| 1. |
- Araya, Zufan, et al.
(författare)
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Metabolism of 25-hydroxyvitamin D3 by microsomal and mitochondrial vitamin D3 25-hydroxylases (CYP2D25 and CYP27A1) : a novel reaction by CYP27A1
- 2003
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - 1388-1981 .- 1879-2618. ; 1632:1-21-3, s. 40-47
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Tidskriftsartikel (refereegranskat)abstract
- The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).
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| 2. |
- Rotticci, D., et al.
(författare)
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An active-site titration method for lipases
- 2000
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - 1388-1981 .- 1879-2618. ; 1483:1, s. 132-140
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Tidskriftsartikel (refereegranskat)abstract
- A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate, This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.
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| 3. |
- Svartz, Jesper, 1972-, et al.
(författare)
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Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer
- 2003
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier. - 1388-1981 .- 1879-2618. ; 1633:2, s. 90-95
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Tidskriftsartikel (refereegranskat)abstract
- Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC4 and its metabolites LTD4 and LTE4 stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC4 is formed by addition of glutathione to LTA4, catalyzed by the integral membrane protein, LTC4 synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.
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| 4. |
- Lundell, Kerstin, et al.
(författare)
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Gene structure of pig sterol 12alpha-hydroxylase (CYP8B1) and expression in fetal liver : comparison with expression of taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21)
- 2003
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981 .- 1879-2618. ; 1634:3, s. 86-96
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Tidskriftsartikel (refereegranskat)abstract
- Cholic acid is the major trihydroxy bile acid formed in most mammals. The domestic pig (Sus scrofa) is an exception. The bile of adult pig is devoid of cholic acid whereas hyocholic acid is found in amounts equal to that of cholic acid in humans. The pathway leading to formation of hyocholic acid is believed to be species-specific and to have evolved in the pig to compensate for a nonexistent or deficient cholic acid biosynthesis. However, a high level of cholic acid has recently been found in the bile of fetal pig. Here we describe that a gene encoding the key enzyme in cholic acid biosynthesis, the sterol 12alpha-hydroxylase (CYP8B1), is in fact present in the pig genome. The deduced amino acid sequence shows 81% identity to the human and rabbit orthologues. CYP8B1 mRNA is expressed at significant levels in fetal pig liver. Both CYP8B1 and the key enzyme in hyocholic acid formation, taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21), were found to be expressed in pig liver in a developmental-dependent but opposite fashion.
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| 5. |
- Ekblad, Lars, et al.
(författare)
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Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains
- 2001
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - 1388-1981. ; 1531:3, s. 209-221
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Tidskriftsartikel (refereegranskat)abstract
- The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.
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| 6. |
- Raclot, Thierry, et al.
(författare)
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A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids
- 2001
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - 1388-1981. ; 1532:1-2, s. 88-96
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Tidskriftsartikel (refereegranskat)abstract
- The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most(18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.
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| 7. |
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| 8. |
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| 9. |
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| 10. |
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| 11. |
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| 12. |
- Andersson, Mats X., 1977, et al.
(författare)
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The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope :
- 2004
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Ingår i: Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981. ; 1684:1-3, s. 46-53
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Tidskriftsartikel (refereegranskat)abstract
- Here we report that cytosolic phospholipases are involved in the utilization of phosphatidylcholine (PC) as substrate for chloroplast-localized synthesis of monogalactosyldiacylglycerol (MGDG). Isolated chloroplasts were pre-incubated with lysoPC and [C-14]18:0-CoA to form [C-14]PC. When soluble plant proteins (cytosol) and UDP-galactose were added, [C-14] MGDG was formed. An inhibitor of phospholipase D markedly lowered the formation of [C-14]MGDG, whereas thermolysin pretreatment of the chloroplasts was without effect. The cytosolic activity resided in the >100-kDa fraction. In a second approach, [C-14]PC-containing lipid mixtures were incubated with cytosol. Degradation of [C-14]PC to [C-14]diacylglycerol was highest when the lipid composition of the mixture mimicked that of the outer chloroplast envelope. We also investigated whether PC of extraplastidic origin could function as substrate for MGDG synthesis. Isolated chloroplasts were incubated with enriched endoplasmic reticulum containing radiolabelled acyl lipids. In the presence of cytosol and UDPgalactose, there was a time-dependent transfer of [C-14]PC from this fraction to chloroplasts, where [C-14]MGDG was formed. We conclude that chloroplasts recruit cytosolic phospholipase D and phosphatidic acid phosphatase to convert PC to diacylglycerol. Apparently, these lipases do not interact with chloroplast surface proteins, but rather with outer membrane lipids, either for association to the envelope or for substrate presentation. (C) 2004 Elsevier B.V. All rights reserved.
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| 13. |
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| 14. |
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| 16. |
- Massih, Ali R, et al.
(författare)
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Phase ordering under quenching : a case of Zr-alloy
- 2004
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Ingår i: Journal of Physics and Chemistry of Solids. - : Elsevier. - 0022-3697 .- 1879-2553. ; 65:6, s. 1193-1198
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Tidskriftsartikel (refereegranskat)abstract
- The Lifshitz–Cahn–Allen kinetic theory of second-order phase transition has been successfully applied to the case of microstructure development of Zr alloy subjected to different cooling rates under the allotropic β→α transformation. In particular, the dependence of α-Zr lamella width on quenching rate observed in the Widmanstätten structure of the material is captured. The effect of the interstitial element oxygen, acting as a driving conveyor for transformation, is considered as an external field within the Ginzburg-Landau free energy framework.
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| 17. |
- Xu, Ning, et al.
(författare)
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Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells.
- 2004
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981. ; 1683:1-3, s. 33-37
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo. (C) 2004 Elsevier B.V. All rights reserved.
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