Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum

The role of prostaglandin E2 (PGE2) in basal and noradrenaline (NA)-stimulated utilization of high density lipoprotein (HDL) as a source of cholesterol for progesterone synthesis was examined. In Experiment 1, a cannula was inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery) in mature heifers, to facilitate infusion of NA (4 mg/30 min; n = 3) on day 10 of the estrous cycle. Three other heifers were similarly cannulated to serve as control. Before, during, and after NA or saline infusion, blood samples from the vena cava were collected every 5-15 min for analysis of PGE2, progesterone, and cholesterol. Each NA infusion stimulated (P < 0.01) secretion of both hormones in heifers. Short-duration increases (P < 0.05) in progesterone were observed due to the infusion of NA while cholesterol was not altered significantly. In addition, increases in PGE2 concentrations (P < 0.05) compared to controls were seen after NA infusion. Therefore, we used an in vitro model to verify the effect of PGE2 on HDL utilization by luteal cells from day 5 to 10 of the estrous cycle. In the preliminary experiment, 10(-6) M of PGE2 out of four different doses examined was selected for further studies, since it evoked the highest release of progesterone. In the next experiment, it was found that HDL increases progesterone secretion by luteal cells and both PGE2 and LH increased (P < 0.05) the response to HDL while NA did not. In the last in vitro experiment, progesterone stimulated PGE2 secretion by luteal cells. In conclusion, PGE2 may be directly involved in the utilization of cholesterol from HDL for progesterone synthesis. Furthermore, PGE2 may influence NA-stimulated progesterone secretion by the corpus luteum (CL). It is concluded that there is a positive feedback loop between progesterone and luteal PGE2 during days 5-10 of the estrous cycle.     

Non-genomic effect of steroids on oxytocin-stimulated intracellular mobilization of calcium and on prostaglandin F2alpha and E2 secretion from bovine endometrial cells

Progesterone (P4) was found to interfere directly with the interaction of oxytocin (OT) with its own receptor in bovine endometrium. The aim of these studies was to investigate whether other steroids have a similar effect. Endometrial slices and epithelial endometrial cells from days 14 to 18 of the estrous cycle were used. Progesterone (P4), pregnenolone (P5), 17beta-hydroxyprogesterone (17-OHP4), the P4 receptor antagonist (aP4), and testosterone (T4) did not affect (P > 0.01) basal secretion of PGE2 and PGF 2alpha during 4h of incubation but all steroids inhibited (P < 0.05) OT-stimulated PGF2alpha secretion both from endometrial slices and from dispersed cells. None of the steroids used affected OT-stimulated PGE2 secretion from the cells (P > 0.01). In the next experiment it was studied whether P5, 17-OHP4 and P4 pretreatment for 30min modifies intracellular mobilization of Ca(2+) in response to OT. Oxytocin induced a rapid increase in intracellular Ca(2+)concentrations within 15s, while cells pretreated with steroids this increase occurred later. The total amount of intracellular Ca(2+)concentrations was lower (P < 0.05) in cells preincubated with steroids compared to controls. We conclude that steroids and aP4 are able to suppress OT-stimulated endometrial PGE2 and PGF2alpha secretion via a non-genomic pathway.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.