| 1. |
- Backmark, Anna, 1979, et al.
(författare)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 2. |
- Agemark, Maria, et al.
(författare)
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Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.
- 2012
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2736. ; 1818:3, s. 839-850
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Tidskriftsartikel (refereegranskat)abstract
- Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of one human aquaporin (HsAQP5) has been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.
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| 3. |
- Ampah-Korsah, Henry, et al.
(författare)
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The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain
- 2016
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JUNE2016
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.
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| 4. |
- Austeng, Dordi, et al.
(författare)
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Incidence of and risk factors for neonatal morbidity after active perinatal care : extremely preterm infants study in Sweden (EXPRESS)
- 2010
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Ingår i: Acta Paediatrica. - : Wiley. - 0803-5253 .- 1651-2227. ; 99:7, s. 978-992
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Tidskriftsartikel (refereegranskat)abstract
- Aims: The aim of this study was to determine the incidence of neonatal morbidity in extremely preterm infants and to identify associated risk factors. Methods: Population based study of infants born before 27 gestational weeks and admitted for neonatal intensive care in Sweden during 2004-2007. Results: Of 638 admitted infants, 141 died. Among these, life support was withdrawn in 55 infants because of anticipation of poor long-term outcome. Of 497 surviving infants, 10% developed severe intraventricular haemorrhage (IVH), 5.7% cystic periventricular leucomalacia (cPVL), 41% septicaemia and 5.8% necrotizing enterocolitis (NEC); 61% had patent ductus arteriosus (PDA) and 34% developed retinopathy of prematurity (ROP) stage >= 3. Eighty-five per cent needed mechanical ventilation and 25% developed severe bronchopulmonary dysplasia (BPD). Forty-seven per cent survived to one year of age without any severe IVH, cPVL, severe ROP, severe BPD or NEC. Tocolysis increased and prolonged mechanical ventilation decreased the chances of survival without these morbidities. Maternal smoking and higher gestational duration were associated with lower risk of severe ROP, whereas PDA and poor growth increased this risk. Conclusion: Half of the infants surviving extremely preterm birth suffered from severe neonatal morbidities. Studies on how to reduce these morbidities and on the long-term health of survivors are warranted.
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| 5. |
- Bagdonaite, I., et al.
(författare)
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A Strategy for O-Glycoproteomics of Enveloped Viruses-the O-Glycoproteome of Herpes Simplex Virus Type 1
- 2015
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Ingår i: Plos Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Glycosylation of viral envelope proteins is important for infectivity and interaction with host immunity, however, our current knowledge of the functions of glycosylation is largely limited to N-glycosylation because it is difficult to predict and identify site-specific O-glycosylation. Here, we present a novel proteome-wide discovery strategy for O-glycosylation sites on viral envelope proteins using herpes simplex virus type 1 (HSV-1) as a model. We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins. Two of the identified glycosites found in glycoprotein B were previously implicated in virus attachment to immune cells. We show that HSV-1 infection distorts the secretory pathway and that infected cells accumulate glycoproteins with truncated O-glycans, nonetheless retaining the ability to elongate most of the surface glycans. With the use of precise gene editing, we further demonstrate that elongated O-glycans are essential for HSV-1 in human HaCaT keratinocytes, where HSV-1 produced markedly lower viral titers in HaCaT with abrogated O-glycans compared to the isogenic counterpart with normal O-glycans. The roles of O-linked glycosylation for viral entry, formation, secretion, and immune recognition are poorly understood, and the O-glycoproteomics strategy presented here now opens for unbiased discovery on all enveloped viruses.
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| 6. |
- Bengtsson, Stefan, et al.
(författare)
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Mind the gap! Moving from awareness to action : Showcasing emergent research from the Swedish Graduate School in Education for Sustainable Development (GRESD)
- 2015
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Ingår i: Abstract list of WEEC 2015. - : WEEC.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: The main purpose of the symposium is to showcase some recent research findings produced by PhD students accepted by or affiliated with the Swedish Graduate School in Education for Sustainable development (GRESD). Objectives: GRESD started as a state sponsored one-time research capacity development project that accepted 9 post-graduate student and included additional 9 post-graduate students all focusing on ESD in their PhD projects. With the project coming to an end and having produced a number of dissertations targeting an international research audience, it is the intention to showcase some of the central contributions made and to receive feedback on from practitioners and researchers on how existent research projects can tie into and contribute to existent demands in environmental education (EE) practice and practice. The presentations of research results are aimed to cover a wide range of issues, including topics such as evaluation of classroom practices, students qualifications, globalization and teachers’ ethical reflections the role of place-specific artifacts in learning. As GRESD is a collaboration between eight universities with their specific traditions and approaches to educational research, approaches show a creative variety of theoretical backgrounds. This variation is also reflected in the presentations that are putting into play Lacanian psychoanalysis, discourse theory, pragmatist theory and phenomenography in order to shed new light on critical areas of environmental education. Methods: The symposium will consist of an introduction (10 minutes) brief presentations (10-15 minutes each) of central research findings in the context of their overarching research projects, followed by a synthesis and suggestions by a selected commentator (20 minutes) and general discussions with the audience (20 minutes). The dialogue following the presentations is intended to outline possible future research projects as well as emerging areas topics in the portrayed GRESD research that could feed into existing demands in EE practice and research.
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| 7. |
- Elfving, Kristina, et al.
(författare)
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Pneumococcal concentration and serotype distribution in preschool children with radiologically confirmed pneumonia compared to healthy controls prior to introduction of pneumococcal vaccination in Zanzibar : an observational study
- 2022
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Ingår i: BMC Infectious Diseases. - : BioMed Central (BMC). - 1471-2334. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The World Health Organization recommends pneumococcal vaccination (PCV) in the first year of life. We investigated pneumococcal serotypes in children with clinical or radiologically confirmed pneumonia and healthy controls prior to PCV13 vaccine introduction in Zanzibar. Methods: Children (n = 677) with non-severe acute febrile illness aged 2-59 months presenting to a health centre in Zanzibar, Tanzania April-July 2011 were included. Nasopharyngeal swabs collected at enrolment were analysed by real-time PCR to detect and quantify pneumococcal serotypes in patients (n = 648) and in healthy asymptomatic community controls (n = 161). Children with clinical signs of pneumonia according to the Integrated Management of Childhood illness guidelines ( "IMCI pneumonia ") were subjected to a chest-X-ray. Consolidation on chest X-ray was considered "radiological pneumonia ". Results: Pneumococcal DNA was detected in the nasopharynx of 562/809 (69%) children (70% in patients and 64% in healthy controls), with no significant difference in proportions between patients with or without presence of fever, malnutrition, IMCI pneumonia or radiological pneumonia. The mean pneumococcal concentration was similar in children with and without radiological pneumonia (Ct value 26.3 versus 27.0, respectively, p = 0.3115). At least one serotype could be determined in 423 (75%) participants positive for pneumococci of which 33% had multiple serotypes detected. A total of 23 different serotypes were identified. One serotype (19F) was more common in children with fever (86/648, 13%) than in healthy controls (12/161, 7%), (p = 0.043). Logistic regression adjusting for age and gender showed that serotype 9A/V [aOR = 10.9 (CI 2.0-60.0, p = 0.006)] and 14 [aOR = 3.9 (CI 1.4-11.0, p = 0.012)] were associated with radiological pneumonia. The serotypes included in the PCV13 vaccine were found in 376 (89%) of the 423 serotype positive participants. Conclusion: The PCV13 vaccine introduced in 2012 targets a great majority of the identified serotypes. Infections with multiple serotypes are common. PCR-determined concentrations of pneumococci in nasopharynx were not associated with radiologically confirmed pneumonia.
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| 8. |
- Fant, Kristina, 1979, et al.
(författare)
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DNA condensation by PAMAM dendrimers: Self-assembly characteristics and effect on transcription
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 47:6, s. 1732-1740
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Tidskriftsartikel (refereegranskat)abstract
- Electrostatic shielding and steric blocking by histones are two significant factors that participate in the control of the local rates of transcription in chromatin. As a simple model system to determine how the degree of DNA condensation affects enzyme accessibility and gene expression, we have used generation 5 polyamidoamine (G5 PAMAM) cationic dendrimer particles (size 5.4 nm) as a synthetic histone model together with an in vitro transcription assay. The degree of compaction, conformation, and binding availability of the dendrimer-DNA complexes is characterized by linear and circular dichroism, dynamic light scattering, and competitive binding of ethidium. Using ultracentrifugation we are able to show explicitly, for the first time, that dendrimer particles bind to DNA in a highly cooperative manner, and that the dendrimer-induced condensation of the DNA strongly attenuates transcription. Two fractions with different properties can be identified: a low-density fraction which behaves very similar to uncondensed DNA and a high-density fraction which is condensed to a high extent and where binding availability and transcription are strongly reduced. Circular dichroism gives clues to the structure of the condensed DNA indicating long-range order between the helices such as in polymer-salt-induced cholesteric liquid crystalline domains, one possible shape being a toroidal structure. On the basis of the experimental data, we propose a model for the self-assembly of the dendrimer-DNA system.
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| 9. |
- Fant, Kristina, 1979, et al.
(författare)
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Effects of PEGylation and acetylation of PAMAM dendrimers on DNA binding, cytotoxicity and in vitro transfection efficiency
- 2010
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8392 .- 1543-8384. ; 7:5, s. 1734-1746
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Tidskriftsartikel (refereegranskat)abstract
- Poly(amidoamine) (PAMAM) dendrimers are promising multipotent gene delivery vectors, providing favourable DNA condensation properties also in combination with the possibility of conjugation of different targeting ligands to their surface. They have been used for transfection both in vitro and in vivo, but their application is currently somewhat limited due to inherent cytotoxicity. In this work we investigate how two types of surface modification, acetylation and PEGylation, affect the DNA binding characteristics, the cytotoxicity and the in vitro transfection efficiency of generation 4 and 5 PAMAM dendrimers. Particularly, we address how the morphology of DNA-dendrimer complexes, formed under low salt conditions, changes upon dilution in cell growth medium, an event that inevitably occurs before the complexes reach the cell surface in any transfection experiment. We find that acetylation and PEGylation essentially eliminates the inherent dendrimer cytotoxicity. However, the transfection efficiency of the modified dendrimers is lower than that of the corresponding unmodified dendrimers, which can be rationally understood by our observations that DNA is less condensed when complexed with these modified dendrimers. Although small DNA-dendrimer particles are formed, the availability for ethidium intercalation and nuclease degradation is significantly higher in the modified DNA-dendrimer complexes than in unmodified ones. Dilution in cell growth medium has a drastic effect on these electrostatically assembled complexes, resulting in increase in size and DNA availability. Our results strongly add to the notion that it is of importance to perform a biophysical characterization under conditions as close to the transfection situation as possible, to enable conclusions regarding structure- activity relations of gene delivery vectors.
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| 10. |
- Fant, Kristina, 1979, et al.
(författare)
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Using Ethidium To Probe Nonequilibrium States of DNA Condensed for Gene Delivery
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 50:7, s. 1125-1127
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Tidskriftsartikel (refereegranskat)abstract
- Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic equilibrium. Moreover, dendrimer surface modification through PEGylation appears to make the interaction with DNA more reversible, which is favorable from the perspective of vector unpacking. Probing the nonequilibrium state of DNA during condensation processes is thus important for developing novel vectors, and further, it could also be useful in the study of chromatin folding.
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