commercially available aryl iodides 6a-f, six potential VAChT PET tracers, [C-11]-(+/-)5a-f, were C-11-labeled by a palladium (0)-mediated aminocarbonylation, utilizing a standard protocol. The labeled compounds [C-11]-(+/-)5a-f were obtained in radiochemical purities >95% with decay-corrected radiochemical yields and specific radioactivities between 4-25% and 124-597 GBq/mu mol, respectively. Autoradiography studies were then conducted to assess the compounds binding selectivity for VAChT. Labeled compounds [C-11]-(+/-)5d and [C-11]-(+/-)5e showed specific binding but not enough to permit further preclinical studies. To conclude, a general method for a facile synthesis and labeling of a small piperazine-based library of potential PET tracers for imaging of VAChT was shown, and in upcoming work, another scaffold will be explored using this approach.

  • 111.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Proteomics to Understand the Degenerative Matter2014In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 75, p. S10-S10Article in journal (Other academic)
  • 112.
    Bergquist, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Baykut, Gökhan
    Bruker Daltonik GmbH, 28359 Bremen, Germany.
    Bergquist, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology.
    Witt, Matthias
    Bruker Daltonik GmbH, 28359 Bremen, Germany.
    Mayer, Franz-Josef
    Bruker Daltonik GmbH, 28359 Bremen, Germany.
    Baykut, Doan
    Institute of Biophysics, University of Frankfurt, 60438 Frankfurt/M, Germany.
    Human Myocardial Protein Pattern Reveals Cardiac Diseases2012In: International Journal of Proteomics, ISSN 2090-2174, Vol. 2012, p. 342659-Article in journal (Refereed)
    Abstract [en]

    Proteomic profiles of myocardial tissue in two different etiologies of heart failure were investigated using high performance liquid chromatography (HPLC)/Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Right atrial appendages from 10 patients with hemodynamically significant isolated aortic valve disease and from 10 patients with isolated symptomatic coronary heart disease were collected during elective cardiac surgery. As presented in an earlier study by our group (Baykut et al., 2006), both disease forms showed clearly different pattern distribution characteristics. Interesting enough, the classification patterns could be used for correctly sorting unknown test samples in their correct categories. However, in order to fully exploit and also validate these findings there is a definite need for unambiguous identification of the differences between different etiologies at molecular level. In this study, samples representative for the aortic valve disease and coronary heart disease were prepared, tryptically digested, and analyzed using an FT-ICR MS that allowed collision-induced dissociation (CID) of selected classifier masses. By using the fragment spectra, proteins were identified by database searches. For comparison and further validation, classifier masses were also fragmented and analyzed using HPLC-/Matrix-assisted laser desorption ionization (MALDI) time-offlight/time-of-flight (TOF/TOF) mass spectrometry. Desmin and lumican precursor were examples of proteins found in aortic samples at higher abundances than in coronary samples. Similarly, adenylate kinase isoenzyme was found in coronary samples at a higher abundance. The described methodology could also be feasible in search for specific biomarkers in plasma or serum for diagnostic purposes.

  • 113.
    Bergquist, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Turner, Charlotta
    Lund Univ, Dept Chem, Ctr Anal & Synth, Lund, Sweden..
    Analytical chemistry for a sustainable society - trends and implications2018In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 410, no 14, p. 3235-3237Article in journal (Other academic)
  • 114.
    Bergquist, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology.
    Huss, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Plastic Surgery.
    Hästbacka, Johanna
    Lindholm, Catharina
    Martijn, Cecile
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Rylander, Christian
    Hedenstierna, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Physiology.
    Fredén, Filip
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    Glucocorticoid receptor expression and binding capacity in patients with burn injury2016In: Acta Anaesthesiologica Scandinavica, ISSN 0001-5172, E-ISSN 1399-6576, Vol. 60, no 2, p. 213-221Article in journal (Refereed)
    Abstract [en]

    Background

    Burn injuries are associated with strong inflammation and risk of secondary sepsis which both may affect the function of the glucocorticoid receptor (GR). The aim of this study was to determine GR expression and binding capacity in leucocytes from patients admitted to a tertiary burn center.

    Methods

    Blood was sampled from 13 patients on admission and days 7, 14 and 21, and once from 16 healthy subjects. Patients were grouped according to the extent of burn and to any sepsis on day 7. Expression and binding capacity of GR were determined as arbitrary units using flow cytometry.

    Results

    GR expression and binding capacity were increased compared to healthy subjects in most circulating leucocyte subsets on admission irrespective of burn size. Patients with sepsis on day 7 displayed increased GR expression in T lymphocytes (51.8%, < 0.01) compared to admission. There was a negative correlation between GR binding capacity in neutrophils and burn size after 14 days (< 0.05).

    Conclusions

    GR expression and binding capacity are increased in most types of circulating leucocytes of severely burned patients on their admission to specialized burn care. If sepsis is present after 1 week, it is associated with higher GR expression in T lymphocytes and NK cells.

  • 115.
    Bergsaker, H.
    et al.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;KTH Royal Inst Technol, Sch Elect Engn, Dept Fus Plasma Phys, SE-10405 Stockholm, Sweden..
    Bykov, I.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;KTH Royal Inst Technol, Sch Elect Engn, Dept Fus Plasma Phys, SE-10405 Stockholm, Sweden..
    Zhou, Y.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;KTH Royal Inst Technol, Sch Elect Engn, Dept Fus Plasma Phys, SE-10405 Stockholm, Sweden..
    Petersson, P.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;KTH Royal Inst Technol, Sch Elect Engn, Dept Fus Plasma Phys, SE-10405 Stockholm, Sweden..
    Possnert, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, För teknisk-naturvetenskapliga fakulteten gemensamma enheter, Tandem Laboratory. EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England..
    Likonen, J.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;VTT Tech Res Ctr Finland, POB 1000, FI-02044 Espoo, Finland..
    Pettersson, Jean
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England..
    Koivuranta, S.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;VTT Tech Res Ctr Finland, POB 1000, FI-02044 Espoo, Finland..
    Widdowson, A. M.
    EUROfus Consortium, JET, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England.;CCFE, Culham Sci Ctr, Abingdon OX14 3DB, Oxon, England..
    Deep deuterium retention and Be/W mixing at tungsten coated surfaces in the JET divertor2016In: Physica Scripta, ISSN 0031-8949, E-ISSN 1402-4896, Vol. T167, article id 014061Article in journal (Refereed)
    Abstract [en]

    Surface samples from a full poloidal set of divertor tiles exposed in JET through operations 2010-2012 with ITER-like wall have been investigated using SEM, SIMS, ICP-AES analysis and micro beam nuclear reaction analysis (mu-NRA). Deposition of Be and retention of D is microscopically inhomogeneous. With careful overlaying of mu-NRA elemental maps with SEM images, it is possible to separate surface roughness effects from depth profiles at microscopically flat surface regions, without pits. With (He-3, p) mu-NRA at 3-5 MeV beam energy the accessible depth for D analysis in W is about 9 mu m, sufficient to access the W/Mo and Mo/W interfaces in the coatings and beyond, while for Be in W it is about 6 mu m. In these conditions, at all plasma wetted surfaces, D was found throughout the whole accessible depth at concentrations in the range 0.2-0.7 at% in W. Deuterium was found to be preferentially trapped at the W/Mo and Mo/W interfaces. Comparison is made with SIMS profiling, which also shows significant D trapping at the W/Mo interface. Mixing of Be and W occurs mainly in deposited layers.

  • 116.
    Bergstrand, Nill
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical Chemistry.
    Bohl, Erika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical Chemistry.
    Ghaneolhosseine, Hadi
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical Chemistry.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Jonsson, Markus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical Chemistry.
    Silvander, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical Chemistry.
    Sjöberg, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Stabilised Liposomes with Double Targeting for Use in BNCT2000In: Contemporary Boron Chemistry / [ed] Matthew Davidson, Cambridge, UK: Royal Society of Chemistry, 2000, p. 131-134Chapter in book (Other academic)
  • 117.
    Bergström, L. Magnus
    et al.
    Department of Chemistry, Surface, and Corrosion Science, School of Chemical Science and Engineering, KTH Royal Institute of Technology, SE-10044 Stockholm, Sweden.
    Skoglund, Sara
    Department of Chemistry, Surface, and Corrosion Science, School of Chemical Science and Engineering, KTH Royal Institute of Technology, SE-10044 Stockholm, Sweden.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Eriksson, Jonny
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Grillo, Isabelle
    Institut Laue Langevin, DS/LSS, 6 rue Jules Horowitz, BP156, 38042 Grenoble Cedex 9, France.
    Self-Assembly in Mixtures of an Anionic and a Cationic Surfactant: A Comparison between Small-Angle Neutron Scattering and Cryo-Transmission Electron Microscopy2013In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 29, no 38, p. 11834-11848Article in journal (Refereed)
    Abstract [en]

    The self-assembly in SOS-rich mixtures of the anionic surfactant sodium octyl sulfate (SOS) and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) has been investigated with the complementary techniques small-angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM). Both techniques confirm the simultaneous presence of open and closed bilayer structures in highly diluted samples as well as the existence of small globular and large elongated micelles at higher concentrations. However, the two techniques sometimes differ with respect to which type of aggregates is present in a particular sample. In particular, globular or wormlike micelles are sometimes observed with cryo-TEM in the vicinity of the micelle-to-bilayer transition, although only bilayers are present according to SANS and the samples appear bluish to the eye. A similar discrepancy has previously been reported but could not be satisfactorily rationalized. On the basis of our comparison between in situ (SANS) and ex situ (cryo-TEM) experimental techniques, we suggest that this discrepancy appears mainly as a result of the non-negligible amount of surfactant adsorbed at interfaces of the thin sample film created during the cryo-TEM specimen preparation. Moreover, from our detailed SANS data analysis, we are able to observe the unusually high amount of free surfactant monomers present in SOS-rich mixtures of SOS and CTAB, and the experimental results give excellent agreement with model calculations based on the Poisson?Boltzmann mean field theory. Our careful comparison between model calculations and experiments has enabled us to rationalize the dramatic microstructural transformations frequently observed upon simply diluting mixtures of an anionic and a cationic surfactant.

  • 118.
    Bergström, L. Magnus
    et al.
    School of Chemical Science and Engineering, Department of Chemistry, Surface and Corrosion Science, KTH Royal Institute of Technology, Sweden.
    Skoglund, Sara
    School of Chemical Science and Engineering, Department of Chemistry, Surface and Corrosion Science, KTH Royal Institute of Technology, Sweden.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Eriksson, Jonny
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Grillo, Isabelle
    Institut Laue Langevin, DS/LSS, 6 rue Jules Horowitz, B.P. 156, 38042 Grenoble Cedex 9, France.
    Spontaneous Transformations between Surfactant Bilayers of Different Topologies Observed in Mixtures of Sodium Octyl Sulfate and Hexadecyltrimethylammonium Bromide2014In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 30, no 14, p. 3928-3938Article in journal (Refereed)
    Abstract [en]

    The influence of adding salt on the self-assembly in sodium octyl sulfate (SOS)-rich mixtures of the anionic surfactant SOS and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been investigated with the two complementary techniques, small-angle neutron scattering (SANS) and cryo-transmission electron microscopy. We are able to conclude that addition of a substantial amount of inert salt, NaBr, mainly has three effects on the structural behaviors: (i) the micelles become much larger at the transition from micelles to bilayers, (ii) the fraction of bilayer disks increases at the expense of vesicles, and (iii) bilayer aggregates perforated with holes are formed in the most diluted samples. A novel form factor valid for perforated bilayer vesicles and disks is introduced for the first time and, as a result, we are able to directly observe the presence of perforated bilayers by means of fitting SANS data with an appropriate model. Moreover, we are able to conclude that the morphology of bilayer aggregates changes according to the following sequence of different bilayer topologies, vesicles ? disks ? perforated bilayers, as the electrolyte concentration is increased and surfactant mole fraction in the bilayer aggregates approaches equimolarity. We are able to rationalize this sequence of transitions as a result of a monotonous increase of the bilayer saddle-splay constant (k?cbi) with decreasing influence from electrostatics, in agreement with theoretical predictions as deduced from the Poisson?Boltzmann theory.

  • 119.
    Bergström Lind, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artemenko, Konstantin A
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elfineh, Lioudmila
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zhao, Yanhong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The phosphoproteome of the adenovirus type 2 virion2012In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 433, no 1, p. 253-261Article in journal (Refereed)
    Abstract [en]

    We have used a proteomics approach to identify sites of phosphorylation in the structural proteins of the Adenovirus type 2 particle. This protein modification might play an important role during infection. Peptides from highly purified virus were enriched for phosphorylations and analyzed by liquid chromatography-high-resolving mass spectrometry. Phosphorylations were identified in 11 structural peptides and 29 non-redundant phosphorylation sites were unambiguously assigned to specific amino acid. An unexpected result was the finding of phosphotyrosine in two of the viral polypeptides. The most highly phosphorylated protein was pIIIa with 12 identified phosphorylation sites. An identified preference for proline or leucine residue flanking the phosphorylation sites downstream suggests that cellular kinases are involved in many of the phosphorylations. Structural modeling showed that one site in the hexon is located on the outer side of the virus and could be of importance for the virus when attaching and entering cells.

  • 120.
    Bergström Lind, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Artemenko, Konstantin A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    A strategy for identification of protein tyrosine phosphorylation2012In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 56, no 2, p. 275-283Article in journal (Refereed)
    Abstract [en]

    To develop methods for studying phosphorylation of protein tyrosine residues is an important task since this protein modification regulates many cellular functions and often is involved in oncogenesis. An optimal protocol includes enrichment of tyrosine phosphorylated (pTyr) peptides or proteins, followed by a high resolving analytical method for identification of the enriched components. In this Methods paper, we describe a working strategy on how immunoaffinity enrichments, using anti-pTyr antibodies, combined with mass spectrometric (MS) analysis can be used to study the pTyr proteome. We describe in detail how our procedure was used to characterize the pTyr proteome of K562 leukemia cells. Important questions concerning the use of different anti-pTyr antibodies, enrichments performed at the peptide and/or the protein level, pooling of enrichments and requirements for the MS characterization are discussed.

  • 121. Bergvall, Ulrika A.
    et al.
    Co, Michelle
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Bergstrom, Roger
    Sjöberg, Per J. R.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Waldebäck, Monica
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Turner, Charlotta
    Anti-browsing effects of birch bark extract on fallow deer2013In: European Journal of Forest Research, ISSN 1612-4669, E-ISSN 1612-4677, Vol. 132, no 5-6, p. 717-725Article in journal (Refereed)
    Abstract [en]

    A major problem within forest industry is unwanted browsing on seedlings from mammalian herbivores. The aim of this study was to evaluate the effects of birch bark extracts as repellents towards fallow deer. Birch bark was extracted in a conventional way with ethanol as solvent at ambient temperature and with a new method, liquid CO2 extraction. An analysis of the ethanol-extracted birch bark showed that it contained large amounts of terpenoids, of which the most abundant was betulin. In seven different treatment trials, we used 15 individually handled fallow deer. To investigate the binary taste preferences, birch bark extract was added to food and presented in two bowls in typical two-choice tests. We found that the amount of a food type consumed during a trial and the number of shifts between food bowls were dependent on the amount of the birch extract the food contained. Concentrations of above 1 % by dry weight of birch extract acted as a repellent. In addition, such concentrations produced shorter feeding bouts by a greater willingness to change bowls. Therefore, our conclusion is that birch bark extract acts as a repellent towards fallow deer and is therefore likely to act as a repellent against other deer species. In addition, we show that birch bark extract produced by the new and more environmentally sustainable method employing liquid CO2 mixed with ethanol has the same repellent effect as the traditional ethanol extraction.

  • 122.
    Berndtson, Emma
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Qualitative analysis of LGD-4033 and its metabolites in equine plasma using UHPLC-MS(MS) for doping control purposes2017Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    A new class of drugs has been developed for treatment of muscle and bone mass wasting diseases called non-steroidal selective androgen receptor modulators (SARMs). Because of their positive androgenic effects such as muscle gain, they are desirable as performance enhancers.

    One of those substances is LGD-4033 (4-[(2R)-2-[(1R)-2,2,2-trifluoro-1-hydroxyethyl]pyrrolidin-1-yl]-2-(trifluoromethyl)- benzonitrile). It has been detected in human samples in routine doping control and another SARM has been detected in an equine blood sample in routine doping control. It is therefore indicated that SARMs need to be screened for in routine testing in equestrian sport.

    The aim of this project was to identify what metabolites were found in equine plasma after an intra venous administration of LGD-4033 using UHPLC coupled with QToF-MS and determine whether the parent compound or any of its metabolites were most suitable for doping control.

    With the sample preparation method protein precipitation, six possible metabolites were identified in samples from three horses. Two of the metabolites were identified as phase I-metabolites (monohydroxylated and dihydroxylated). Four of the metabolites were identified as phase II-metabolites, where glucuronidation had occurred.

    The most suitable species for doping control were determined based on a semi- quantification and were M1a, M2 and M3a. 

  • 123.
    Berrier, Audrey
    et al.
    Universität Stuttgart, Physikalisches Institut, Germany.
    Schaafsma, Martijn C.
    FOM Institute AMOLF, Centre for Nanophotonics, c/o Philips Research Laboratories, Eindhoven, Netherlands.
    Nonglaton, Guillaume
    CEA Leti, MINATEC Campus, Department of microtechnologies for Biology and Healthcare, France.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Rivas, Jaime Gómez
    FOM Institute AMOLF, Centre for Nanophotonics, c/o Philips Research Laboratories AND COBRA Research Institute, Eindhoven University of Technology, Netherlands .
    Selective detection of bacterial layers with terahertz plasmonic antennas2012In: Biomedical Optics Express, ISSN 2156-7085, E-ISSN 2156-7085, Vol. 3, no 11, p. 2937-2949Article in journal (Refereed)
    Abstract [en]

    Current detection and identification of micro-organisms is based on either rather unspecific rapid microscopy or on more accurate complex, time-consuming procedures. In a medical context, the determination of the bacteria Gram type is of significant interest. The diagnostic of microbial infection often requires the identification of the microbiological agent responsible for the infection, or at least the identification of its family (Gram type), in a matter of minutes. In this work, we propose to use terahertz frequency range antennas for the enhanced selective detection of bacteria types. Several microorganisms are investigated by terahertz time-domain spectroscopy: a fast, contactless and damage-free investigation method to gain information on the presence and the nature of the microorganisms. We demonstrate that plasmonic antennas enhance the detection sensitivity for bacterial layers and allow the selective recognition of the Gram type of the bacteria.

  • 124.
    Bertilsson, Sarah
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Glycanmapping of glycoproteins with UPLC-FLR-MALDI/TOF-MS2014Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
  • 125.
    Billinger, Erika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    An investigation of protective formulations containing enzyme inhibitors: Model experiments of trypsin2012Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS-­activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized. 

  • 126.
    Billinger, Erika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Light scattering determination of the stoichiometry for protease-potato serine protease inhibitor complexes2019In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 582, p. 113357-Article in journal (Refereed)
    Abstract [en]

    The interaction between pancreatic proteases and a serine protease inhibitor purified from potato tubers was investigated by chromatography-coupled light scattering measurements. The molar mass distribution in the chromatogram was compared to theoretical values calculated for the different possible combinations of complexes and free components by three different approaches, namely section analyses of the chromatograms, full mass average determination and mass distribution analysis. This revealed that the inhibitor was able to bind trypsin in a 2:1 complex, whereas the data for chymotrypsin clearly showed a limitation to 1:1 complex regardless of the molar ratio in the injected samples. The same experiment carried out with elastase and the potato inhibitor gave only weak indications of complex formation under the conditions used.

  • 127.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Characterization of Serine Protease Inhibitor from Solanum tuberosum Conjugated to Soluble Dextran and Particle Carriers2019In: ACS Omega, Vol. 4, p. 18456-18464Article, review/survey (Refereed)
    Abstract [en]

    A serine protease inhibitor was extracted from potato tubers. The inhibitor was conjugated to soluble, prefractionated dextran and titanium dioxide and zinc oxide nanoparticles. Conjugation to dextran was achieved by periodate oxidation of the dextran, followed by Schiff base coupling to inhibitor amino groups, and finally reduction, whereas the conjugation to the oxide particles was carried out by aminosilanization and carbonyldiimidazole activation. The inhibitory effect of the conjugated inhibitor was compared to that of free inhibitor in solution and with gelatin gel as a direct substrate. A certain degree of inhibitory activity was retained for both the dextran-conjugated and particle-conjugated inhibitors. In particular, the apparent Ki value of the dextran-conjugated inhibitor was found to be in the same range as that for free inhibitor. The dextran conjugate retained a higher activity than the free inhibitor after 1 month of storage at room temperature.

  • 128.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems: Diffusion through an inhibitor barrier2018In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 546, p. 43-49Article in journal (Refereed)
    Abstract [en]

    A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

  • 129.
    Biswas, Srijit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Dahlstrand, Christian
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Watile, Rahul A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Kalek, Marcin
    Himo, Fahmi
    Samec, Joseph S. M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Atom-Efficient Gold(I)-Chloride-Catalyzed Synthesis of alpha-Sulfenylated Carbonyl Compounds from Propargylic Alcohols and Aryl Thiols: Substrate Scope and Experimental and Theoretical Mechanistic Investigation2013In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 19, no 52, p. 17939-17950Article in journal (Refereed)
    Abstract [en]

    Gold(I)-chloride-catalyzed synthesis of -sulfenylated carbonyl compounds from propargylic alcohols and aryl thiols showed a wide substrate scope with respect to both propargylic alcohols and aryl thiols. Primary and secondary aromatic propargylic alcohols generated -sulfenylated aldehydes and ketones in 60-97% yield. Secondary aliphatic propargylic alcohols generated -sulfenylated ketones in yields of 47-71%. Different gold sources and ligand effects were studied, and it was shown that gold(I) chloride gave the highest product yields. Experimental and theoretical studies demonstrated that the reaction proceeds in two separate steps. A sulfenylated allylic alcohol, generated by initial regioselective attack of the aryl thiol on the triple bond of the propargylic alcohol, was isolated, evaluated, and found to be an intermediate in the reaction. Deuterium labeling experiments showed that the protons from the propargylic alcohol and aryl thiol were transferred to the 3-position, and that the hydride from the alcohol was transferred to the 2-position of the product. Density functional theory (DFT) calculations showed that the observed regioselectivity of the aryl thiol attack towards the 2-position of propargylic alcohol was determined by a low-energy, five-membered cyclic protodeauration transition state instead of the strained, four-membered cyclic transition state found for attack at the 3-position. Experimental data and DFT calculations supported that the second step of the reaction is initiated by protonation of the double bond of the sulfenylated allylic alcohol with a proton donor coordinated to gold(I) chloride. This in turn allows for a 1,2-hydride shift, generating the final product of the reaction.

  • 130.
    Biswas, Srijit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Samec, Joseph
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    A Gold(I)-Catalyzed Route to α-Sulfenylated Carbonyl Compounds from Propargylic Alcohols and Aryl Thiols2012In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 48, no 52, p. 6586-6588Article in journal (Refereed)
    Abstract [en]

    A one-step atom efficient gold(I)-catalyzed route to α-sulfenylated ketones and aldehydes from propargylic alcohols and aryl thiols is described.

  • 131.
    Biswas, Srijit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Samec, Joseph
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    The Efficiency of the Metal Catalysts in the Nucleophilic Substitution of Alcohols is Dependent on the Nucleophile and Not on the Electrophile2013In: Chemistry - An Asian Journal, ISSN 1861-4728, E-ISSN 1861-471X, Vol. 8, no 5, p. 974-981Article in journal (Refereed)
    Abstract [en]

    In this study, we investigate the effect of the electrophiles and the nucleophiles for eight catalysts in the catalytic SN1 type substitution of alcohols with different degree of activation by sulfur-, carbon-, oxygen-, and nitrogen-centered nucleophiles. The catalysts do not show any general variance in efficiency or selectivity with respect to the alcohols and follow the trend of alcohol reactivity. However, when it comes to the nucleophile, the eight catalysts show general and specific variances in the efficiency and selectivity to perform the desired substitution. Interestingly, the selectivity of the alcohols to produce the desired substitution products was found to be independent of the electrophilicity of the generated carbocations but highly dependent on the ease of formation of the cation. Catalysts based on iron(III), bismuth(III), and gold(III) show higher conversions for S-, C-, and N-centered nucleophiles, and BiIII was the most efficient catalyst in all combinations. Catalysts based on rhenium(I) or rhenium(VII), palladium(II), and lanthanum(III) were the most efficient in performing the nucleophilic substitution on the various alcohols with the O-centered nucleophiles. These catalysts generate the symmetrical ether as a by-product from the reactions of S-, C-, and N-centered nucleophiles as well, resulting in lower chemoselectivity.

  • 132.
    Biswas, Srijit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Samec, Joseph S. M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Gold-catalyzed route to alpha-sulfenylated carbonyl compounds from propargylic alcohols and thiophenol: Scope, limitations, and mechanism2013In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 245, p. 382-ORGN-Article in journal (Other academic)
  • 133.
    Biswas, Srijit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Watile, Rahul A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Samec, Joseph S. M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Synthetical Organic Chemistry.
    Tandem Pd/Au-Catalyzed Route to alpha-Sulfenylated Carbonyl Compounds from Terminal Propargylic Alcohols and Thiols2014In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 20, no 8, p. 2159-2163Article in journal (Refereed)
    Abstract [en]

    An efficient and highly atom-economical tandem Pd/Au-catalyzed route to -sulfenylated carbonyl compounds from terminal propargylic alcohols and thiols has been developed. This one-step procedure has a wide substrate scope with respect to substituents at the -position of the alcohol. Both aromatic and aliphatic thiols generated the -sulfenylated carbonyl products in good to excellent yields. A mechanism is proposed in which the reaction proceeds through a Pd-catalyzed regioselective hydrothiolation at the terminal triple bond of the propargyl alcohol followed by an Au-catalyzed 1,2-hydride migration.

  • 134.
    Bivehed, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Strömvall, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bakalkin, Georgy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry2017In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 87, p. 20-27Article in journal (Refereed)
    Abstract [en]

    Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region-and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections. (C) 2016 The Authors. Published by Elsevier Inc.

  • 135.
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Oxidation of 1,2-Diols Using Alcohol Dehydrogenases: From Kinetic Characterization to Directed Evolution2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The use of enzymes as catalysts for chemical transformations has emerged as a “greener” alternative to traditional organic synthesis. An issue to solve though, is that enzymes are designed by nature to catalyze reactions in a living cell and therefore, in many cases, do not meet the requirements of a suitable biocatalyst. By mimicking Darwinian evolution these problems can be addressed in vitro by different types of directed evolution strategies.

    α-Hydroxy aldehydes and α-hydroxy ketones are important building blocks in the synthesis of natural products, fine chemicals and pharmaceuticals. In this thesis, two alcohol dehydrogenases, FucO and ADH-A, have been studied. Their potentials to serve as useful biocatalysts for the production of these classes of molecules have been investigated, and shown to be good. FucO for its strict regiospecificity towards primary alcohols and that it strongly prefers the S-enantiomer of diol substrates. ADH-A for its regiospecificity towards secondary alcohols, its enantioselectivity and that is has the ability to use a wide variety of bulky substrates. The kinetic mechanisms of these enzymes were investigated using pre-steady state kinetics, product inhibition, kinetic isotope effects and solvent viscosity effects, and in both cases, the rate limiting steps were pin-pointed to conformational changes occurring at the enzyme-nucleotide complex state. These characterizations provide an important foundation for further studies on these two enzymes.  

    FucO is specialized for activity with small aliphatic substrates but is virtually inactive with aryl-substituted compounds. By the use of iterative saturation mutagenesis, FucO was re-engineered and several enzyme variants active with S-3-phenylpropane-1,2-diol and phenylacetaldehyde were obtained. It was shown that these variants capability to act on larger substrates are mainly due to an enlargement of the active site cavity. Furthermore, several amino acids which are important for catalysis and specificity were identified. Phe254 interacts with aryl-substituted substrates through π-π stacking and may be essential for activity with these larger substrates. One mutation caused a loss in the interactions made between the enzyme and the nucleotide and thereby enhanced the turnover number for the preferred substrate

    List of papers
    1. Functional characterization of a stereospecific diol dehydrogenase, FucO, from Escherichia coli: substrate specificity, pH dependence, kinetic isotope effects and influence of solvent viscosity
    Open this publication in new window or tab >>Functional characterization of a stereospecific diol dehydrogenase, FucO, from Escherichia coli: substrate specificity, pH dependence, kinetic isotope effects and influence of solvent viscosity
    2010 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 66, no 1-2, p. 148-155Article in journal (Refereed) Published
    Abstract [en]

    FucO, (S)-1,2-propanediol oxidoreductase, from Escherichia coli is involved in the anaerobic catabolic metabolism of L-fucose and L-rhamnose, catalyzing the interconversion of lactaldehyde to propanediol. The enzyme is specific for the S-enantiomers of the diol and aldehyde suggesting stereospecificity in catalysis. We have studied the enzyme kinetics of FucO with a spectrum of putative alcohol and aldehyde substrates to map the substrate specificity space. Additionally, for a more detailed analysis of the kinetic mechanism, pH dependence of catalysis, stereochemistry in hydride transfer, deuterium kinetic isotope effect of hydride transfer and effect of increasing solvent viscosity were also analyzed. The outcome of this study can be summarized as follows: FucO is highly stereospecific with the highest E-value measured to be 320 for the S-enantiomer of 1,2-propanediol. The enzyme is strictly regiospecific for oxidation of primary alcohols. The enzyme prefers short-chained (2-4 carbons) substrates and does not act on bulkier compounds such as phenyl-substituted alcohols. FucO is an 'A-side' dehydrogenase transferring the pro-R-hydrogen of NADH to the aldehyde substrate. The deuterium KIEs of kcat and kcat/KM were 1.9 and 4.2, respectively, illustrating that hydride transfer is partially rate-limiting but also that other reaction steps contribute to rate limitation of catalysis. Combining the KIE results with the observed effects of increasing medium viscosity proposed a working model for the kinetic mechanism involving slow, rate-limiting, product release and on-pathway conformational changes in the enzyme-nucleotide complexes.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-123057 (URN)10.1016/j.molcatb.2010.04.010 (DOI)000280928200020 ()
    Available from: 2010-04-22 Created: 2010-04-22 Last updated: 2017-12-12Bibliographically approved
    2. Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
    Open this publication in new window or tab >>Kinetic characterization of Rhodococcus ruber DSM 44541 alcohol dehydrogenase A
    2014 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 99, p. 68-78Article in journal (Refereed) Published
    Abstract [en]

    An increasing interest in biocatalysis and the use of stereoselective alcohol dehydrogenases in synthetic asymmetric catalysis motivates detailed studies of potentially useful enzymes such as alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber. This enzyme is capable of catalyzing enantio-, and regioselective production of phenyl-substituted α-hydroxy ketones (acyloins) which are precursors for the synthesis of a range of biologically active compounds. In this study, we have determined the enzyme activity for a selection of phenyl-substituted vicinal diols and other aryl- or alkyl-substituted alcohols and ketones. In addition, the kinetic mechanism for the oxidation of (R)- and (S)-1-phenylethanol and the reduction of acetophenone has been identified as an Iso Theorell-Chance (hit and run) mechanism with conformational changes of the enzyme-coenzyme binary complexes as rate-determining for the oxidation of (S)-1-phenylethanol and the reduction of acetophenone. The underlying cause of the 270-fold enantiopreference for the (S)-enantiomer of 1-phenylethanol has been attributed to non-productive binding of the R-enantiomer. We have also shown that it is possible to tune the direction of the redox chemistry by adjusting pH with the oxidative reaction being favored at pH values above 7.

    Keywords
    alcohol dehydrogenase, kinetic mechanism, pre-steady state kinetics, product inhibition
    National Category
    Other Chemistry Topics Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-207474 (URN)10.1016/j.molcatb.2013.10.023 (DOI)000331340500010 ()
    Available from: 2013-09-15 Created: 2013-09-15 Last updated: 2017-12-06Bibliographically approved
    3.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    4. Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution
    Open this publication in new window or tab >>Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution
    2014 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 10, p. 2387-2398Article in journal (Refereed) Published
    Abstract [en]

    We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single muta