Isoprostanes, prostaglandins and tocopherols in pre-eclampsia, normal pregnancy and non-pregnancy

This study is designed to evaluate whether oxidative stress and inflammation are involved in severe pre-eclampsia compared to normal pregnancy and non-pregnancy. We have measured plasma and urinary levels of 8-iso-PGF2alpha, a major isoprostane as an indicator of oxidative stress; plasma and urinary 15-keto-dihydro-PGF2alpha, a major metabolite of cyclooxygenase-catalysed PGF2alpha as an indicator of inflammatory response, and plasma -alpha-and -gamma-tocopherol in 18 pre-eclamptic, 19 normal pregnancy and 20 non-pregnant women. Pregnant women had significantly higher levels of 8-iso-PGF2alpha and PGF2alpha metabolite as compared to the non-pregnancy. Levels of 8-iso-PGF2alpha in the pre-eclamptic women did not differ from the normal pregnancy but PGF2alpha metabolite levels were significantly higher in normal pregnancy. On the other hand, gamma-tocopherol levels were significantly lower in pre-eclampsia than normal pregnancy. In contrast, the concentration of alpha-tocopherol was very similar between the groups. alpha-and gamma-tocopherol levels were significantly lower in pregnancy compared to non-pregnancy. Although no direct evidence of oxidative stress and inflammatory response was observed in severe pre-eclampsia, a reduction of gamma-tocopherol suggests the possible precedence of oxidative stress in this condition. Higher levels of isoprostanes and prostaglandin metabolite in late pregnancy suggest the importance of both free radicals and cyclooxygenase-catalysed oxidation products in normal biological processes of pregnancy.     

Nicotine enhances neovascularization and promotes tumor growth

Solid tumors require vascularization for their growth. Bone marrow-derived endothelial progenitor cells participate in tumor angiogenesis. Here, we show that nicotine markedly accelerated growth of colon cancer cells inoculated subcutaneously in mice but had no effect on proliferation of carcinoma cells in vitro. We found that the tumor growth was associated with increased vascularization of the tumor and that bone marrow-derived cells contributed to the formation of the new blood vessels. Our findings show that nicotine promotes tumor growth, at least in part, by stimulating tumor-associated neovascularization.     

Up-regulation of Rnd1 during pregnancy serves as a negative-feedback control for Ca2+ sensitization of contractile elements in rat myometrium

We investigated the role of Rnd1, a member of the small GTP-binding Rho protein family, in the change in Ca(2+) sensitivity of contractile element in rat myometrium at estrus, gestation, and postpartum stages. In the permeabilized muscles, GTPgammaS or carbachol with GTP increased Ca(2+) sensitivity of contractile force in non-pregnant myometrium at the estrus stage, whereas these stimuli were ineffective in pregnant myometrium at day 21. After postpartum, the reduced Ca(2+) sensitization was recovered. Semi-quantitative RT-PCR analysis indicated that the expressions of RhoA, ROCKI, and ROCKII were not significantly different between non-pregnant and pregnant myometria. In contrast, the expression of Rnd1 was increased during the course of pregnancy, reaching a maximal at day 21, and rapidly declined after the delivery. On the other hand, Ca(2+) sensitization of contractile elements was decreased during the progress in gestation. These results suggest that Rnd1 may have an important role as a negative-feedback control of uterine contraction during gestation through the inhibition of RhoA-mediated increase in the Ca(2+) sensitivity of contractile elements.     

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.     

Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.     

Disruption of adiponectin causes insulin resistance and neointimal formation

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.     

Cutting edge: a novel role for Fas ligand in facilitating antigen acquisition by dendritic cells

Fas ligand (FasL)-expressing tumor cells are found to effectively mediate rejection of the coinoculated FasL negative parental cells while having no effect on the growth of histologically distinct tumor cells. These observations indicate that FasL induces a specific immune response against Ag derived from FasL-bearing tumors and suggest a possible role for FasL in tumor Ag presentation. Indeed, tumor cells expressing FasL can efficiently interact with dendritic cells (DCs) and this interaction requires the expression of membrane-bound FasL on tumors and Fas on DCs. Moreover, DCs cocultured with FasL-expressing tumors are able to elicit a tumor-specific immune response in vivo, suggesting that DCs acquire tumor Ag during the Fas/FasL-mediated DC-tumor contact. These results identify a novel role for FasL in augmenting tumor-DC interactions and subsequent tumor Ag acquisition by DCs, and suggest that FasL-expressing tumor cells could be used to generate tumor-specific DC vaccines.     

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.