| 1. |
- Gu, Chungang, et al.
(författare)
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Lipid Peroxide-Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples
- 2015
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Ingår i: Drug Metabolism And Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 43:10, s. 1441-1449
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Tidskriftsartikel (refereegranskat)abstract
- This study focused on the mechanistic interpretation of ex vivo oxidation of a candidate drug in blood plasma samples. An unexpected lipid peroxide-mediated epoxidation followed by a dramatic rearrangement led to production of a five-membered oxazole derivative from the original six-membered pyrazinone-carboxamide core of a human neutrophil elastase inhibitor, 6-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-N-ethyl-5-methyl-3-oxo-4-(3-(trifluoromethyl) phenyl)-3,4-dihydropyrazine-2-carboxamide (AZD9819). The rearranged oxidation product 2-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-5-(N-ethylacetamido)-N-(3-(trifluoromethyl)phenyl)oxazole-4-carboxamide was characterized by accurate-mass tandem mass spectrometry fragmentations, by two-dimensional NMR and X-ray crystallography of an authentic standard, and by incorporation of an O-18 atom from molecular O-18(2) to the location predicted by our proposed mechanism. The lipid peroxide-mediated oxidation was demonstrated by using human low-density lipoprotein (LDL) in pH 7.4 phosphate buffer and by inhibiting the oxidation with ascorbic acid or L-glutathione, two antioxidants effective in both plasma and the LDL incubation. A nucleophilic mechanism for the epoxidation of AZD9819 by lipid hydroperoxides explains the prevention of its ex vivo oxidation by acidification of the plasma samples. The discovery of the lipid peroxide-dependent oxidation of an analyte and the means of prevention could provide valuable information for biotransformation and bioanalysis.
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| 2. |
- Johnsson, Eskil
(författare)
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Studies on streptococcal M proteins. Interactions with IgA and human complement regulators
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human pathogenic bacterium Streptococcus pyogenes (group A Streptococcus) expresses several different virulence factors. Of these, the M protein is regarded as one of the most important, because it confers resistance to phagocytosis, allowing the bacterium to multiply in blood. A remarkable property of M proteins is their ability to bind different human plasma proteins, in particular components of the immune system. This property is believed to be of importance for the biological functions of M protein. This thesis describes studies of two such interactions: binding of IgA and binding of regulators of complement activation. Many M proteins bind to the Fc part of IgA, the major immunoglobulin on mucosal surfaces. The biological role of this binding is not yet known, but the IgA-binding proteins are valuable model systems for studies of IgA. Alignment of the amino acid residue sequences of five different IgA-binding M proteins identified a putative IgA-binding region. Further analysis of one M protein demonstrated that a 29-residue region was necessary and sufficient for IgA-binding. Subsequent work showed that the IgA-binding region can be studied in the form of a synthetic peptide. Indeed, a 50-residue peptide including the IgA-binding region binds IgA with high specificity, and the properties of this peptide indicate that it corresponds to an isolated IgA-binding domain. An important property of M proteins is the presence of an N-terminal hypervariable region, which probably plays a major role in pathogenesis. However, the function of the hypervariable region has remained unknown. We have found that the hypervariable region of many M proteins specifically binds a ligand, the human complement regulator C4BP. Alignment of the amino acid residue sequences of several C4BP-binding regions did not reveal any identities sufficient to explain the binding of C4BP. Thus, hypervariable regions with highly divergent primary sequences all bind the same ligand, C4BP. This ability to bind C4BP may explain the capacity of these M proteins to confer phagocytosis resistance, since bound C4BP may protect the bacterium against complement attack. However, some M proteins do not bind C4BP. Analysis of two such M proteins demonstrated that their hypervariable regions bind another human complement regulator, FHL-1. Thus, the studies with C4BP and FHL-1 made it possible to attribute a specific function to the hypervariable region of many M proteins: the binding of a human complement regulator.
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| 3. |
- Krais, Annette M., et al.
(författare)
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Excretion of Urinary Metabolites of the Phthalate Esters DEP and DEHP in 16 Volunteers after Inhalation and Dermal Exposure
- 2018
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Ingår i: International Journal of Environmental Research and Public Health. - : MDPI AG. - 1660-4601. ; 15:11
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Tidskriftsartikel (refereegranskat)abstract
- Phthalate esters are suspected endocrine disruptors that are found in a wide range of applications. The aim of this study was to determine the excretion of urinary metabolites in 16 individuals after inhalation and/or dermal exposure to 100⁻300 µg/m³ of deuterium-labelled diethyl phthalate (D₄-DEP) and bis(2-ethylhexyl) phthalate (D₄-DEHP). Dermal exposure in this study represents a case with clean clothing acting as a barrier. After inhalation, D₄-DEP and D₄-DEHP metabolites were excreted rapidly, though inter-individual variation was high. D₄-DEP excretion peaked 3.3 h (T½ of 2.1 h) after combined inhalation and dermal exposure, with total excreted metabolite levels ranging from 0.055 to 2.351 nmol/nmol/m³ (nmol of urinary metabolites per phthalates air concentration in (nmol/m³)). After dermal exposure to D₄-DEP, metabolite excretion peaked 4.6 h (T½ of 2.7 h) after exposure, with excreted metabolite levels in between 0.017 and 0.223 nmol/nmol/m³. After combined inhalation and dermal exposure to D₄-DEHP, the excretion of all five analysed metabolites peaked after 4.7 h on average (T½ of 4.8 h), and metabolite levels ranged from 0.072 to 1.105 nmol/nmol/m³ between participants. No dermal uptake of particle phase D₄-DEHP was observed. In conclusion, the average excreted levels of metabolites after combined inhalation and dermal exposure to D₄-DEP was three times higher than after combined exposure to D₄-DEHP; and nine times higher than after dermal exposure of D₄-DEP. This study was made possible due to the use of novel approaches, i.e., the use of labelled phthalate esters to avoid the background concentration, and innovative technique of phthalate generation, both in the particle and the gas phase.
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| 4. |
- Morfeldt, E, et al.
(författare)
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Isolated hypervariable regions derived from streptococcal M proteins specifically bind human C4b-binding protein : implications for antigenic variation.
- 2001
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 167:7, s. 3870-7
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Tidskriftsartikel (refereegranskat)abstract
- Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the approximately 50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HVRs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function.
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| 5. |
- Stålhammar-Carlemalm, Margaretha, et al.
(författare)
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Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine.
- 2007
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Ingår i: Cell Host and Microbe. - : Elsevier BV. - 1934-6069 .- 1931-3128. ; 2:6, s. 427-434
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Tidskriftsartikel (refereegranskat)abstract
- Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development.
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| 6. |
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