13 research outputs found

    Castellon - Brunelleschis runtomsvängade kran

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    Kandidatuppsats 20 p, Bygghantverksprogrammet vid Institutionen för kulturvård, HT 200

    The Carboxyl Ester Lipase Gene. Organization, Evolution and Transcriptional Regulation in Exocrine Pancreas and Mammary Gland

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    The carboxyl ester lipase (CEL) is an important enzyme for the intestinal absorption of dietary lipids. In humans the gene is highly expressed in exocrine pancreas and in the lactating mammary gland. It is secreted in an inactive form and becomes activated by bile salts in the intestine. Since the infants own capacity to digest milk lipids is low during the first months, due to a poorly developed pancreatic function, the presence of CEL in milk seems to secure fat absorption. In this work we have cloned and characterized the human CEL gene. The gene spans 9,832 bp and contains 11 exons interrupted by introns. We also cloned a CEL pseudogene, which due to a large deletion lacks the corresponding CEL gene exons 2 to 7. Both genes were assigned to separate loci at chromosome 9q34-qter. Further analysis of a 30 kb cosmid fragment revealed that the genes are arranged head to tail approximately 11 kb apart with the CEL gene as the most 5\u27 one. Analysis suggests that this is the result of gene duplication in which an 11,719 bp large fragment, containing the functional CEL gene has been duplicated. The molecular mechanism seems to involve over-replication and a transposition-like event. Analysis of the regulation in pancreatic cells revealed that high level, tissue specific, expression is established by the proximal 839 base pairs of the 5\ub4-flanking region. This is due to a new, strong, pancreas specific enhancer, located at -672. By further dissection we could show that the enhancer is composed of two closely located cis-elements. The proximal element mediates a positive effect whereas the distal element exerts a silencing effect on the positive element. The functional enhancer complex is composed of ubiquitously expressed factors and the net activity is the result of a tissue specific balance between factors interacting with the two elements. In mouse mammary gland endogenous CEL gene expression was detected at day 14 of pregnancy. STAT5 isoforms interact with GAS sites in the promoter but without affecting the activity. The enhanced CEL gene expression is not directly induced by lactogenic hormone but instead indirectly by correlation to the increase in number of differentiated epithelial cells. This demonstrates that prolactin/Stat5 signaling is not the general and direct mechanism explaining the activation of all milk protein genes. Instead, we could identify a major positive element at ^1792 that is required for high level, tissue specific expression and further analysis revealed that this element is a palindromic CTF/NF1 motif. Since this element was inactive in pancreatic cells we suggests that the activity is due to a mammary gland specific CTF/NF-1 complex. Together these two studies show that the expression of the CEL gene in the mammary gland and pancreas respectively, is due to two different regulatory systems

    Cell adhesion on supported lipid bilayers

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    The cell and protein repellent properties of supported phospholipid bilayer (SPB) membranes were investigated. The SPBs were prepared by vesicle adsorption on SiO(2) surfaces. The vesicles of phosphatidylcholine fuse and rupture, and form a supported bilayer covering the surface. We carried out cell culture experiments on several surfaces, including SPBs, using two types of epithelial cells to address the cell adhesional properties. The Quartz Crystal Microbalance Dissipation (QCM-D) technique was used to monitor the SPB formation and subsequent protein adsorption. Neither cell type adhered or proliferated on SiO(2) surfaces coated with SPBs, whereas both cell types adhered and proliferated on the three control surfaces of SiO(2), tissue culture glass, and TiO(2). The QCM-D measurements showed that about two orders of magnitude less mass adsorbed on a SPB surface compared to a TiO(2) surface, from serum-containing media (10% fetal bovine serum). The reduced adsorption on the SPB is a likely explanation for the nondetectable epithelial cell adhesion on the SPB surface. Biomembranes are therefore attractive candidate systems to achieve alternating cell-resistant and cell-interacting regions on surfaces, by including specific cell-binding proteins in the latter regions
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