| 1. |
- Domino, Steven E, et al.
(författare)
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Cervical mucins carry alpha(1,2)fucosylated glycans that partly protect from experimental vaginal candidiasis.
- 2009
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Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 26:9, s. 1125-34
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Tidskriftsartikel (refereegranskat)abstract
- Cervical mucins are glycosylated proteins that form a protective cervical mucus. To understand the role of mucin glycans in Candida albicans infection, oligosaccharides from mouse cervical mucins were analyzed by liquid chromatography-mass spectrometry. Cervical mucins carry multiple alpha(1-2)fucosylated glycans, but alpha(1,2)fucosyltransferase Fut2-null mice are devoid of these epitopes. Epithelial cells in vaginal lavages from Fut2-null mice lacked Ulex europaeus agglutinin-1 (UEA-I) staining for alpha(1-2)fucosylated glycans. Hysterectomy to remove cervical mucus eliminated UEA-I and acid mucin staining in vaginal epithelial cells from wild type mice indicating the cervix as the source of UEA-I positive epithelial cells. To assess binding of alpha(1-2) fucosylated glycans on C. albicans infection, an in vitro adhesion assay was performed with vaginal epithelial cells from wild type and Fut2-null mice. Vaginal epithelial cells from Fut2-null mice were found to bind increased numbers of C. albicans compared to vaginal epithelial cells obtained from wild type mice. Hysterectomy lessened the difference between Fut2-null and wild type mice in binding of C. ablicans in vitro and susceptibility to experimental C. albicans vaginitis in vivo. We generated a recombinant fucosylated MUC1 glycanpolymer to test whether the relative protection of wild type mice compared to Fut2-null mice could be mimicked with exogenous mucin. While a small portion of the recombinant MUC1 epitopes displayed alpha(1-2)fucosylated glycans, the predominant epitopes were sialylated due to endogenous sialyltransferases in the cultured cells. Intravaginal instillation of recombinant MUC1 glycanpolymer partially reduced experimental yeast vaginitis suggesting that a large glycanpolymer, with different glycan epitopes, may affect fungal burden.
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| 2. |
- Ambort, Daniel, 1978, et al.
(författare)
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Function of the CysD domain of the gel-forming MUC2 mucin.
- 2011
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Ingår i: Biochem Journal. ; 436, s. 61-70
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Tidskriftsartikel (refereegranskat)abstract
- The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.
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| 3. |
- Bäckström, Malin, 1967, et al.
(författare)
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Increased understanding of the biochemistry and biosynthesis of MUC2 and other gel-forming mucins through the recombinant expression of their protein domains.
- 2013
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Ingår i: Molecular biotechnology. - : Springer Science and Business Media LLC. - 1559-0305 .- 1073-6085. ; 54:2, s. 250-6
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Tidskriftsartikel (refereegranskat)abstract
- The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins.
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| 4. |
- Ekendahl, Susanne, 1965, et al.
(författare)
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Characterisation of Yeasts isolated from deep igneous rock aquifers of the fennoscandian shield
- 2003
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Ingår i: Microbial Ecology. - : Springer Science and Business Media LLC. - 0095-3628 .- 1432-184X. ; 46:4, s. 416-428
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Tidskriftsartikel (refereegranskat)abstract
- The diversity of prokaryotes in the groundwater deep below the surface of the Baltic Sea at the Aspo Hard Rock Laboratory (HRL) in southeast Sweden is well documented. In addition, there is some evidence that eukaryotes, too, are present in the deep groundwater at this site, although their origins are uncertain. To extend the knowledge of eukaryotic life in this environment, five yeast, three yeastlike, and 17 mold strains were isolated from Aspo HRL groundwater between 201 and 444 m below sea level. Phenotypic testing and phylogenetic analysis of 18S rDNA sequences of the five yeast isolates revealed their relationships to Rhodotorula minuta and Cryptococcus spp. Scanning and transmission electron microscopy demonstrated that the strains possessed morphological characteristics typical for yeast, although they were relatively small, with an average length of 3 mum. Enumeration through direct counting and most probable number methods showed low numbers of fungi, between 0.01 and 1 cells mL(-1), at some sites. Five of the strains were characterized physiologically to determine whether they were adapted to life in the deep biosphere. These studies revealed that the strains grew within a pH range of 4-10, between temperatures of 4degreesC and 25-30degreesC, and in NaCl concentrations from 0 to 70 g L-1. These growth parameters suggest a degree of adaptation to the groundwater at Aspo HRL. Despite the fact that these eukaryotic microorganisms may be transient members of the deep biosphere microbial community, many of the observations of this study suggest that they are capable of growing in this extreme environment.
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| 5. |
- Grahn, Anna, 1973, et al.
(författare)
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Varicella-Zoster Virus (VZV) Glycoprotein E Is a Serological Antigen for Detection of Intrathecal Antibodies to VZV in Central Nervous System Infections, without Cross-Reaction to Herpes Simplex Virus 1
- 2011
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Ingår i: Clinical and Vaccine Immunology. - 1556-6811 .- 1556-679X. ; 18:8, s. 1336-1342
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Tidskriftsartikel (refereegranskat)abstract
- Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) cause serious central nervous system (CNS) diseases that are diagnosed with PCR using samples of cerebrospinal fluid (CSF) and, during later stages of such infections, with assays of intrathecal IgG antibody production. However, serological diagnoses have been hampered by cross-reactions between HSV-1 and VZV IgG antibodies and are commonly reported in patients with herpes simplex encephalitis (HSE). In this study we have evaluated VZV glycoprotein E (gE) as a new antigen for serological diagnosis of VZV-induced CNS infections. Paired samples of CSF and serum from 29 patients with clinical diagnosis of VZV CNS infection (n = 15) or HSE (n = 14), all confirmed by PCR, were analyzed. VZV gE and whole VZV were compared as antigens in enzyme-linked immunosorbent assays (ELISAs) for serological assays in which the CSF/serum sample pairs were diluted to identical IgG concentrations. With the gE antigen, none of the HSE patients showed intrathecal IgG antibodies against VZV, compared to those shown by 11/14 patients using whole-VZV antigen (P < 0.001). In the patients with VZV infections, significantly higher CSF/serum optical density (OD) ratios were found in the VZV patients using the VZV gE antigen compared to those found using the whole-VZV antigen (P = 0.001). These results show that gE is a sensitive antigen for serological diagnosis of VZV infections in the CNS and that this antigen was devoid of cross-reactivity to HSV-1 IgG in patients with HSE. We therefore propose that VZV gE can be used for serological discrimination of CNS infections caused by VZV and HSV-1. Biosynthesis of ethylene (ethene) is mainly performed by plants and some bacteria and fungi, via two distinct metabolic routes. Plants use two steps, starting with S-adenosylmethionine, while the ethylene-forming microbes perform an oxygen dependent reaction using 2-oxoglutarate and arginine. Introduction of these systems into Saccharomyces cerevisiae was studied in silico. The reactions were added to a metabolic network of yeast and flux over the two networks was optimised for maximal ethylene formation. The maximal ethylene yields obtained for the two systems were similar in the range of 7-8 mol ethylene/10 mol glucose. The microbial metabolic network was used for testing different strategies to increase the ethylene formation. It was suggested that supplementation of exogenous proline, using a solely NAD-coupled glutamate dehydrogenase, and using glutamate as the nitrogen source, could increase the ethylene formation. Comparison of these in silico results with published experimental data for yeast expressing the microbial system confirmed an increased ethylene formation when changing nitrogen source from ammonium to glutamate. The theoretical analysis methods indicated a much higher maximal yield per glucose for ethylene than was experimentally observed. However, such high ethylene yields could only be obtained with a concomitant very high respiration (per glucose). Accordingly, when ethylene production was optimised under the additional constraint of restricted respiratory capacity (i.e. limited to experimentally measured values) the theoretical maximal ethylene yield was much lower at 0.2/10 mol glucose, and closer to the experimentally observed values.
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| 6. |
- Nilsson, Harriet E., et al.
(författare)
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Intestinal MUC2 mucin supramolecular topology by packing and release resting on D3 domain assembly.
- 2014
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 426:14, s. 2567-79
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Tidskriftsartikel (refereegranskat)abstract
- MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.
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| 7. |
- Persson Berg, Linn, 1984, et al.
(författare)
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Recombinant Epstein-Barr virus glycoprotein 350 as a serological antigen
- 2020
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 284
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus (EBV) glycoprotein 350 (gp350) is the most abundant glycoprotein expressed on the EBV envelope, the major target for neutralizing antibodies and also essential for virion attachment to B lymphocytes. Several studies have addressed EBV gp350 as a vaccine candidate, but less commonly as a potential antigen for serological assays. The aim of the current study was to develop a diagnostic tool to quantify EBV gp350-specific IgG in previously EBV-infected individuals. A construct encoding the extracellular domain of EBV gp350 (amino acid (aa) 1-860) was developed for expression in Chinese hamster ovary cells. Serum samples (n = 360) with known IgG serostatus against viral capsid antigen (VCA) and Epstein-Barr nuclear antigen 1 (EBNA1) were divided into three groups based on the differences in their serostatus: VCA + EBNA1+ (n = 120), VCA + EBNA1-(n = 120) and VCA-EBNA1-(n = 120). The samples were analyzed by indirect ELISA using recombinant EBV gp350 aa 1-860 as antigen. A clear majority, 108 of the 120 VCA + EBNA1+ samples, had detectable EBV gp350-specific IgG. Of the 120 VCA + EBNA1-samples, 79 had detectable EBV gp350-specific IgG. Only 2 of the 120 VCA-EBNA1-samples had detectable EBV gp350-specific IgG. The results reported here show that use of the EBV gp350 aa 1-860 ELISA can serve as a sensitive method for EBV-specific IgG detection in serum samples.
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| 8. |
- Persson Berg, Linn, 1984, et al.
(författare)
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Serum IgG levels to Epstein-Barr and measles viruses in patients with multiple sclerosis during natalizumab and interferon beta treatment
- 2022
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Ingår i: Bmj Neurology Open. - : BMJ. - 2632-6140. ; 4:2
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Tidskriftsartikel (refereegranskat)abstract
- Background Patients with multiple sclerosis (MS) demonstrate higher seroprevalence of Epstein-Barr virus (EBV) and increased anti-EBV IgG levels in serum compared with healthy controls. Intrathecal antibody production to measles virus (MeV) is a common finding in patients with MS. Objective To measure serum IgG reactivity to EBV glycoprotein 350 (gp350) and MeV nucleocapsid protein (N-CORE) in patients with MS and healthy controls and to determine if reactivity changed in patients during interferon beta (IFN beta) and/or natalizumab (NAT) treatment. A secondary aim was to determine the seroprevalence of EBV in patients and controls. Methods Patients with MS (n=728) were included from the Swedish pharmacovigilance study for NAT. Paired serum samples from 714 patients drawn before and during NAT treatment and paired samples from 170 patients during prior IFN beta treatment were analysed. In total, 156 patients were included in both groups. Samples from 144 matched blood donors served as controls. Indirect ELISA was applied using recombinant EBVgp350 and MeV N-CORE as antigens. EBVgp350 IgG seronegative samples were also analysed using EBV nuclear antigen 1 and viral capsid antigen (VCA). Results Patients with MS showed higher serum levels of anti-EBVgp350 and anti-MeV N-CORE IgG compared with controls. During NAT treatment, the levels of anti-EBVgp350 and anti-MeV N-CORE IgG declined, compared with the relatively stable levels noted during prior IFN beta treatment. Ten patients failed to demonstrate anti-EBVgp350 IgG but did show detectable anti-VCA IgG, indicating EBV seropositivity. In contrast, 10/144 controls were EBV seronegative. Conclusions Treatment with NAT, which is considered a selective immunosuppressive agent with a compartmentalised effect on the central nervous system, appeared to be associated with a moderate decrease in circulating IgG levels to EBVgp350 and MeV N-CORE. All patients with MS were EBV IgG seropositive, supporting the potential role of EBV in the pathogenesis of MS.
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| 9. |
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| 10. |
- Thomsson, Elisabeth, 1975
(författare)
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Glycolytic flux regulation in Saccharomyces cerevisiae during anaerobic growth and starvation
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The physiology of S. cerevisiae under anaerobic growth conditions is of interest not least during implementation and development of industrial yeast-catalysed ethanol fermentations in order to maintain a productive yeast population. During growth in industrial fermentations the concentration of lactic acid can often reach considerable concentrations as a result of contamination by lactic acid bacteria. The yeast also often faces complex nutritional conditions including nutrient limitation and/or starvation. The effects of lactic or benzoic acid on glycolytic rate and product yields were studied on cells growing under carbon or nitrogen limiting conditions in anaerobic chemostat cultures (D=0.1 h-1). It was shown that during growth under nitrogen limiting conditions compared to growth under carbon limiting conditions and/or in presence of lactic acid or benzoic acid, the flow of glucose was directed to a larger extent from glycogen and biomass formation towards ethanol production. The specific ethanol production rate (mmol/gh) was also stimulated; however, the effect of lactic acid was not as large as that of benzoic acid or as the effect of growth under nitrogen limitation compared to growth under carbon limitation. High glycolytic rates/specific ethanol production rates were obtained by the weak organic acids and/or nitrogen limitation. Based on comparisons of exhibited glycolytic rate with levels of allosteric effectors of the glycolytic enzymes during the different growth conditions it was suggested that a concerted action of decreased ATP levels and increased fructose-6-phosphate levels may account for at least a part of the increase glycolytic rate during these growth conditions. Studies on the carbon or nitrogen starvation response of anaerobically growing cells were also performed. Carbon starvation of anaerobic exponentially growing cells induced an almost complete loss of fermentative capacity and viability. Nitrogen starvation reduced fermentative capacity by 70-95% depending on strain. The levels of glycolytic enzymes were not altered neither after carbon nor nitrogen starvation, however, a depletion of ATP was seen immediately upon carbon starvation but not nitrogen starvation. Growth into stationary-phase prior to carbon starvation enabled the cells to retain most of their fermentative capacity after carbon starvation. Reduction in protein synthesis prior to starvation of anaerobic exponential-phase cells could also to some extent alleviate the effects of carbon starvation. Further studies on cells grown in chemostat (D=0.1h-1) under different growth conditions that were subsequently exposed to carbon or nitrogen starvation showed that there was a positive relationship between cellular content of endogenous glycogen before carbon starvation and fermentative capacity after carbon starvation. A positive relationship after carbon starvation between the intracellular ATP levels (0-1.5 mM) and the fermentative capacity in cells grown in anaerobic chemostat cultures was also found. From these results it is suggested that anaerobically grown cells not containing any endogenous carbohydrate reserves face a lack of energy upon rapid carbon starvation which renders necessary adjustments to the starvation conditions impossible to perform, thereby reducing viability and fermentative capacity of the cell upon carbon starvation.
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| 11. |
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| 13. |
- Thomsson, Elisabeth, 1975, et al.
(författare)
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Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology.
- 2011
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Ingår i: Journal of virological methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 175:1, s. 53-9
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Tidskriftsartikel (refereegranskat)abstract
- A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV.
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