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  • 1.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Strålin, Kristoffer
    Department of Infectious Diseases, Örebro University Hospital.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Olcén, Per
    Department of Clinical Microbiology, Örebro University Hospital.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction2009In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 64, no 4, p. 366-373Article in journal (Refereed)
    Abstract [en]

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  • 2.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Homologous recombination between the tuf genes of Salmonella typhimurium1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 260, no 4, p. 506-522Article in journal (Refereed)
    Abstract [en]

    The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichiacoli suggest that within each species the two tuf genes are evolving inconcert. Here we show that each of the S. typhimurium tuf genes cantransfer genetic information to the other. In our genetic system thetransfers are seen as non-reciprocal, i.e. as gene conversion events.However, the mechanism of recombination could be reciprocal, with sisterchromosome segregation and selection leading to the isolation of aparticular class of recombinant. The amount of sequence informationtransferred in individual recombination events varies, but can be close tothe entire length of the gene. The recombination is RecABCD-dependent,and is opposed by MutSHLU mismatch repair. In the wild-type, this typeof recombination occurs at a rate that is two or three orders of magnitudegreater than the nucleotide substitution rate. The rate of recombinationdiffers by six orders of magnitude between a recA and a mutS strain.Mismatch repair reduces the rate of this recombination 1000-fold. The rateof recombination also differs by one order of magnitude depending onwhich tuf gene is donating the sequence selected for. We discuss threeclasses of model that could, in principle, account for the sequencetransfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocalrecombination involving sister chromosomes; (3) non-allelic geneconversion involving sister chromosomes, initiated by a double-strandbreak close to one tuf gene. Although the mechanism remains to bedetermined, the effect on the bacterial cells is tuf gene sequencehomogenisation. This recombination phenomenon can account for theconcerted evolution of the tuf genes.

  • 3.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu.GTP1994In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 352, p. 118-122Article in journal (Refereed)
    Abstract [en]

    The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis.We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at sevendifferent sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu’GTP and EF-Tu.GDP. In the active GTP form of EF-Tu themutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

  • 4.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Tuohy, TMF
    Buckingham, RH
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Missense substitutions lethal to essential functions of EF-Tu1991In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 73, no 12, p. 1457-1464Article in journal (Refereed)
    Abstract [en]

    We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.

  • 5.
    Alberti, Jean-Christophe
    et al.
    Univ Corse, Lab Biochim & Biol Mol Vegetales, CNRS SPE UMR6134, Campus Grimaldi,BP52, F-20250 Corte, France.;Univ Toulouse, INSA, UPS, INP,LISBP, 135 Ave Rangueil, F-31077 Toulouse, France..
    Mariani, Magali
    Univ Corse, Lab Biochim & Biol Mol Vegetales, CNRS SPE UMR6134, Campus Grimaldi,BP52, F-20250 Corte, France..
    de Caraffa, Virginie Brunini-Bronzini
    Univ Corse, Lab Biochim & Biol Mol Vegetales, CNRS SPE UMR6134, Campus Grimaldi,BP52, F-20250 Corte, France..
    Gambotti, Claude
    Univ Corse, Lab Biochim & Biol Mol Vegetales, CNRS SPE UMR6134, Campus Grimaldi,BP52, F-20250 Corte, France..
    Oliw, Ernst H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Berti, Liliane
    Univ Corse, Lab Biochim & Biol Mol Vegetales, CNRS SPE UMR6134, Campus Grimaldi,BP52, F-20250 Corte, France..
    Maury, Jacques
    Univ Corse, Lab Biochim & Biol Mol Vegetales, CNRS SPE UMR6134, Campus Grimaldi,BP52, F-20250 Corte, France..
    A functional role identified for conserved charged residues at the active site entrance of lipoxygenase with double specificity2016In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 123, p. 167-173Article in journal (Refereed)
    Abstract [en]

    Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of free polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and alpha-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13- hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the "double specificity" character of these LOXs. In this study, we used the olive LOX1 with double specificity to investigate the implication of the charged residues R265, R268, and K283 in the orientation of the substrate into the active site. These residues are present in a conserved pattern around the entrance of the active site. Our results show that these residues are involved in the penetration of the substrate into the active site: this positive patch could capture the carboxylate end of the substrate, and then guide it into the active site. Due to its position on alpha 2 helix, the residue K283 could have a more important role, its interaction with the substrate facilitating the motions of residues constituting the "cork of lipoxygenases" or the alpha 2 helix, by disrupting putative hydrogen and ionic bonds.

  • 6.
    Altman, S., Kirsebom, L.A.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. MIKROBIOLOGI.
    Ribonuclease P1999In: RNA World (second edition), Cold Spring Harbor Press, NY , 1999, p. 351-Chapter in book (Other scientific)
  • 7.
    Altuvia, S.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wagner, E.G.H.
    Switching on and off with RNA.2000In: Proc. Natl. Acad. Sci. USA, Vol. 97, no 18, p. 9824-9826Article in journal (Refereed)
  • 8.
    Anagandula, Mahesh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Studies of Enterovirus Infection and Induction of Innate Immunity in Human Pancreatic Cells2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Several epidemiological and clinical studies have indicated a possible role of Enterovirus (EV) infection in type 1 diabetes (T1D) development. However, the exact casual mechanism of these viruses in T1D development is not known. The aim of this thesis is to study various EVs that have been shown to differ in their immune phenotype, lytic ability, association with induction of islet autoantibodies, ability to replicate, cause islet disintegration and induce innate antiviral pathways in infected pancreatic cells in vitro. Furthermore, EV presence and pathogenic process in pancreatic tissue and isolated islets of T1D patients was also studied.

    Studies in this thesis for first time show the detection of EV RNA and protein in recent onset live T1D patients supporting the EV hypothesis in T1D development. Further all EV serotypes studied were able to replicate in islets, causing variable amount of islet disintegration ranging from extensive islet disintegration to not affecting islet morphology at all. However, one of the EV serotype replicated in only two out of seven donors infected, highlighting the importance of individual variation between donors. Further, this serotype impaired the insulin response to glucose stimulation without causing any visible islet disintegration, suggesting that this serotype might impaired the insulin response by inducing a functional block. Infection of human islets with the EV serotypes that are differentially associated with the development of islet autoantibodies showed the islet cell disintegration that is comparable with their degree of islet autoantibody seroconversion. Suggesting that the extent of the epidemic-associated islet autoantibody induction may depend on the ability of the viral serotypes to damage islet cells. Furthermore, one of the EV strains showed unique ability to infect and replicate both in endo and exocrine cells of the pancreas. EV replication in both endo and exocrine cells affected the genes involved in innate and antiviral pathways and induction of certain genes with important antiviral activity significantly varied between different donors. Suggesting that the same EV infection could result in different outcome in different individuals. Finally, we compared the results obtained by lytic and non lytic EV strains in vitro with the findings reported in fulminant and slowly progressing autoimmune T1D and found some similarities. In conclusion the results presented in this thesis further support the role of EV in T1D development and provide more insights regarding viral and host variation.  This will improve our understanding of the possible causative mechanism by EV in T1D development.

    List of papers
    1. Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes
    Open this publication in new window or tab >>Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes
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    2015 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 64, no 5, p. 1682-1687Article in journal (Refereed) Published
    Abstract [en]

    The Diabetes Virus Detection study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes for the presence of viruses. Minimal pancreatic tail resection was performed 3-9 weeks after onset of type 1 diabetes in 6 adult patients (age 24-35 years). The presence of enteroviral capsid protein 1 (VP1) and the expression of class I HLA were investigated by immunohistochemistry. Enterovirus RNA was analyzed from isolated pancreatic islets and from fresh frozen whole pancreatic tissue using PCR and sequencing. Non-diabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetes patients (2 of 9 controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (1 of 9 controls). Enterovirus specific RNA sequences were detected in 4 of 6 cases (0 of 6 controls). The results were confirmed in different laboratories. Only 1.7 % of the islets contained VP1 positive cells and the amount of enterovirus RNA was low. The results provides evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, being consistent with the possibility that a low grade enteroviral infection in the pancreatic islets contribute to disease progression in humans.

    National Category
    Endocrinology and Diabetes Microbiology
    Identifiers
    urn:nbn:se:uu:diva-239513 (URN)10.2337/db14-1370 (DOI)000353431200023 ()25422108 (PubMedID)
    Available from: 2014-12-29 Created: 2014-12-29 Last updated: 2017-12-05
    2. Infection of Human Islets of Langerhans With Two Strains of Coxsackie B Virus Serotype 1: Assessment of Virus Replication, Degree of Cell Death and Induction of Genes Involved in the Innate Immunity Pathway
    Open this publication in new window or tab >>Infection of Human Islets of Langerhans With Two Strains of Coxsackie B Virus Serotype 1: Assessment of Virus Replication, Degree of Cell Death and Induction of Genes Involved in the Innate Immunity Pathway
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    2014 (English)In: Journal of Medical Virology, ISSN 0146-6615, E-ISSN 1096-9071, Vol. 86, no 8, p. 1402-1411Article in journal (Refereed) Published
    Abstract [en]

    Type 1 diabetes mellitus is believed to be triggered, in part, by one or more environmental factors and human enteroviruses (HEVs) are among the candidates. Therefore, this study has examined whether two strains of HEV may differentially affect the induction of genes involved in pathways leading to the synthesis of islet hormones, chemokines and cytokines in isolated, highly purified, human islets. Isolated, purified human pancreatic islets were infected with strains of Coxsackievirus B1. Viral replication and the degree of CPE/islet dissociation were monitored. The expression of insulin, glucagon, CXCL10, TLR3, IF1H1, CCL5, OAS-1, IFN beta, and DDX58 was analyzed. Both strains replicated in islets but only one of strain caused rapid islet dissociation/CPE. Expression of the insulin gene was reduced during infection of islets with either viral strain but the gene encoding glucagon was unaffected. All genes analyzed which are involved in viral sensing and the development of innate immunity were induced by Coxsackie B viruses, with the notable exception of TLR3. There was no qualitative difference in the expression pattern between each strain but the magnitude of the response varied between donors. The lack of virus induced expression of TLR3, together with the differential regulation of IF1H1, OAS1 and IFN beta, (each of which has polymorphic variants influence the predisposition to type 1 diabetes), that might result in defective clearance of virus from islet cells. The reduced expression of the insulin gene and the unaffected expression of the gene encoding glucagon by Coxsackie B1 infection is consistent with the preferential beta-cell tropism of the virus.

    Keyword
    enterovirus, type 1 diabetes, innate immunity, human pancreatic islets, RNA sensors
    National Category
    Infectious Medicine Microbiology
    Identifiers
    urn:nbn:se:uu:diva-231113 (URN)10.1002/jmv.23835 (DOI)000339486200015 ()24249667 (PubMedID)
    Available from: 2014-09-05 Created: 2014-09-04 Last updated: 2017-12-05
    3. Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity
    Open this publication in new window or tab >>Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity
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    2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. e77850-Article in journal (Refereed) Published
    Abstract [en]

    Three large-scale Echovirus (E) epidemics (E4, E16, E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-212334 (URN)10.1371/journal.pone.0077850 (DOI)000326499300010 ()
    Available from: 2013-12-10 Created: 2013-12-09 Last updated: 2017-12-06Bibliographically approved
    4. Field strains of Echovirus 6 infect human endocrine and exocrine pancreatic cells and induce pro-inflammatory innate immune responses.
    Open this publication in new window or tab >>Field strains of Echovirus 6 infect human endocrine and exocrine pancreatic cells and induce pro-inflammatory innate immune responses.
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    (English)Article in journal (Refereed) In press
    Abstract [en]

    Increasing evidence suggests that type 1 diabetes (T1D) is a combined endocrine-exocrine disease. Human enteroviruses (HEV) have been suggested to induce T1D, but so far evidence on HEV infection in human pancreas has been reported only in islets and ductal cells. Aim of this study was to investigate the capability of HEV strains to infect primary human endocrine and exocrine pancreatic cells and to induce the expression of innate immunity genes in both cell types. Isolated human pancreatic islets and exocrine cells were either mock-infected or inoculated with seven field isolates of Echovirus 6 (E6). Beta-cell tropic strains of E4, E16 and E30 were assayed in primary exocrine cells. Viral infection, replication, virus-induced cytopathic effect (CPE) and expression of innate immunity genes were measured. All the seven strains of E6 replicated in both pancreatic endocrine and exocrine cells with infectious progeny production and appearance of CPE. By contrast, no virus titer increase or CPE were observed in exocrine cells exposed to E4, E16 and E30. Virus particles were found in E6-infected acinar cells, both free in cytoplasm and enclosed in vacuoles. Insulin granules accumulation in proximity to virus particles and beta cells functional impairment were demonstrated in E6-infected islets. Endocrine and exocrine cells responded to E6 infection by upregulating the transcription of genes involved in viral recognition (IF1H1), antiviral defense (OAS1, IFN-β) and inflammation (CXCL10, CCL5). Our results indicate that islets and exocrine pancreatic cells productively support the E6 infection and suggest that HEV-associated T1D may involve both endocrine and exocrine pancreas.

    National Category
    Clinical Medicine Microbiology
    Identifiers
    urn:nbn:se:uu:diva-283276 (URN)
    Available from: 2016-04-12 Created: 2016-04-12 Last updated: 2016-06-01
    5. Gene expression analysis of human islets in a subject at onset of type 1 diabetes
    Open this publication in new window or tab >>Gene expression analysis of human islets in a subject at onset of type 1 diabetes
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    2014 (English)In: Acta Diabetologica, ISSN 0940-5429, E-ISSN 1432-5233, Vol. 51, no 2, p. 199-204Article in journal (Refereed) Published
    Abstract [en]

    Swollen islet cells have been repeatedly described at onset of type 1 diabetes, but the underlying mechanism of this observation, termed hydropic degeneration, awaits characterization. In this study, laser capture microdissection was applied to extract the islets from an organ donor that died at onset of type 1 diabetes and from an organ donor without pancreatic disease. Morphologic analysis revealed extensive hydropic degeneration in 73 % of the islets from the donor with type 1 diabetes. Expression levels of genes involved in apoptosis, ER stress, beta cell function, and inflammation were analyzed in isolated and laser-captured islets by qPCR. The chemokine MCP-1 was expressed in islets from the donor with type 1 diabetes while undetectable in the control donor. No other signs of inflammation were detected. There were no signs of apoptosis on the gene expression level, which was also confirmed by negative immunostaining for cleaved caspase-8. There was an increased expression of the transcription factor ATF4, involved in transcription of ER stress genes, in the diabetic islets, but no further signs of ER stress were identified. In summary, on the transcription level, islets at onset of type 1 diabetes in which many beta cells display hydropic degeneration show no obvious signs of apoptosis, ER stress, or inflammation, supporting the notion that these cells are responding normally to high glucose and eventually succumbing to beta cell exhaustion. Also, this study validates the feasibility of performing qPCR analysis of RNA extracted from islets from subjects with recent onset of T1D and healthy controls by laser capture microdissection.

    National Category
    Endocrinology and Diabetes
    Identifiers
    urn:nbn:se:uu:diva-202844 (URN)10.1007/s00592-013-0479-5 (DOI)000334054200004 ()23624551 (PubMedID)
    Available from: 2013-06-28 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
    6. Enterovirus-induced changes in explanted human islet of Langerhans resemble findings in islets of fulminant and conventional type 1 diabetes
    Open this publication in new window or tab >>Enterovirus-induced changes in explanted human islet of Langerhans resemble findings in islets of fulminant and conventional type 1 diabetes
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Hypothesis: Fulminant Type 1 diabetes is a unique subtype of T1D, mostly reported in the Japanese population, which is characterized by extensive beta cell death already at onset, often without any insulitis. Enterovirus (EV) infections are associated with the etiology of both fulminant and conventional T1D. However the causative mechanism is not known for any of these diseases. EVs capability to cause lytic vs non-lytic infection in explanted human islets may have implications on the pathogenesis of these two types of T1D.

    Aim: To study the effect of infection of explanted human pancreatic islets with lytic (CBV-1) and non-lytic (CBV-4) Coxsackie B virus strains on cytopathic effect/islet disintegration and to what extent genes involved in viral sensing, antiviral defense and encoding of islet auto-antigens are affected by the viral replication. Also, to compare these findings with the findings reported in fulminant and conventional T1D.

    Methods: Degree of cytopathic effect/islet disintegration was studied and viral replication was measured. Genes involved in viral sensing (NOD2, TLR7 and TLR4), antiviral pathways (OAS2, MX1, PKR, and IRF7), genes coding for known islet auto antigens (GAD65, ZNT8) and the islet hormones, insulin and glucagon, were studied. Mock-infected explanted islet served as controls.

    Results: All CBV strains replicated in the explanted islets but only the CBV-1 strains caused cytopathic effect/islet cell disintegration. Infection with all CBV strains resulted in the induction of genes encoding OAS2 and MX1. In contrast, mRNA expression levels of the gene encoding insulin was reduced. The gene encoding PKR was induced by one of the lytic strains (CBV-1-11) and also by the non-lytic CBV4 strain, while the mRNA expression levels of genes encoding glucagon, NOD2, TLR7, TLR4, MCL1, GAD65 and ZNT8 were not significantly affected.

    National Category
    Endocrinology and Diabetes Microbiology
    Identifiers
    urn:nbn:se:uu:diva-283281 (URN)
    Available from: 2016-04-12 Created: 2016-04-12 Last updated: 2016-06-01
  • 9.
    Andersson, D. I., Björkman, J. and Hughes, D.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fitness and virulence of antibiotic resistant bacteria.2001In: Antibiotic Development and Resistance., p. 155-162Article, book review (Other scientific)
  • 10.
    Andersson, Dan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkman, Johanna
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Antibiotikaresistens: är den reversibel?1998In: Smittskydd: Smittskyddsinstitutets tidskrift, ISSN 1401-0690, Vol. 4, no 1, p. 3-5Article, book review (Other academic)
  • 11.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Antibiotic resistance and its cost: is it possible to reverse resistance?2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 4, p. 260-271Article, review/survey (Refereed)
    Abstract [en]

    Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.

  • 12.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Microbiological effects of sublethal levels of antibiotics2014In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 12, no 7, p. 465-478Article, review/survey (Refereed)
    Abstract [en]

    The widespread use of antibiotics results in the generation of antibiotic concentration gradients in humans, livestock and the environment. Thus, bacteria are frequently exposed to non-lethal (that is, subinhibitory) concentrations of drugs, and recent evidence suggests that this is likely to have an important role in the evolution of antibiotic resistance. In this Review, we discuss the ecology of antibiotics and the ability of subinhibitory concentrations to select for bacterial resistance. We also consider the effects of low-level drug exposure on bacterial physiology, including the generation of genetic and phenotypic variability, as well as the ability of antibiotics to function as signalling molecules. Together, these effects accelerate the emergence and spread of antibiotic-resistant bacteria among humans and animals.

  • 13.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Persistence of antibiotic resistance in bacterial populations2011In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 35, no 5, p. 901-911Article, review/survey (Refereed)
    Abstract [en]

    Unfortunately for mankind, it is very likely that the antibiotic resistance problem we have generated during the last 60 years due to the extensive use and misuse of antibiotics is here to stay for the foreseeable future. This view is based on theoretical arguments, mathematical modeling, experiments and clinical interventions, suggesting that even if we could reduce antibiotic use, resistant clones would remain persistent and only slowly (if at all) be outcompeted by their susceptible relatives. In this review, we discuss the multitude of mechanisms and processes that are involved in causing the persistence of chromosomal and plasmid-borne resistance determinants and how we might use them to our advantage to increase the likelihood of reversing the problem. Of particular interest is the recent demonstration that a very low antibiotic concentration can be enriching for resistant bacteria and the implication that antibiotic release into the environment could contribute to the selection for resistance.

  • 14.
    Andersson, D.I
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkman, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Antibiotikaresistens här för att stanna?1998In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 95, no 37, p. 3940-3944Article, review/survey (Other academic)
  • 15.
    Andersson, Jan O
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bacterial DNA in the human genome2003In: Encyclopedia of the Human Genome, Nature Publishing Group; London; UK , 2003Chapter in book (Refereed)
  • 16.
    Andersson, Jan O
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Lateral gene transfer in eukaryotes.2005In: Cell Mol Life Sci, ISSN 1420-682X, Vol. 62, no 11, p. 1182-97Article in journal (Refereed)
    Abstract [en]

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular.

  • 17.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Roger, Andrew J.
    Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes2003In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 3, p. 14-Article in journal (Refereed)
    Abstract [en]

    Background

    Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH), to evaluate and compare the patterns and rates of lateral gene transfer (LGT) in prokaryotes and eukaryotes.

    Results

    We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists).

    Conclusion

    LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

  • 18.
    Andersson, Leif
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Swedish University of Agricultural Sciences.
    Archibald, Alan L.
    Bottema, Cynthia D.
    Brauning, Rudiger
    Burgess, Shane C.
    Burt, Dave W.
    Casas, Eduardo
    Cheng, Hans H.
    Clarke, Laura
    Couldrey, Christine
    Dalrymple, Brian P.
    Elsik, Christine G.
    Foissac, Sylvain
    Giuffra, Elisabetta
    Groenen, Martien A.
    Hayes, Ben J.
    Huang, LuSheng S.
    Khatib, Hassan
    Kijas, James W.
    Kim, Heebal
    Lunney, Joan K.
    McCarthy, Fiona M.
    McEwan, John C.
    Moore, Stephen
    Nanduri, Bindu
    Notredame, Cedric
    Palti, Yniv
    Plastow, Graham S.
    Reecy, James M.
    Rohrer, Gary A.
    Sarropoulou, Elena
    Schmidt, Carl J.
    Silverstein, Jeffrey
    Tellam, Ross L.
    Tixier-Boichard, Michele
    Tosser-Klopp, Gwenola
    Tuggle, Christopher K.
    Vilkki, Johanna
    White, Stephen N.
    Zhao, Shuhong
    Zhou, Huaijun
    Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project2015In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 16Article in journal (Refereed)
    Abstract [en]

    We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

  • 19.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Baker, Louise
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Melbourne, Vic, Australia..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 398Article in journal (Refereed)
    Abstract [en]

    Giardia duodenalis is an intestinal parasite that causes 200-300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate: ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID(10), 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid a alpha-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID(10) diverged from those in 713-r and WB-r (r <= 0.2), which were more similar to each other (r = 0.47). In 106-2ID(10), a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

  • 20.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Bio21 Mol Sci & Biotechnol Inst, Melbourne, Vic, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Svärd, Staffan G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis2016In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, no 10, p. 6034-6045Article in journal (Refereed)
    Abstract [en]

    Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and alpha-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.

  • 21.
    Argaman, L., Hershberg, R., Vogel, J., Bejerano, G., Wagner, E.G.H., Margalit, H. and Altuvia, S.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Novel small RNA-encoding genes in the intergenic regions of Escherichia coli.2001In: Curr. Biol., Vol. 11, no 12, p. 941-950Article in journal (Refereed)
    Abstract [en]

    Background: Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life. These RNAs carry out diverse functions, and many of them are regulators of gene expression. Genes encoding small, untra

  • 22.
    Assadian, Farzaneh
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandström, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Laurell, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Otolaryngology and Head and Neck Surgery.
    Svensson, Catharina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils2015In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, Vol. 105, article id e53374Article in journal (Refereed)
    Abstract [en]

    Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

  • 23. Athlin, Simon
    et al.
    Kaltoft, Margit
    Slotved, Hans-Christian
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Holmberg, Hans
    Konradsen, Helle Bossen
    Stralin, Kristoffer
    Association between Serotype-Specific Antibody Response and Serotype Characteristics in Patients with Pneumococcal Pneumonia, with Special Reference to Degree of Encapsulation and Invasive Potential2014In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 21, no 11, p. 1541-1549Article in journal (Refereed)
    Abstract [en]

    We studied the immunoglobulin (Ig) response to causative serotype-specific capsular polysaccharides in adult pneumococcal pneumonia patients. The serotypes were grouped according to their degree of encapsulation and invasive potential. Seventy patients with pneumococcal pneumonia, 20 of whom were bacteremic, were prospectively studied. All pneumococcal isolates from the patients were serotyped, and the Ig titers to the homologous serotype were determined in acute-and convalescent-phase sera using a serotype-specific enzyme-linked immunosorbent assay. The Ig titers were lower in bacteremic cases than in nonbacteremic cases (P < 0.042). The Ig titer ratio (convalescent/acute titer) was >= 2 in 33 patients, 1 to 1.99 in 20 patients, and < 1 in 17 patients. Patients >= 65 years old had a lower median Ig titer ratio than did younger patients (P < 0.031). The patients with serotypes with a thin capsule (1, 4, 7F, 9N, 9V, and 14) and medium/high invasive potential (1, 4, 7F, 9N, 9V, 14, and 18C) had higher Ig titer ratios than did patients with serotypes with a thick capsule (3, 6B, 11A, 18C, 19A, 19F, and 23F) and low invasive potential (3, 6B, 19A, 19F, and 23F) (P < 0.05 for both comparisons after adjustment for age). Ig titer ratios of <1 were predominantly noted in patients with serotypes with a thick capsule. In 8 patients with pneumococcal DNA detected in plasma, the three patients with the highest DNA load had the lowest Ig titer ratios. In conclusion, a high antibody response was associated with serotypes with a thin capsule and medium/high invasive potential, although a low antibody response was associated with serotypes with a thick capsule and a high pneumococcal plasma load.

  • 24.
    Backström, Ellenor
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Regulation of Adenoviral Gene Expression by the L4-33K and L4-22K Proteins2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The splicing pattern during an adenovirus infection is shifted at the late phase towards using weaker splice sites, splicing out larger introns. Splicing of weak 3´ splice sites usually requires recognition of the 3´AG dinucleotide before the first catalytic step of splicing. Such splicing events are said to be AG-dependent and requires an interaction of both subunits of the cellular splicing factor U2AF with the 3´ splice site. We show that splicing of transcripts that are AG-dependent in uninfected nuclear extracts (NE) becomes AG-independent in nuclear extracts prepared form adenovirus late-infected HeLa cells (Ad-NE). Further we demonstrate that the first step in splicing of a model transcript, IgM, becomes completely U2AF-independent in Ad-NE. This finding supports our working model that 3´ splice site recognition in Ad-NE is altered, and in fact might be U2AF-independent.

    We further show that the adenovirus late protein L4-33K acts as a virus encoded alternative splicing factor. L4-33K activates splicing of both cellular and viral transcripts containing weak 3´ splice sites. This supports the hypothesis that adenovirus alter splicing during the infection to favour usage of weak, suboptimal 3´ splice sites. However, we were unable to find an alternative U2AF-related factor that could stimulate L4-33K splicing enhancer activity. Furthermore, we demonstrate that the serine residues in the C-terminal part of L4-33K are important for the splicing enhancer activity but also for its nuclear localisation.

    The adenovirus major late promoter is highly activated after the onset of viral genome replication. Protein complexes binding to downstream elements of the promoter are required for full enhancement of this promoter. We show that an L4-33K-related protein, L4-22K, stimulates transcription from the major late promoter. This stimulation is mainly via the downstream elements and does not require the viral IVa2 protein, which is a transcription factor of the major late promoter.

    List of papers
    1. Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts.
    Open this publication in new window or tab >>Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts.
    2005 (English)In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 27, p. 25478-84Article in journal (Refereed) Published
    Keyword
    Adenoviridae/*genetics, Adenoviridae Infections/*genetics/*metabolism, Cell Extracts, Hela Cells, Humans, Immunoglobulin M/genetics, Introns/physiology, Nuclear Proteins/*metabolism, RNA Precursors/*metabolism, RNA Splice Sites/physiology, RNA Splicing/*physiology, RNA; Small Nuclear/metabolism, Research Support; Non-U.S. Gov't, Ribonucleoproteins/*metabolism
    Identifiers
    urn:nbn:se:uu:diva-80306 (URN)15899895 (PubMedID)
    Available from: 2006-05-05 Created: 2006-05-05 Last updated: 2011-01-11
    2. L4-33K, an adenovirus-encoded alternative RNA splicing factor
    Open this publication in new window or tab >>L4-33K, an adenovirus-encoded alternative RNA splicing factor
    2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 48, p. 36510-36517Article in journal (Refereed) Published
    Abstract [en]

    Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3' splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3' splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus late-infected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-22559 (URN)10.1074/jbc.M607601200 (DOI)000242220800006 ()17028184 (PubMedID)
    Available from: 2007-01-18 Created: 2007-01-18 Last updated: 2017-12-07Bibliographically approved
    3. Regulation of Adenovirus Late Region 1 Splicing
    Open this publication in new window or tab >>Regulation of Adenovirus Late Region 1 Splicing
    (English)Manuscript (Other academic)
    Abstract [en]

    The major late transcription unit (MLTU) produces one pre-mRNA that is processed into more than 20 cytoplasmic mRNAs by alternative polyadenylation and extensive alternative 3' splice site usage. The alternative splicing of the MLTU is temporally regulated, resulting in the expression of only one mRNA, the L1 52,55K, before the onset of viral genome replication. The L1 unit also encodes the IIIa mRNA, the expression of which is highly regulated at the level of splicing. We have previously shown that the adenoviral L4-33K protein enhances IIIa splicing via the IIIa virus infection-dependent splicing enhancer element. In this study we show that serine to glycine mutations in the tiny RS domain of the L4-33K protein retain more activity in vivo compared to results obtained previously in vitro. In addition, it is also clear that these mutations in the RS domain affect the sub-cellular localization of L4-33K. Thus, the RS domain appears to contain a nuclear localisation signal that is dependent on the serine residues. In a previous report we showed that, in extracts prepared from adenovirus-infected cells, splicing is independent of the general splicing factor U2AF. In this study we also demonstrate that none of the tested U2AF-replacement candidate proteins (PUF60, Caper α, and Caper β) collaborate with L4-33K in the activation of IIIa splicing. It has been suggested that regulation of L1 alternative splicing does not require cis-competition between the 52.55K and IIIa 3' splice sites. We find that activity of the IIIa splice site increases considerably in the absence of cis-competition with the 52,55K splice site. Interestingly, this cis-competition is not virus-specific since this observation is reproducible in a transcription unit where the β-globin 3' splice site replaces the natural 52,55K 3' splice site. We conclude that L1 alternative splicing conforms to the general rule in that it ordinarily makes use of the proximal 3' splice site (52,55K), whereas activation of distal 3' splice site usage requires active intervention. In adenovirus this intervention is achieved by production of the L4-33K protein.

    Keyword
    Adenovirus, L4-33K, Splicing, 3VDF
    Identifiers
    urn:nbn:se:uu:diva-101308 (URN)
    Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2011-06-28
    4. Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site
    Open this publication in new window or tab >>Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site
    2010 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 151, no 2, p. 220-228Article in journal (Refereed) Published
    Abstract [en]

    The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Here we have searched for a possible DEF-A candidate protein. The adenovirus L4-33K protein functions as a virus-encoded alternative RNA splicing factor, stimulating cytoplasmic accumulation of most late viral mRNAs. Interestingly, the L4 region also encodes for a second related protein, L4-22K, which share the 105 amino-terminal amino acids with L4-33K. Here we show that L4-22K both in vivo and in vitro stimulates transcription from the MLP in a DE sequence dependent manner. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Interestingly, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K, as a BPV E2 DNA binding domain fusion protein either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.

    Keyword
    Adenovirus, L4-22K, MLP, transcription, DE elements
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-101310 (URN)10.1016/j.virusres.2010.05.013 (DOI)000280210000015 ()20621673 (PubMedID)
    Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2017-12-13Bibliographically approved
  • 25.
    Badrul, Hasan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Järhult, Josef D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Absence of vancomycin-resistant enterococci among highly ESBL-positive crows (Corvus splendens) foraging on hospital waste in Bangladesh2015In: Infection Ecology & Epidemiology, ISSN 2000-8686, E-ISSN 2000-8686, Vol. 5, article id 29761Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    Vancomycin-resistant enterococci (VRE) have emerged as a growing problem in hospitals; however, domesticated animals, poultry, and wild birds are acting as potential reservoirs. There is a knowledge gap in the Epidemiology of VRE from Bangladesh.

    METHODS:

    To study the prevalence of VRE and the mechanisms of resistance implicated among wild birds, 238 fecal samples were collected in 2010 from house crows (Corvus splendens) foraging on hospital waste in Bangladesh. Fecal samples were screened by analyzing color change in broth and screening for vanA and vanB resistant genes by PCR.

    RESULTS:

    Neither vanA nor vanB genes were detected from the fecal samples. The house crow does not seem to constitute a reservoir for VRE.

    CONCLUSION:

    The zero prevalence is an indication that foraging on hospital waste does not constitute a major risk of VRE carriage in house crows and this is the first study to focus on the prevalence of VRE from wild birds in Bangladesh.

  • 26.
    Beckman-Sundh, Ulla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ek, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zetterqvist, Örjan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ek, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A screening method for phosphohistidine phosphatase 1 activity2011In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 116, no 3, p. 161-168Article in journal (Refereed)
    Abstract [en]

    Introduction. Research in the field of protein-bound phosphohistidine phosphorylation has been hampered by the difficulties in analysis and detection of phosphohistidine. Therefore a screening method was developed primarily for the analysis of phosphohistidine phosphatase 1 (PHPT1) activity. Methods. A highly positively charged substrate, Ac-Val-Arg-Leu-Lys-His-Arg-Lys-Leu-Arg-pNA, containing the peptide surrounding the phosphorylated histidine in ion channel KCa3.1 was chemically phosphorylated using phosphoramidate. Excess phosphoramidate was removed by anion exchange chromatography using a micro spin column. After incubation of the eluate with PHPT1, the removed phosphate was bound on a consecutive anion exchange spin column. The eluate was assayed in a micro plate format for remaining phosphate in the substrate Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA. Histone H4, also highly positive in charge, was subjected to the same procedure to explore the possibility to use other substrates to PHPT1 in this assay format. Results. It was found that Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA and phosphohistone H4 were dephosphorylated by PHPT1. The apparent K(m) for Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA was in the order of 10 mu M. Using this method, phosphohistidine phosphatase activity was detected in mouse liver cell sap with Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA as substrate. Discussion. The described method for determination of PHPT1 activity is comparably much easier and faster than presently used methods for detection of phosphohistidine phosphatase activity. It is also sensitive, since the lower activity limit was 5 pmol phosphate released per min. It has the potential to be used both for more rapid screening for inhibitors and activators to phosphohistidine phosphatases and for screening of histidine kinases.

  • 27.
    Benachenhou, Farid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Retroviral long Terminal Repeats; Structure, Detection and Phylogeny2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Long terminal repeats (LTRs) are non-coding repeats flanking the protein-coding genes of LTR retrotransposons. The variability of LTRs poses a challenge in studying them. Hidden Markov models (HMMs), probabilistic models widely used in pattern recognition, are useful in dealing with this variability. The aim of this work was mainly to study LTRs of retroviruses and LTR retrotransposons using HMMs.

    Paper I describes the methodology of HMM modelling applied to different groups of LTRs from exogenous retroviruses (XRVs) and endogenous retroviruses (ERVs). The detection capabilities of HMMs were assessed and were found to be high for homogeneous groups of LTRs. The alignments generated by the HMMs displayed conserved motifs some of which could be related to known functions of XRVs. The common features of the different groups of retroviral LTRs were investigated by combining them into a single alignment. They were the short inverted terminal repeats TG and CA and three AT-rich stretches which provide retroviruses with TATA boxes and AATAAA polyadenylation signals.

    In Paper II, phylogenetic trees of three groups of retroviral LTRs were constructed by using HMM-based alignments. The LTR trees were consistent with trees based on other retroviral genes suggesting co-evolution between LTRs and these genes.

    In Paper III, the methods in Paper I and II were extended to LTRs from other retrotransposon groups, covering much of the diversity of all known LTRs. For the first time an LTR phylogeny could be achieved. There were no major disagreement between the LTR tree and trees based on three different domains of the Pol gene. The conserved LTR structure of paper I was found to apply to all LTRs. Putative Integrase recognition motifs extended up to 12 bp beyond the short inverted repeats TG/CA.

    Paper IV is a review article describing the use of sequence similarity and structural markers for the taxonomy of ERVs. ERVs were originally classified into three classes according to the length of the target site duplication. While this classification is useful it does not include all ERVs. A naming convention based on previous ERV and XRV nomenclature but taking into account newer information is advocated in order to provide a practical yet coherent scheme in dealing with new unclassified ERV sequences.

    Paper V gives an overview of bioinformatics tools for studies of ERVs and of retroviral evolution before and after endogenization. It gives some examples of recent integrations in vertebrate genomes and discusses pathogenicity of human ERVs including their possible relation to cancers.

    In conclusion, HMMs were able to successfully detect and align LTRs. Progress was made in understanding their conserved structure and phylogeny. The methods developed in this thesis could be applied to different kinds of non-coding DNA sequence element.

    List of papers
    1. The phylogeny of orthoretroviral long terminal repeats (LTRs)
    Open this publication in new window or tab >>The phylogeny of orthoretroviral long terminal repeats (LTRs)
    2009 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 448, no 2, p. 134-138Article in journal (Refereed) Published
    Abstract [en]

    LTRs are sequence elements in retroviruses and retrotransposons which are difficult to align due to their variability. One way of handling such cases is to use Hidden Markov Models (HMMs). In this work HMMs of LTRs were constructed for three groups of orthoretroviruses: the betaretroviruslike human MMTV-like (HML) endogenous retroviruses, the lentiviruses, including HIV, and gammaretroviruslike human endogenous retroviruses (HERVs). The HMM-generated LTR alignments and the phylogenetic trees constructed from them were compared with trees based on alignments of the pol gene at the nucleic acid level. The majority of branches in the LTR and pol based trees had the same order for the three retroviral genera, showing that HMM methods are successful in aligning and constructing phylogenies of LTRs. The HML LTR tree deviated somewhat from the pol tree for the groups HML3, HML7 and HML6. Among the gammaretroviruslike proviruses, the exogenous Mouse Leukemia Virus (MLV) was highly related to HERV-T in the pol based tree, but not in the LTR based tree. Aside from these differences, the similarity between the trees indicates that LTRs and pol coevolved in a largely monophyletic way.

    Place, publisher, year, edition, pages
    Amsterdam: Elsevier, 2009
    Keyword
    Hidden Markov models, Long terminal repeats, phylogeny, virology, retroviruses
    National Category
    Medical and Health Sciences
    Research subject
    Medical Science
    Identifiers
    urn:nbn:se:uu:diva-119775 (URN)10.1016/j.gene.2009.07.002 (DOI)000271972200005 ()19595747 (PubMedID)
    Available from: 2010-03-02 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
    2. Evolutionary Conservation of Orthoretroviral Long Terminal Repeats (LTRs) and ab initio Detection of Single LTRs in Genomic Data
    Open this publication in new window or tab >>Evolutionary Conservation of Orthoretroviral Long Terminal Repeats (LTRs) and ab initio Detection of Single LTRs in Genomic Data
    Show others...
    2009 (English)In: PLos ONE, ISSN 1932-6203, Vol. 4, no 4, p. e5179-Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND: Retroviral LTRs, paired or single, influence the transcription of both retroviral and non-retroviral genomic sequences. Vertebrate genomes contain many thousand endogenous retroviruses (ERVs) and their LTRs. Single LTRs are difficult to detect from genomic sequences without recourse to repetitiveness or presence in a proviral structure. Understanding of LTR structure increases understanding of LTR function, and of functional genomics. Here we develop models of orthoretroviral LTRs useful for detection in genomes and for structural analysis. PRINCIPAL FINDINGS: Although mutated, ERV LTRs are more numerous and diverse than exogenous retroviral (XRV) LTRs. Hidden Markov models (HMMs), and alignments based on them, were created for HML- (human MMTV-like), general-beta-, gamma- and lentiretroviruslike LTRs, plus a general-vertebrate LTR model. Training sets were XRV LTRs and RepBase LTR consensuses. The HML HMM was most sensitive and detected 87% of the HML LTRs in human chromosome 19 at 96% specificity. By combining all HMMs with a low cutoff, for screening, 71% of all LTRs found by RepeatMasker in chromosome 19 were found. HMM consensus sequences had a conserved modular LTR structure. Target site duplications (TG-CA), TATA (occasionally absent), an AATAAA box and a T-rich region were prominent features. Most of the conservation was located in, or adjacent to, R and U5, with evidence for stem loops. Several of the long HML LTRs contained long ORFs inserted after the second A rich module. HMM consensus alignment allowed comparison of functional features like transcriptional start sites (sense and antisense) between XRVs and ERVs. CONCLUSION: The modular conserved and redundant orthoretroviral LTR structure with three A-rich regions is reminiscent of structurally relaxed Giardia promoters. The five HMMs provided a novel broad range, repeat-independent, ab initio LTR detection, with prospects for greater generalisation, and insight into LTR structure, which may aid development of LTR-targeted pharmaceuticals.

    Place, publisher, year, edition, pages
    Public Library of Science (PLoS), 2009
    Keyword
    Long terminal repeats, hidden markov models, virology, bioinformatics
    National Category
    Microbiology in the medical area
    Research subject
    Medical Science
    Identifiers
    urn:nbn:se:uu:diva-119767 (URN)10.1371/journal.pone.0005179 (DOI)000265509900009 ()19365549 (PubMedID)
    Available from: 2010-03-02 Created: 2010-03-01 Last updated: 2018-01-12Bibliographically approved
    3. Universal structure and phylogeny of Long Terminal Repeats (LTRs)
    Open this publication in new window or tab >>Universal structure and phylogeny of Long Terminal Repeats (LTRs)
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Long terminal repeats (LTRs) are important sequence elements of retroviruses and related retrotransposons. They are however difficult to analyse due to their variability.

    The aim of this work was to construct models of LTRs from all known groups of LTR retrotransposons and retroviruses, representative of the entire LTR diversity, making it possible for the first time to comprehensively study their phylogeny and to compare it to phylogenies of other retrotransposon genes.

    A general HMM describing all LTRs was built. Its associated Viterbi alignment showed a consistent basic structure with inverted repeats starting with TGTT at the 5´end, ending with AACA at the 3´ end, plus two conserved AT-rich areas, the first one often containing the TATA box and the second one containing the polyadenylation signal AATAAA. A less conserved AT-rich stretch was apparent in the likely U3 portion. R was harder to delineate. The polyadenylation signal was followed by a T rich area characteristic of U5. The modular LTR structure previously reported by us, with modules separated by clusters of insert states, was also observed in this pan-LTR setting The result attests to the highly conserved basic structure of LTRs, which must date over a billion years back.

    Hidden Markov models (HMM) were also created for 14 subgroups of LTRs. The HMMs yielded consensus sequences which were aligned to a "Superviterbi" alignment. The Superviterbi alignment yielded a phylogenetic tree which was consistent with a tree based on an alignment of concatenated RT, RNAse H and INT proteins. In particular it gave further support for the monophyly of retroviral LTRs.

    The phylogenetic reconstruction now allows inferences regarding the origin of LTR retrotransposons.

    Keyword
    Long terminal repeats, hidden Markov models, phylogeny, alignment, detection
    National Category
    Microbiology in the medical area
    Research subject
    Clinical Virology
    Identifiers
    urn:nbn:se:uu:diva-119977 (URN)
    Available from: 2010-03-04 Created: 2010-03-04 Last updated: 2018-01-12
    4. Evolution of human endogenous retroviral sequences: a conceptual account
    Open this publication in new window or tab >>Evolution of human endogenous retroviral sequences: a conceptual account
    2008 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 65, no 21, p. 3348-3365Article, review/survey (Refereed) Published
    Abstract [en]

    Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic 'junk'.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a multi-author review).

    Keyword
    Bioinformatics, Endogenous retrovirus, Evolution, Lateral transfer, Sequence recognition, Vertebrate genome, Virulence
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-102543 (URN)10.1007/s00018-008-8495-2 (DOI)000260684200003 ()18818874 (PubMedID)
    Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved
    5. Classification and nomenclature of endogenous retroviral sequences (ERVs): problems and recommendations
    Open this publication in new window or tab >>Classification and nomenclature of endogenous retroviral sequences (ERVs): problems and recommendations
    Show others...
    2009 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 448, no 2, p. 115-123Article, review/survey (Refereed) Published
    Abstract [en]

    The genomes of many species are crowded with repetitive mobile sequences. In the case of endogenous retroviruses (ERVs) there is, for various reasons, considerable confusion regarding names assigned to families/groups of ERVs as well as individual ERV loci. Human ERVs have been studied in greater detail, and naming of HERVs in the scientific literature is somewhat confusing not just to the outsider. Without guidelines, confusion for ERVs in other species will also probably increase if those ERVs are studied in greater detail. Based on previous experience, this review highlights some of the problems when naming and classifying ERVs, and provides some guidance for detecting and characterizing ERV sequences. Because of the close relationship between ERVs and exogenous retroviruses (XRVs) it is reasonable to reconcile their classification with that of XRVs. We here argue that classification should be based on a combination of similarity, structural features, (inferred) function, and previous nomenclature. Because the RepBase system is widely employed in genome annotation, RepBase designations should be considered in further taxonomic efforts. To lay a foundation for a phylogenetically based taxonomy, further analyses of ERVs in many hosts are needed. A dedicated, permanent, international consortium would best be suited to integrate and communicate our current and future knowledge on repetitive, mobile elements in general to the scientific community.

    Place, publisher, year, edition, pages
    Amsterdam: Elsevier, 2009
    Keyword
    Endogenous retrovirus, taxonomy, nomenclature, phylogeny
    National Category
    Microbiology in the medical area
    Research subject
    Clinical Virology
    Identifiers
    urn:nbn:se:uu:diva-119976 (URN)10.1016/j.gene.2009.06.007 (DOI)000271972200003 ()19540319 (PubMedID)
    Available from: 2010-03-04 Created: 2010-03-04 Last updated: 2018-01-12Bibliographically approved
  • 28.
    Benachenhou, Farid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Jern, Patric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Oja, Merja
    Helsinki University of Technology.
    Sperber, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Blikstad, Vidar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Somervuo, Panu
    Helsinki Institute for Information Technology, Department of Computer Science, University of Helsinki.
    Kaski, Samuel
    Helsinki Institute for Information Technology, Department of Computer Science, University of Helsinki.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Evolutionary Conservation of Orthoretroviral Long Terminal Repeats (LTRs) and ab initio Detection of Single LTRs in Genomic Data2009In: PLos ONE, ISSN 1932-6203, Vol. 4, no 4, p. e5179-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Retroviral LTRs, paired or single, influence the transcription of both retroviral and non-retroviral genomic sequences. Vertebrate genomes contain many thousand endogenous retroviruses (ERVs) and their LTRs. Single LTRs are difficult to detect from genomic sequences without recourse to repetitiveness or presence in a proviral structure. Understanding of LTR structure increases understanding of LTR function, and of functional genomics. Here we develop models of orthoretroviral LTRs useful for detection in genomes and for structural analysis. PRINCIPAL FINDINGS: Although mutated, ERV LTRs are more numerous and diverse than exogenous retroviral (XRV) LTRs. Hidden Markov models (HMMs), and alignments based on them, were created for HML- (human MMTV-like), general-beta-, gamma- and lentiretroviruslike LTRs, plus a general-vertebrate LTR model. Training sets were XRV LTRs and RepBase LTR consensuses. The HML HMM was most sensitive and detected 87% of the HML LTRs in human chromosome 19 at 96% specificity. By combining all HMMs with a low cutoff, for screening, 71% of all LTRs found by RepeatMasker in chromosome 19 were found. HMM consensus sequences had a conserved modular LTR structure. Target site duplications (TG-CA), TATA (occasionally absent), an AATAAA box and a T-rich region were prominent features. Most of the conservation was located in, or adjacent to, R and U5, with evidence for stem loops. Several of the long HML LTRs contained long ORFs inserted after the second A rich module. HMM consensus alignment allowed comparison of functional features like transcriptional start sites (sense and antisense) between XRVs and ERVs. CONCLUSION: The modular conserved and redundant orthoretroviral LTR structure with three A-rich regions is reminiscent of structurally relaxed Giardia promoters. The five HMMs provided a novel broad range, repeat-independent, ab initio LTR detection, with prospects for greater generalisation, and insight into LTR structure, which may aid development of LTR-targeted pharmaceuticals.

  • 29.
    Benachenhou, Farid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Sperber, Goran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Universal structure and phylogeny of Long Terminal Repeats (LTRs)Manuscript (preprint) (Other academic)
    Abstract [en]

    Long terminal repeats (LTRs) are important sequence elements of retroviruses and related retrotransposons. They are however difficult to analyse due to their variability.

    The aim of this work was to construct models of LTRs from all known groups of LTR retrotransposons and retroviruses, representative of the entire LTR diversity, making it possible for the first time to comprehensively study their phylogeny and to compare it to phylogenies of other retrotransposon genes.

    A general HMM describing all LTRs was built. Its associated Viterbi alignment showed a consistent basic structure with inverted repeats starting with TGTT at the 5´end, ending with AACA at the 3´ end, plus two conserved AT-rich areas, the first one often containing the TATA box and the second one containing the polyadenylation signal AATAAA. A less conserved AT-rich stretch was apparent in the likely U3 portion. R was harder to delineate. The polyadenylation signal was followed by a T rich area characteristic of U5. The modular LTR structure previously reported by us, with modules separated by clusters of insert states, was also observed in this pan-LTR setting The result attests to the highly conserved basic structure of LTRs, which must date over a billion years back.

    Hidden Markov models (HMM) were also created for 14 subgroups of LTRs. The HMMs yielded consensus sequences which were aligned to a "Superviterbi" alignment. The Superviterbi alignment yielded a phylogenetic tree which was consistent with a tree based on an alignment of concatenated RT, RNAse H and INT proteins. In particular it gave further support for the monophyly of retroviral LTRs.

    The phylogenetic reconstruction now allows inferences regarding the origin of LTR retrotransposons.

  • 30. Bengtsson, S.
    et al.
    Bjelkenbrant, C.
    Kahlmeter, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Validation of EUCAST zone diameter breakpoints against reference broth microdilution2014In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 20, no 6, p. O353-O360Article in journal (Refereed)
    Abstract [en]

    The European Committee on Antimicrobial Susceptibility Testing (EUCAST) began harmonizing clinical breakpoints in Europe 2002. In 2009, work to develop a disc diffusion method began and the first disc diffusion breakpoints calibrated to EUCAST clinical MIC breakpoints were published in December 2009. In this study we validated EUCAST clinical zone diameter breakpoints against the International Standard Organization (ISO) reference broth microdilution. A collection of 544 isolates (238 Gram-negative and 306 Gram-positive) were tested against a panel of antimicrobial agents. Antimicrobial susceptibility testing was performed with broth microdilution as described by ISO and disc diffusion in accordance with EUCAST methodology. Inhibition zone diameters and MIC values were interpreted and categorized (S, I and R) according to EUCAST clinical breakpoint table version 2.0. Categorical agreement (CA) as well as minor (mD), major (MD) and very major (VMD) discrepancies were determined. There was in general good correlation between susceptibility test results obtained with disc diffusion and broth microdilution. Overall CA was 97.3% for all combinations of organisms and antimicrobial agents (n = 5231) and the overall discrepancy rates were 110 (2.1%) mD, 24 (0.5%) MD and 7 (0.1%) VMD. The overall CA for Gram-positive and Gram-negative organisms were 98.7% (2346 tests) and 96.2% (2942 tests), respectively. Seven VMD were observed, five for Gram-positive organisms (coagulase negative staphylococci (n = 2) and Staphylococcus aureus (n = 3)) and two for Gram-negative organisms (Pseudomonas aeruginosa). Minor discrepancies were mainly observed in Gram-negatives and were related to different antimicrobial agents and species.

  • 31.
    Bergman, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ding, Zhoujie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heyman, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Antigen-Specific IgM Causes Deposition of C3 on Sheep Red Blood Cells Within Seconds After Immunization2014In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 79, no 6, p. 442-442Article in journal (Other academic)
  • 32.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Genetics and Growth Regulation in Salmonella enterica2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications.

    Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response.

    To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E.

    Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA.

    Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria. 

    List of papers
    1. Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
    Open this publication in new window or tab >>Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
    2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, p. e90486-Article in journal (Other academic) Published
    Abstract [en]

    Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.

    Keyword
    tufA; ppGpp; RelA; Salmonella enterica; growth regulation
    National Category
    Microbiology
    Research subject
    Microbiology; Molecular Cellbiology
    Identifiers
    urn:nbn:se:uu:diva-159663 (URN)10.1371/journal.pone.0090486 (DOI)000332396200210 ()
    Note

    Jessica M. Bergman and Disa L. Hammarlöf contributed equally to this work.

    Available from: 2011-10-06 Created: 2011-10-05 Last updated: 2017-12-08Bibliographically approved
    2. Turnover of mRNAs is an essential function of RNase E
    Open this publication in new window or tab >>Turnover of mRNAs is an essential function of RNase E
    (English)Manuscript (preprint) (Other academic)
    Keyword
    rpsA, vacB, RNaseR, RelBE, RNA turnover
    National Category
    Microbiology in the medical area
    Research subject
    Biology with specialization in Microbiology; Microbiology; Molecular Genetics
    Identifiers
    urn:nbn:se:uu:diva-235199 (URN)
    Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2018-01-11
    3. Autoregulation of the tufB operon in Salmonella
    Open this publication in new window or tab >>Autoregulation of the tufB operon in Salmonella
    2016 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 100, no 6, p. 1004-1016Article in journal (Refereed) Published
    Abstract [en]

    In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3'-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5'-end of the tufB mRNA. Formation of the 'closed' structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.

    Keyword
    Salmonella enterica, tufA, tufB, EF-Tu, Rho, post-transcriptional regulation
    National Category
    Microbiology in the medical area
    Research subject
    Biology with specialization in Microbiology; Microbiology; Molecular Genetics
    Identifiers
    urn:nbn:se:uu:diva-235218 (URN)10.1111/mmi.13364 (DOI)000379687100008 ()26934594 (PubMedID)
    Funder
    Knut and Alice Wallenberg Foundation, KAW 2009.0251Swedish Research Council, 621-2012-2188; 521-2013-2904
    Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2018-01-11Bibliographically approved
    4. Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies
    Open this publication in new window or tab >>Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies
    2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, p. e109255-Article in journal (Refereed) Published
    Abstract [en]

    When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.

    Keyword
    aceBAK, ackA-pta, acs, pka, growth in stationary phase, Salmonella Typhimurium
    National Category
    Microbiology in the medical area
    Research subject
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-234281 (URN)10.1371/journal.pone.0109255 (DOI)000342591500086 ()25275605 (PubMedID)
    Available from: 2014-10-15 Created: 2014-10-15 Last updated: 2018-01-11Bibliographically approved
  • 33.
    Bergman, Jessica M
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, United States of America.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, p. e109255-Article in journal (Refereed)
    Abstract [en]

    When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.

  • 34. Bergqvist, A
    et al.
    Nilsson, M
    Bondeson, K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Magnusson, G
    Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.1990In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 18, no 9, p. 2715-20Article in journal (Refereed)
    Abstract [en]

    A putative zinc finger in polyomavirus large T-antigen was investigated. We were unable to demonstrate unequivocally a requirement for zinc in specific DNA-binding using the chelating agent 1, 10-phenanthroline. An involvement of the putative zinc finger in specific DNA-binding was nevertheless suggested by the properties of a mutant protein with a cys----ser replacement in the finger motif. Probably as a result of the defective DNA-binding, the mutant protein had lost its activity in initiation of viral DNA-replication and in negative regulation of viral early transcription. However, the trans-activation of the viral late promoter was normal. The analysis also revealed a previously unrecognized activity of large T-antigen. The mutant protein trans-activated the viral early promoter. In the wild-type protein this activity is probably concealed by the separate, negative regulatory function.

  • 35.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sundström, Sara
    Karolinska Institutet.
    Dimberg, Lina Y.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Masucci, Maria G.
    Karolinska Institutet.
    The Hepatitis C Virus Core Protein Modulates T Cell Responses by Inducing Spontaneous and Altering T-cell Receptor-triggered Ca2+ Oscillations2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 21, p. 18877-18883Article in journal (Refereed)
    Abstract [en]

    Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infection, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Expression of the HCV core (C) protein modulates transcription of the IL-2 promoter in T lymphocytes by activating the nuclear factor of activated T lymphocyte (NFAT) pathway. Here we report on the effect of HCV C on Ca2+ signaling, which is essential for activation of NFAT. Expression of HCV C correlated with increased levels of cytosolic Ca2+ and spontaneous Ca2+ oscillations in transfected Jurkat cells. Triggering of the T-cell receptor induced a prolonged Ca2+ response characterized by vigorous high frequent oscillations in a high proportion of the responding cells. This was associated with decreased sizes and accelerated emptying of the intracellular calcium stores. The effect of HCV C on calcium mobilization was not dependent on phospholipase C-1 (PLC-) activity or increased inositol 1,4,5-trisphosphate (IP3) production and did not require functional IP3 receptors, suggesting that insertion of the viral protein in the endoplasmic reticulum membrane may be sufficient to promote Ca2+ leakage with dramatic downstream consequences on the magnitude and duration of the response. Our data suggest that expression of HCV C in infected T lymphocytes may contribute to the establishment of persistent infections by inducing Ca2+ oscillations that regulate both the efficacy and information content of Ca2+ signals and are ultimately responsible for induction of gene expression and functional differentiation.

  • 36.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Söderbärg, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Magnusson, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Altered susceptibility to tumor necrosis factor alpha-induced apoptosis of mouse cells expressing polyomavirus middle and small T antigens1997In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 71, no 1, p. 276-283Article in journal (Refereed)
    Abstract [en]

    Infection with some virus types induces susceptibility to the cytotoxic effect of tumor necrosis factor alpha (TNF-alpha). To investigate whether expression of polyomavirus proteins has this effect on cells, the TNF-alpha sensitivity of C127 and L929 mouse cells transfected with viral DNA was analyzed. Expression of all three polyomavirus early proteins, the tumor (T) antigens, had no apparent effect. In contrast, middle T antigen by itself induced hypersensitivity to TNF-alpha. This effect was reversed by retransfection of the cells with DNA encoding small T antigen. Expression of this polypeptide also decreased the sensitivity of bovine papillomavirus type 1-transformed cells to TNF- alpha, showing that the protective function of the polyomavirus small T antigen was not strictly linked to a middle-T-antigen-induced event. Mouse and human TNF-alpha had the same effect on normal and transformed mouse cells, suggesting that this effect was mediated by TNF receptor 1. Consistent with this conclusion, all cell clones used in the experiments expressed TNF receptor 1 at similar levels, while we failed to detect TNF receptor 2. The amount of receptor on the cells was not influenced by binding of the ligand. Addition of TNF-alpha at cytotoxic concentrations to cells expressing middle T antigen by itself resulted in significant fragmentation of chromosomal DNA after only a few hours, indicating induction of apoptosis. Addition of the cytokine to these cells also leads to release of arachidonic acid, showing that phospholipase A2 was activated. However, production of arachidonic acid did not appear to significantly precede loss of cell viability.

  • 37.
    Bergström, Jennie
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nested PCR for distinguishing Haemophilus haemolyticus from Haemophilus influenzae and Cloning and expression of fragmented Moraxella catarrhalis IgD-binding protein in E. coli2007Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    ABSTRACT

    Nontypable Haemophilus influenzae is a common cause of otitis, sinusitis and conjunctivitis. It is the most common bacterial pathogen associated with chronic obstructive pulmonary disease (COPD). Studies have shown that nonpathogenic Haemophilus haemolyticus are often mistaken for Haemophilus influenzae due to an absent hemolytic reaction on blood agar. Distinguishing H. haemolyticus from H. influenzae is important to prevent unnecessary antibiotic use, and to understand the role of H. influenzae in clinical infections. In this study, PCR-primers for amplifying 16S rDNA sequences were used to set up a method for distinguishing H. haemolyticus from H. influenzae. The aim was to use the method for analyzing apparent H. influenzae strains, to investigate if some strains were in fact H. haemolyticus. However, because of problems with unspecific primerannealing,no conclusions could be drawn regarding misclassification of H. haemolyticus.

    Moraxella catarrhalis is the second most common bacterial pathogen associated with COPD. It also causes otitis and sinusitis. An important virulence factor of M. catarrhalis is the outer membrane protein Moraxella catarrhalis IgD-binding protein (MID). One part of the protein; MID764-913 , has been shown to function as an adhesin, and this part has been fragmented to further investigate its adhesive properties. The aim of this second, independent study, was to express some of these proteinfragments by cloning in E. coli. The time spent on this project was too short, and no proteins could be expressed duing this period.

  • 38.
    Bergström, Joakim J E
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Heyman, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    IgG Suppresses Antibody Responses in Mice Lacking C1q, C3, Complement Receptors 1 and 2, or IgG Fc-Receptors.2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11Article in journal (Refereed)
    Abstract [en]

    Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.

  • 39.
    Bergström, Joakim J. E.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Westin, Annika Grahn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heyman, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    IgG-Mediated Suppression of Primary IgG Anti-SRBC Responses is not Dependent on Fc gamma R or Complement2014In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 79, no 6, p. 443-443Article in journal (Other academic)
  • 40. Bergvist, Anders
    et al.
    Nilsson, Mats
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Immunology and Microbiology.
    Magnusson, Göran
    Loss of DNA-binding and new transcriptional trans activation function in polymavirus large T-antigen with mulation of zinc finger motif1990In: Nucleic Acids Research, Vol. 18, no 9, p. 2715-2720Article in journal (Refereed)
  • 41.
    Bernander, Sverker
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences. Sektionen för klinisk mikrobiologi.
    Jacobson, Kerstin
    Lundholm, Monica
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences. Sektionen för klinisk mikrobiologi.
    A hospital-associated outbreak of Legionnaires´ disease caused by Legionella pneumophila serogroups 4 and 10 with a common fingerprinting pattern.2004In: APMIS, Vol. 112, no 3, p. 210-7Article in journal (Refereed)
  • 42.
    Björkman, J., Samuelsson, P., Andersson, D.I. and Hughes, D.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium.1999In: Mol. Microbiol., Vol. 31, p. 53-58Article in journal (Refereed)
  • 43.
    Blomberg, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Benachenhou, Farid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Blikstad, Vidar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Sperber, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Mayer, Jens
    Dpt of Human Genetics, Medical Faculty, University of Saarland, Homburg, Germany.
    Classification and nomenclature of endogenous retroviral sequences (ERVs): problems and recommendations2009In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 448, no 2, p. 115-123Article, review/survey (Refereed)
    Abstract [en]

    The genomes of many species are crowded with repetitive mobile sequences. In the case of endogenous retroviruses (ERVs) there is, for various reasons, considerable confusion regarding names assigned to families/groups of ERVs as well as individual ERV loci. Human ERVs have been studied in greater detail, and naming of HERVs in the scientific literature is somewhat confusing not just to the outsider. Without guidelines, confusion for ERVs in other species will also probably increase if those ERVs are studied in greater detail. Based on previous experience, this review highlights some of the problems when naming and classifying ERVs, and provides some guidance for detecting and characterizing ERV sequences. Because of the close relationship between ERVs and exogenous retroviruses (XRVs) it is reasonable to reconcile their classification with that of XRVs. We here argue that classification should be based on a combination of similarity, structural features, (inferred) function, and previous nomenclature. Because the RepBase system is widely employed in genome annotation, RepBase designations should be considered in further taxonomic efforts. To lay a foundation for a phylogenetically based taxonomy, further analyses of ERVs in many hosts are needed. A dedicated, permanent, international consortium would best be suited to integrate and communicate our current and future knowledge on repetitive, mobile elements in general to the scientific community.

  • 44.
    Bohl Kullberg, Erika
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Tumor Cell Targeting of Stabilized Liposome Conjugates: Experimental studies using boronated DNA-binding agents2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    To further develop cancer therapy, targeted delivery of cell killing agents directly to tumor cells is an interesting approach. This thesis describes the development of PEG-stabilized liposome conjugates targeting either epidermal growth factor receptor (EGFR) using its natural ligand EGF, or human epidermal growth factor receptor 2 (HER-2) using the antibody trastuzumab. Both receptors are known to be overexpressed on a variety of tumors. The liposomes were loaded with the boronated compounds water soluble boronated acridine (WSA) or water soluble boronated phenantridine (WSP), compounds primarily developed for boron neutron capture therapy, BNCT.

    The liposome conjugates bound specifically to their receptors in cell culture. Because the WSA conjugates exhibited the most favorable boron uptake this compound was chosen for further study. The WSA-loaded liposome conjugates was internalized, an important characteristic for BNCT, and had a long retention inside the cells. The cellular localization of WSA, studied using fluorescence was found to be mainly cytoplasmic.

    To increase the boron uptake studies comparing different incubation methods was performed. It was shown for both EGF and trastuzumab targeted liposomes the uptake could be increased over 10 times by changing from incubation in monolayer culture to incubation in cell suspension in roller flasks. With this treatment the boron concentrations reached after 24 h incubation time was 90 ppm for EGF-liposomes and 132 ppm for trastuzumab-liposomes, levels that are clinically interesting.

    To study the cell-killing efficacy of the liposome-conjugates an experimental BNCT study was performed using EGF-liposome-WSA on cultured glioma cells. About half the number of thermal neutron was needed to inactivate 90% of the cells if the cells had been incubated with EGF-liposome-WSA compared to control cells. When comparing the survival to dose it was shown that to inactivate 90% of the cells 2.9 Gy was needed for EGF-liposome-WSA and neutrons compared to 5.6 Gy with 137Cs gamma.

    The biodistribution of EGF-liposomes was also studied in mice. It was compared to EGF and it was found that the addition of a PEG-stabilized liposome to EGF significantly reduced EGF uptake in liver and kidneys, the circulation time in blood was prolonged as well. The reduced liver uptake might be due to inability of the 100 nm liposomes to pass the sinusoidal fenestrations of the liver and bind to the EGFR-rich hepatocytes. The reduced liver uptake potentates the use of EGF-liposome conjugates for systemic injection.

  • 45.
    Bondeson, K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Frostell-Karlsson, A
    Fägerstam, L
    Magnusson, G
    Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.1993In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 214, no 1, p. 245-51Article in journal (Refereed)
    Abstract [en]

    Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.

  • 46.
    Bondeson, K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rönn, O
    Magnusson, G
    DNA binding of polyomavirus large T-antigen: kinetics of interactions with different types of binding sites.1998In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 423, no 3, p. 307-13Article in journal (Refereed)
    Abstract [en]

    Polyomavirus large T-antigen binds to GRGGC sites in double-stranded viral DNA, regulating transcription and replication. Using surface plasmon resonance to record interactions of large T-antigen with different types of binding sites, we found that the configuration of recognition motifs influenced both the association and dissociation rates. Particularly, the complex formed at the origin of DNA replication was labile. A comparison of the interactions between large T-antigen and binding sites with one, two and four GRGGC motifs in tandem showed a strong preference for dimer binding, without detectable co-operativity between dimers. Sodium chloride stabilised the complexes, whereas the dissociation increased rapidly by increasing pH above 7.0.

  • 47.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Immunology and Microbiology.
    Mouse polyomavirus large T-antigen: DNA-binding and related activities1995Book (Other academic)
  • 48.
    Bondeson, Kåre
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Immunology and Microbiology.
    Frostellkarlsson, Aa
    Fagerstam, L
    Magnusson, Göran
    Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor1993In: Analytical biochemistry, Vol. 214, no 1, p. 245-251Article in journal (Refereed)
  • 49.
    Bondeson, Kåre
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Immunology and Microbiology.
    Rönn, Ola
    Magnusson, Göran
    DNA binding of polyomavirus large T-antigen: kinetics of interactions with different types of binding sites1998In: FEBS letters, Vol. 423, no 3, p. 307-313Article in journal (Refereed)
  • 50.
    Bondeson, Kåre
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Immunology and Microbiology.
    Rönn, Ola
    Magnusson, Göran
    Preferred DNA-binding-Sites of Polyomavirus Large T-antigen1995In: European Journal of Biochemistry, Vol. 227, no 1-2, p. 359-366Article in journal (Refereed)
1234567 1 - 50 of 372
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