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- Erlandsson, Ann, et al.
(författare)
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Studies of the interactions between the anticytokeratin 8 monoclonal antibody TS1, its antigen and its anti-idiotypic antibody αTS1
- 2003
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 16:3, s. 157-163
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Tidskriftsartikel (refereegranskat)abstract
- The monoclonal antibody TS1 against cytokeratin 8 and its antiidiotype αTS1 have been used for immunotargeting and therapy of carcinomas in experimental tumor model systems. The interaction surfaces between mab TS1, the cytokeratin 8 epitope, and its anti-idiotypic antibody, αTS1, were studied in detail in order to make future veneering of the interactions possible. The V-genes of TS1 and αTS1 were cloned and sequenced and the CDRs and the framework residues were identified. Amino acids participating in the interactions were identified following chemical modifications of residues in non-protected and protected molecules of cytokeratin 8, αTS1 and TS1. From the sequences, the three-dimensional structures were generated using computer modelling of the antibody variable regions. Several charged amino acid, histidine and tyrosine residues were displayed in the antibody surfaces implicated in the interactions and chemical modification confirmed the importance of these amino acids. The cytokeratin 8 epitope has previously been identified by Johansson et al. and it displays negatively charged amino acid residues which could be identified in the chemical modification. It was also revealed that the TS1 binding to cytokeratin 8 and αTS1 respectively are partly overlapping; a histidine identified in TS1 is probably involved only in the interaction with αTS1. Furthermore, the chemical modification demonstrated that exchanging aspartic–glutamic acids to asparagine–glutamine residues in TS1 increased the binding of TS1 to cytokeratin 8, indicating that there is at least one acidic amino acid that is an obstacle in the TS1–CK8 binding. The detailed assembly of the interaction surfaces will facilitate the future use of site directed mutagenesis to improve the TS1–CK8 association rate and the clearing of TS1 with αTS1 in vivo.
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- Olofsson, Charlotta, et al.
(författare)
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Palmitate Stimulation of Glucagon Secretion in Mouse Pancreatic {alpha}-Cells Results From Activation of L-Type Calcium Channels and Elevation of Cytoplasmic Calcium.
- 2004
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 53:11, s. 2836-2843
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic α-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid–free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1–15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K+ or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca2+ when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca2+ current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca2+ channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca2+-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a ∼50% inhibition of somatostatin release. We conclude that palmitate increases α-cell exocytosis principally by enhanced Ca2+ entry via l-type Ca2+ channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring δ-cells.
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