An Adaptable Phospholipid Membrane Mimetic System for Solution NMR Studies of Membrane Proteins.

Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy

Cryo-EM studies of class A GPCRs: the thyroid-stimulating hormone receptor and the β1-adrenoceptor

G protein-coupled receptors (GPCRs) are integral membrane proteins with seven transmembrane domains. They are essential in intercellular communication in human physiology and represent important drug targets in human disease. While methodological advances, particularly in cryogenic electron microscopy (cryo-EM), have made more GPCRs amenable to structural studies, they remain challenging targets. During this project, the thyroid-stimulating hormone receptor (TSHR) and the β1-adrenoceptor (β1AR) were studied. The TSHR is a class A GPCR that belongs to the family of glycoprotein hormone receptors. It carries a large extracellular domain (ECD) that binds to the endogenous ligand, the thyroid stimulating hormone (TSH). In autoimmune diseases, antibodies bind to the ECD and can act as either agonists or antagonists. The aim was to investigate the structure of the previously uncharacterised full-length TSHR and gain insight into the activation mechanism. A lentivirus-based expression system for the TSHR in mammalian cells to produce sufficient quantities for structural studies by cryo-EM was developed. The purification conditions were optimised, so that the TSHR remained stable throughout the purification. Antibody fragments that activate the TSHR were purified. These components were used to assemble a stable active-state complex of antibody fragment, TSHR and heterotrimeric G protein. Vitrification of the complex was optimised, cryo-EM data were collected, and a low-resolution structure was determined. The β1AR is a class A GPCR, bearing little homology to the TSHR, which has been intensely studied and is structurally well characterised. The β1AR is of particular importance in the treatment of heart disease by beta blockers (receptor antagonists). However, current β1AR antagonists have off-target effects on the β2AR that worsens the symptoms of respiratory diseases. Novel, highly β1AR-selective antagonists have been developed, but the structural basis for their selectivity is unknown. Using these ligands, a humanised turkey β1AR was expressed and purified in complex with antibody fragments to increase the size of the complex and make it amenable to cryo-EM studies was. By using a new grid type, all-gold HexAuFoil grids, cryo-EM data of this complex could be collected and analysed.School of Clinical Medicine; MRC Laboratory of Molecular Biolog

Redox control in cancer development and progression

Cancer is the leading cause of death worldwide after cardiovascular diseases. This has been the case for the last few decades despite there being an increase in the number of cancer treatments. One reason for the apparent lack of drug effectiveness might be, at least in part, due to unspecificity for tumors; which often leads to substantial side effects. One way to improve the treatment of cancer is to increase the specificity of the treatment in accordance with the concept of individualized medicine. This will help to prevent further progression of an existing cancer or even to reduce the tumor burden. Alternatively it would be much more attractive and efficient to prevent the development of cancer in the first place. Therefore, it is important to understand the risk factors and the mechanisms of carcinogenesis in detail. One such risk factor, often associated with tumorigenesis and tumor progression, is an increased abundance of reactive oxygen species (ROS) arising from an imbalance of ROS-producing and -eliminating components. A surplus of ROS can induce oxidative damage of macromolecules including proteins, lipids and DNA. In contrast, ROS are essential for an adequate signal transduction and are known to regulate crucial cellular processes like cellular quiescence, differentiation and even apoptosis. Therefore, regulated ROS-formation at physiological levels can inhibit tumor formation and progression. With this review we provide an overview on the current knowledge of redox control in cancer development and progression

Public policy and hydroelectric development in the Canadian North : the case of the Snare Forks Project

This study of the Snare Forks hydroelectric development in the Northwest Territories has two basic objectives. The first is to provide a critical assessment of the institutional framework (both structural and procedural) within which resource planning decisions are taken in the Canadian North, with reference to the cirteria of 1) democratic accountability, and 2) technical and economic efficiency. The second objective is to suggest (if shortcomings in institutional design are found) means of upgrading structures and procedures to conform with the normative criteria. The Snare Forks (or Strutt Lake) hydroelectric development was first conceived during 1971, when the Northern Canada Power Commission (NCPC) began to consider construction of a third hydro dam on the Snare River, which flows into the north arm of Great Slave Lake, to meet the growing demands of the City of Yellowknife and adjacent mining operations, 130 kilometres to the southwest. A water use licence, required under the Northern Inland Waters Act, was obtained by NCPC after one public hearing before the Northwest Territories Water Board in February 1974. Construction commenced during the spring of that year. However, shortly afterwards the commission was notified of a mining claim existing within the area designated to be flooded. It also became evident that bedrock and permafrost conditions at the site would not support the dyke structures. In September, 1974, the commission decided to relocate the dam 1.4 kilometres downstream, away from the mining claim area, and at a lower elevation (173.5 m above sea level, as opposed to 183m), to eliminate the need for dyking on permafrost. Peak power output was thus reduced from 14 megawatts to 9.6 megawatts. An amendment to the original water licence facilitating the design changes was approved by the Water Board in March, 1975. This was done without the normally required (under NIWA) public hearings, as the Board ruled that an emergency existed. It was not until after the amendment was approved that a press release was issued revealing the construction problems to the general public. The Snare Forks plant was commissioned in November, 1976, one year behind schedule. The final cost of the project was $27.1 million, as opposed to the original estimate of$14.1 million. As a consequence of the Snare Forks cost overruns, electric power rates in Yellowknife and other Northwest Territories communities were immediately raised by as much as ninety per cent. The account of the Snare Forks job history and planning process is based almost entirely on primary sources, including documents and correspondence of the Department of Indian Affairs and Northern Development, Environment Canada, the Northern Canada Power Commission, and the Northwest Territories Water Board. The criteria against which the decision-making system is assessed are derived from the survey of the leading contemporary literature on public administration and organizational behaviour, with an attempt to relate the theories thus obtained to the conditions prevalent in the Canadian North. It appears that circumstances over which decision-makers concerned had no direct control, particularly inflation and adverse weather conditions during construction, contributed in a major way to cost overruns. However, a review of the administrative and planning process reveals a failure by NCPC to adequately take into account possibilities regarding design and scheduling, as well as a closed, secretive decision-making process that effectively excluded local community interests. The failure of existing institutional mechanisms to prevent serious conceptual flaws in a public project and secrecy in decisionmaking gave rise to a widespread sense of alienation and mistrust among Snare system consumers in the aftermath of the project. At the same time, the project history points to a number of social and economic conditions present in the North that act as barriers to the attainment of optimal levels of democratic accountability and technical/economic efficiency in decision-making; and cannot be fully addressed by any set of institutional prescriptions.Arts, Faculty ofPolitical Science, Department ofGraduat

The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes

The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes

Syntheses of the sulfoconjugated isomers of norepinephrine and dopamine, controlled by HPLC with ultraviolet detection

The physiological significance of sulfoconjugated catecholamines and their involvement in clinical disorders, e.g. hypertension and Parkinsonism, is poorly investigated. For this reason, the sulfoconjugated isomers of dopamine as well as of norepinephrine were synthesized by modified methods. All isomers and their intermediates could be detected by a reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC-UV) with short retention times and a good reproducibility. Ion-exchange chromatography with an extended column length improved the separation of the reaction products, and the immediate control by HPLC-UV enabled precise cutting of the fractions. The selection of the fractions with the optimum ratios of product/by-product resulted in improved yields and highest purity. All by-products, e.g. dopamine sulfonic acids, were less than 0.04%, as detected by HPLC-U V and, in addition, the contamination by free catecholamines was only 41×10−4 -87×104%, as measured by HPLC with electrochemical detection (HPLC-ED). The purity was further demonstrated in two highly sensitive biological assays: cAMP production in human mononuclear leukocytes and aggregation of human platelets. The sulfoconjugated catecholamines were characterized by melting point, thin-layer chromatography, infrared spectrum, HPLC-UV, elemental analysis, and unequivocally identified by 1H-NMR