Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Purification and characterization of receptors for myoinositol trisphosphate and myoinositol tetrakis phosphate from Entamoeba histolytica.

The microsomal fraction from the log phase of Entamoeba histolytica cells contains Ins(1,4,5)P3 and Ins(1,3,4,5)P4 binding activity. The binding proteins/receptors for both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were purified and found to be specific for each ligand. The molecular masses for native proteins for InsP3 and InsP4 are 138 kDa and 130 kDa respectively having subunits of 69 kDa and 64 kDa respectively. That these proteins are associated with Ca2+ release was confirmed by including these proteins separately in proteoliposomes and adding InsP3 and InsP4 in both the cases.

Correlation between the lack of mononuclear cell cytotoxicity against schistosomula in vitro with schistosomiasis

A comparison was made of the reactivity of mononuclear cells from subjects and from S. mansoni-infected patients. The following parameters were evaluated: 1) ability of mononuclear cells to kill schistosoma in the presence of complement; 2) [3H]-inositol incorporation into phosphatidylinositol (PI) and the rate of inositolphosphates (IPx) released. Cells from normal subjects, but not from S. mansoni infected patients, were able to kill schistosomula in vitro. A decrease in inositolpolyphosphates (IPx) was observed for phytohemagglutinin (PHA)-stimulated mononuclear cells from infected patients when compared with mononuclear cells from normal subjects after 24 h of incubation. The results suggest that the reactivity of mononuclear cells from infected patients is altered under conditions of nonspecific stimulation with PHA when compared with normal cells