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Sérové markery aktivity cholesterol 7α hydroxylasy. / Serum markers of cholesterol 7α hydroxylase activityBohdanecká, Alena January 2017 (has links)
Cholesterol 7-hydroxylase (CYP7A1) is the rate limiting enzyme of the classical pathway of bile acid (BA) synthesis, which catabolizes approximately half of cholesterol in man. Determination of CYP7A enzymatic activity is a key subject of lipid metabolism research. Direct determination of CYP7A1 activity in hepatic biopsy is mostly not allowed for ethical reasons, so indirect methods are used with serum markers such as 7α-hydroxy-4-cholestene-3- one (C4). The first, methodical aim of the work was to convert the introduced HPLC method for the determination of C4 to LC-MS in order to increase the sensitivity. We focused on the solid phase extraction step, adjusting the composition and volumes of the washing and elution solution. By converting the method from HPLC to LC-MS, the sensitivity was increased approximately 7 times (LD = 1.39 ng/ml). In the second, clinical part of our work, we attempted to confirm the preliminary results of our laboratory on the distribution of C4 in lipoprotein fractions (LPP) in order to find parameter that would correlate with CYP7A1 activity better than C4 level itself. Preliminary results (performed in healthy individuals) showed that most of C4 is carried on HDL, and that the C4 distribution within LPP fractions is similar among examined subjects. We repeated the...
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Transcriptional Activation of the Cholesterol 7α-Hydroxylase Gene (CYP7A) by Nuclear Hormone ReceptorsCrestani, Maurizio, Sadeghpour, Azita, Stroup, Diane, Galli, Giovanni, Chiang, John Y.L. 01 November 1998 (has links)
The gene encoding cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt - 149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139- AGTTCAaggccGGGTAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter- transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.
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