| 1. |
- Garcia-Serrano, Ainhoa, et al.
(författare)
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Assessment of bacterial and viral gut communities in healthy and tumoral colorectal tissue using RNA and DNA deep sequencing
- 2023
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Ingår i: Cancer Medicine. - 2045-7634. ; 12:18, s. 19291-19300
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Tidskriftsartikel (refereegranskat)abstract
- Background: Colorectal cancer (CRC) is known to present a distinct microbiome profile compared to healthy mucosa. Non-targeted deep-sequencing strategies enable nowadays full microbiome characterization up to species level. Aim: We aimed to analyze both bacterial and viral communities in CRC using these strategies. Materials & Methods: We analyzed bacterial and viral communities using both DNA and RNA deep-sequencing (Novaseq) in colorectal tissue specimens from 10 CRC patients and 10 matched control patients. Following taxonomy classification using Kraken 2, different metrics for alpha and beta diversities as well as relative and differential abundance were calculated to compare tumoral and healthy samples. Results: No viral differences were identified between tissue types, but bacterial species Polynucleobacter necessarius had a highly increased presence for DNA in tumors (p = 0.001). RNA analyses showed that bacterial species Arabia massiliensis had a highly decreased transcription in tumors (p = 0.002) while Fusobacterium nucleatum transcription was highly increased in tumors (p = 0.002). Discussion: Sequencing of both DNA and RNA enables a wider perspective of micriobiome profiles. Lack of RNA transcription (Polynucleobacter necessarius) casts doubt on possible role of a microorganism in CRC. The association of F. nucleatum mainly with transcription, may provide further insights on its role in CRC. Conclusion: Joint assessment of the metagenome (DNA) and the metatranscriptome (RNA) at the species level provided a huge coverage for both bacteria and virus and identifies differential specific bacterial species as tumor associated.
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| 2. |
- Jonsson Rudsander, Ulla, et al.
(författare)
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Comparative NMR analysis of cellooligosaccharide hydrolysis by GH9 bacterial and plant endo-1,4-ss-glucanases
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:18, s. 5235-5241
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Tidskriftsartikel (refereegranskat)abstract
- H-1 NMR spectroscopy has been used to analyze the product profiles arising from the hydrolysis of cellooligosaccharides by family GH9 cellulases. The product profiles obtained with the wild type and several active site mutants of a bacterial processive endoglucanase, Tf Cel9A, were compared with those obtained by a randomly acting plant endoglucanase, PttCe19A. PttCe19A is an orthologue of the Arabidopsis endocellulase, Korrigan, which is required for efficient cellulose biosynthesis. As expected, poplar PttCe19A was shown to catalyze the degradation of cellooligosaccharides by inversion of the configuration of the anomeric carbon. The product analyses showed that the number of interactions between the glucose units of the substrate and the aromatic residues in the enzyme active sites determines the point of cleavage in both enzymes.
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| 3. |
- Jonsson Rudsander, Ulla, 1973-
(författare)
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Functional studies of a membrane-anchored cellulase from poplar
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details. This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana. Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario. The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus.
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| 4. |
- Kujawa, Magdalena, et al.
(författare)
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Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:46, s. 35104-35115
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose2-oxidase(P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regios-electivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k(cat), and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp(452) in the substrate loop and, possibly, a nearby arginine residue (Arg(472)). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.
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| 5. |
- Lindqvist, Ulla, et al.
(författare)
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IgA antibodies to gliadin and coeliac disease in psoriatic arthritis
- 2002
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Ingår i: Rheumatology (Oxford, England). - 1462-0324. ; 41:1, s. 31-37
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To find out whether patients with psoriatic arthritis (PsoA) have an increased prevalence of antibodies to gliadin (AGA) and of coeliac disease. METHODS: One hundred and fourteen PsoA patients with skin disease of 20+/-13 yr and joint disease of 11+/-10 yr duration answered a questionnaire concerning their medical history and underwent clinical examination, including radiology. Serum IgA AGA and IgG AGA, IgA antibodies to endomysium and immunoglobulins, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) concentration were determined. RESULTS: Five of the 114 patients (4.4%) had coeliac disease. After exclusion of these five patients, the mean IgA AGA concentration was significantly higher (P=0.0005) than that in a reference group. None of the patients had IgA antibodies to endomysium. The mean serum IgA concentration was significantly increased and IgM decreased. Patients with a high concentration of IgA AGA had significantly higher ESR and CRP and a longer duration of morning stiffness than those with a low AGA concentration. CONCLUSIONS: Patients with PsoA have an increased prevalence of raised serum IgA AGA and of coeliac disease. Patients with raised IgA AGA seem to have more pronounced inflammation than those with a low IgA AGA concentration.
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| 6. |
- Master, Emma, et al.
(författare)
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Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:31, s. 10080-10089
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Tidskriftsartikel (refereegranskat)abstract
- PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.
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| 7. |
- Monari, Magda, et al.
(författare)
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Synthesis of New Thiol Derivatives of Os3(CO)12; Crystal Structure of HOs3(CO)10(SBut)
- 1996
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Ingår i: Inorganica Chimica Acta. - : Elsevier BV. - 0020-1693. ; 247:1, s. 131-134
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Tidskriftsartikel (refereegranskat)abstract
- The clusters [Os3(CO) 10(/z-H) {/z-SC(CH3)3} ] (1) and [Os3(CO) to(/z-H) {p-SCeHH } ] (2) have been synthesized by addition of the appropriate thiol RSH (R ffi C(CH3)3, CeHH) to [Os3(CO) it (NCMe) ] or [Os3(CO) io(NCMe)2] in dichloromethane. Clusters 1 and 2 have been characterized by 1R, NMR, mass speetromet~, and, in the case of 1, X-ray crystallography. Compound I crystallizes in space group P2~/n (affi9.507(1), bffi 12.860(1), cffi 17.838(3) A, flffi99.77(1) °, Zffi4). The molecule conforms to an idealized C, symmetry. The bond distances in the osmium triangle average 2.860 A; there is no significant elongation of the Os-Os edge in I that is bridged by the hydride and the thiolate moiety.
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| 8. |
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| 9. |
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| 10. |
- Rudsander, Ulla, et al.
(författare)
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Molecular features of family GH9 cellulases in hybrid aspen and the filamentous fungus Phanerochaete chrysosporium
- 2003
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Ingår i: Journal of applied glycoscience. - 1344-7882. ; 50:2, s. 253-256
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Tidskriftsartikel (refereegranskat)abstract
- Most plant cellulases belong to the glycosyl hydrolase family 9, which is both enzymatically and structurally well characterized in microbes. The microbial enzymes include both randomly acting endoglucanases active on soluble and amorphous substrates and processive endoglucanases, which can also degrade crystalline cellulose. The corresponding plant enzymes have been difficult to purify and express in heterologous hosts and thus their modes of action have remained obscure. Here we have taken a molecular modelling approach and describe some structural features characteristic for soluble and membrane-bound family 9 cellulases in plants and filamentous fungi.
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