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The ability of induced corpora lutea (CL) to prevent induction of porcine parturition by dexamethasone or prostaglandin F2α (PGF2α) was tested in two experiments. In the first experiment, 19 pregnant crossbred sows were injected intramuscularly (IM) with 1,500 IU of Pregnant Mare Serum Gonadotropin (PMSG) on day 104 followed by 1,500 IU of Human Chorionic Gonadotropin (HCG) on day 106. Eight of the 19 sows were also injected IM with 100 mg of dexamethasone/day on days 109 through 112. Five other sows served as untreated controls. Treatment with gonadotropins to induce CL formation increased length of gestation compared to controls (120.0 vs114.8, SD = 2.3, respectively). Following adjustment by covariance (b = .16, P<.05) for number of induced CL (0 to 27), it was revealed that dexamethasone had no effect on length of gestation in sows containing one or more new CL: PMSG-HCG, 119.4 and PMSG-HCG + dexamethasone, 121.8 days. Litter size, live birth rate and number of CL of pregnancy did not differ among control, PMSG-HCG or PMSG-HCG + dexamethasone-treated sows.Twenty-eight pregnant crossbred sows and gilts were utilized in the 2 × 3 factorial design of experiment 2 as follows: eight received no treatment and served as controls; eight received IM injections of 500 mg of progesterone in oil/day on days 107 to 114 of gestation andnew CL were induced in 12 using the same doses of PMSG and HCG as was used in experiment 1 with treatment starting on day 99. In half of the females of each group, 5 mg of PGF2α was infused into a marginal ear vein during a 9-hr period on day 109. Mean lengths of gestation for the six subgroups were: control, 115.5; PGF2α, 110.0; exogenous progesterone, 117.5; exogenous progesterone + PGF2α, 117.5; PMSG-HCG, 117.5; PMSG-HCG + PGF2α, 118.5.The proportion of piglets born live was lower (P<.01) in females with an additional source of progesterone. Infusion of PGF2α resulted in a further decrease (P<.05) when premature parturition was prevented. The weight of the CL of pregnancy in pigs with an additional source of progesterone was decreased (P<.01). A further decrease in weight of CL (P<.01) was observed in females infused with PGF2α. Weight of the induced CL was not affected by PGF2α. Serum progestin concentrations of 25 to 30 ng/ml were measured in the PMSG-HCG-treated females on days 107 and 109 and the exogenous progesterone-treated pigs on day 109. Between days 109 and 110 progestin concentrations decreased in all infused and PMSG-HCG-treated females; remained the same in controls and increased further in females treated with exogenous progesterone but not infused with PGF2α. The prepartum decline in serum progestins was similar in all groups.

Phosphatidylinositol 3'-kinase (PI 3'-kinase) is activated in insulin-stimulated cells by the binding of the SH2 domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1). We have previously shown that both tyrosyl-phosphorylated IRS-1 and mono-phosphopeptides containing a single YXXM motif activate PI 3'-kinase in vitro. However, activation by the monophosphopeptides was significantly less potent than activation by the multiply phosphorylated IRS-1. We now show that the increased potency of PI 3'-kinase activation by IRS-1 relative to phosphopeptide is not due to tertiary structural features IRS-1, as PI 3'-kinase is activated normally by denatured, reduced, and carboxymethylated IRS-1. Furthermore, activation of PI 3'-kinase by bis-phosphorylated peptides containing two YXXM motifs is 100-fold more potent than the corresponding mono-phosphopeptides and similar to activation by IRS-1. These data suggest that tyrosyl-phosphorylated IRS-1 or bis-phosphorylated peptides bind simultaneously to both SH2 domains of p85. However, these data cannot differentiate between an activation mechanism that requires two-site occupancy for maximal activity as opposed to one in which bivalent binding enhances the occupancy of a single activating site. To distinguish between these possibilities, we produced recombinant PI 3'-kinase containing either wild-type p85 or p85 mutated in its N-terminal, C-terminal, or both SH2 domains. We find that mutation of either SH2 domains significantly reduced phosphopeptide binding and decreased PI 3'-kinase activation by 50%, whereas mutation of both SH2 domains completely blocked binding and activation. These data provide the first direct evidence that full activation of PI 3'-kinase by tyrosylphosphorylated proteins requires occupancy of both SH2 domains in p85.

Intracellular-reduced glutathione (GSH) was removed by thiol-oxidation with diamide during in vitro perfusion of the corneal endothelium. By 15 min the normal mosaic-like pattern of the endothelial cells was disrupted by serpentine-like lines of cell separation at the cell juntions. After 45 min of perfusion, infividual clusters of cells formed cup-shaped islands. The resultant exposure of Descemet's membrane to the perfusion solution resulted in corneal swelling. Transmission electron microscopy revealed that the endothelial cells separated at the apical junctions and that the microfilaments in the apical cytoplasm of cells formed dense bands, whereas the other subcellular organelles were normal in appearance. The change in cellular shape may be due to loss of cellular adhesion which results in the condensation of the microfilaments or contraction of the microfilaments. The addition of glucose to the perfusate prevented the diamide effect, and the diamide effect could be reversed upon removal and perfusion of a glutathione bicarbonate Ringer's solution. These results suggest that the ratio of reduced to oxidized glutathione in the endothelial cells plays a role in the maintenance of the endothelial cell barrier function.