Browsing by Author "Vismer, Michael John"

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    An operational model for estrogenic action in the presence of sex hormone binding globulin (SHBG)
    (Stellenbosch : Stellenbosch University, 2007-03) Vismer, Michael John; Louw, A.; Rohwer, J. M.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The aim of this study was to build a mathematical model that describes the binding of 17- -estradiol (E2) to estrogen receptor (ER- ) and the influence the sex hormone binding globulin (SHBG) has on this interaction. The influence of SHBG on the transactivation of an estrogen response element, via ligand bound ER- , was also studied. COS-1 cells, derived from the kidney of a green african monkey, were used to study the binding of E2 to ER- in the absence of SHBG. The influence of SHBG on the binding of E2 to ER- was studied using Hep89 cells, human hepatacoma carcinoma, which express SHBG endogenously and are stably transfected with the ER- gene. Human pregnancy plasma was used to study the interaction of E2 with SHBG in the absence of ER- . The results of this study have shown that the Kd (E2) for ER- was determined as between 3.4nM and 4.4nM in the absence of SHBG. With respect to the binding of E2 to ER- it was not possible to determine the Kd app and Bmax for ER- using the Hep89 experimental system. The Kd (E2) for SHBG was not determined using the human pregnancy plasma experimental system. With the aid of mathematical modelling, a model of the Hep89 and human pregnancy plasma experimental systems, was built. The results of the numerical modelling, using mathematical modelling, showed that the presence of albumin together with SHBG was the reason that the Kd app (E2) could not be determined in the Hep89 experimental system. With respect to the use of human pregnancy plasma to determine the Kd (E2) for SHBG it was shown that if the plasma was diluted 200 times it would have been possible to determine the Kd app (E2) for SHBG, in the presence of albumin. Ligand independent transactivation of an estrogen response element was shown to be a problem in the COS-1 cell system when promoter reporter gene assays were undertaken. As COS-1 cells were used as a control for the absence of SHBG no further promoter reporter gene assays were undertaken using the Hep89 experimental system.