Acute parathyroid hormone response: sensitivity, relationship to hypocalcemia, and rapidity.
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Biomedical subjects
W Hunziker
Publications and source records attributed to W Hunziker.
Overexpression of the human neonatal Fc-receptor alpha-chain in trophoblast-derived BeWo cells increases cellular retention of beta2-microglobulin.
Major histocompatibility complex (MHC) class I and MHC class I-type molecules such as the neonatal Fcgamma-receptor, FcRn, are heterodimers consisting of a transmembrane alpha-chain non-covalently associated with beta2-microglobulin (beta2m). Human placental villous syncytiotrophoblast (STB) lacks MHC class I molecules, but express hFcRn that mediates materno-fetal transmission of immunoglobulin G (IgG). Trophoblast-derived BeWo cells that are used to study placental IgG transport likewise express beta2m and low levels of hFcRn alpha-chain. The contribution of FcRn alpha-chain in retention and subcellular distribution of beta2m in STB and BeWo cells is unclear. To investigate this issue, we increased expression of hFcRn alpha-chain in BeWo cells (BeWo/hFcRn) by cDNA transfection. Overexpressed hFcRn protein exhibited the characteristic pH-dependent IgG binding and association with beta2m. In comparison to parental BeWo cells, beta2m mRNA levels in BeWo/hFcRn cells were not significantly altered, but total cell-associated beta2m protein was increased by 120%. Treatment of BeWo and BeWo/hFcRn cells with brefeldin A, an inhibitor of the secretory pathway, abrogated this effect, demonstrating that hFcRn alpha-chain expression retained otherwise secreted beta2m. Flow cytometry revealed that beta2m plasma membrane expression was unaffected by alpha-chain overexpression whereas by fluorescence microscopy a preferential staining of beta2m in peripheral endosomes was observed.
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A spin-column procedure for estrogen receptor equilibrium and competition binding analysis.
Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.
Alphavirus Infections↗
Factors affecting the distribution and stability of unoccupied 1,25-dihydroxyvitamin D3 receptors.
Recently our laboratory reported that unoccupied 1,25(OH)2D3 receptors are found in the nuclei/chromatin fraction under low salt conditions in vitro (J. Biol. Chem. 255, 6799-6805, 1980). Additionally, various conditions exert differential effects on receptor solubilization and stability in vitro, potentially leading to confusion when these effects are not fully appreciated. In order to facilitate future studies, we have complied a review of these factors and their effects. The conditions discussed include dilution, ionic strength (KCl and molybdate), protease inhibitors (Trasylol, PMSF and TPCK), temperature, ligand, glycerol, dithiothreitol, and EDTA.
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