Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.

Summary. Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, resolves on delivery of the placenta. Normal pregnancy is itself characterized by systemic inflammation, oxidative stress and alterations in levels of angiogenic factors and vascular reactivity. This is exacerbated in preeclampsia with an associated breakdown of compensatory mechanisms, eventually leading to placental and vascular dysfunction. The underlying pathology of preeclampsia is thought to be a relatively hypoxic or ischemic placenta. Both the placenta and maternal vasculatures are major sources of reactive oxygen and nitrogen species which can interact to produce peroxynitrite a powerful prooxidant that covalently modifies proteins by nitration of tyrosine residues, to possibly alter vascular function in preeclampsia. The linkage between placental hypoxia and maternal vascular dysfunction has been proposed to be via placental syncytiotrophoblast basement membranes shed by the placenta or via angiogenic factors which include soluble flt1 and endoglin secreted by the placenta that bind vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) in the maternal circulation. There is also abundant evidence of altered reactivity of the maternal and placental vasculature and of the altered production of autocoids in preeclampsia. The occurrence of preeclampsia is increased in women with preexisting vascular disease and confers a longterm risk for development of cardiovascular disease. The vascular stress test of pregnancy thus identifies those women with a previously unrecognized at risk vascular system and promotes the development of preeclampsia. Preexisting maternal vascular dysfunction intensified by placental factors is possibly responsible for the individual pathologies of preeclampsia.

Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a “molecular fingerprint” of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings.

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Peroxynitrite, a potent pro-oxidant formed from the interaction of superoxide and nitric oxide, has been widely reported to be nitrating tyrosine residues in proteins resulting in the formation of nitrotyrosine. Biological nitration of tyrosine, a footprint of oxidative injury, has been found to occur in various pathological states including pre-eclampsia, a leading cause of maternal mortality and increased perinatal mortality. Oxidative stress is a major contributor to endothelial dysfunction in pre-eclampsia. Previously, we have demonstrated increased nitrotyrosine immunostaining in placental villous vascular endothelium, surrounding vascular smooth muscle and villous stroma from pre-eclamptic or diabetic pregnancies. Immunoprecipitation (IP) with antinitrotyrosine antibodies followed by immunoblot analysis identified increased nitration of phospho-p38 mitogen-activated protein kinase (MAPK) in the pre-eclamptic placenta. The catalytic activity of p38 MAPK and concentration of phospho-p38 MAPK was also found to be reduced in placentae from pre-eclamptic pregnancies. Comparison of peptide masses of a 42-kDa protein obtained by mass spectrometry with masses of a theoretical tryptic digest of p38 MAPK that was modified by phosphorylation and nitration identified the protein to be p38 MAPK.

This study examines the regulation of major enzymes in prostaglandin E(2) (PGE(2)) synthesis by glucocorticoids in separate cultures of human amnion epithelial and fibroblast cells at term. Cytosolic phospholipase A(2) (cPLA(2)), cytosolic PGES (cPGES), and microsomal PGES (mPGES) mRNA were expressed at similar levels in both cell types, whereas a greater prostaglandin H synthase type 2 (PGHS-2) mRNA expression was observed in amnion fibroblasts than in epithelial cells. Amnion fibroblasts produced 50-fold more PGE(2) per cell than epithelial cells. Dexamethasone (0.01-1 microM) increased PGE(2) production in amnion fibroblasts in a concentration-dependent manner but did not affect PGE(2) production in amnion epithelial cells. Both mRNA and protein expression of cPLA(2) and PGHS-2 but not cPGES and mPGES were increased in a dose-dependent manner by dexamethasone (0.01-1 microM) in amnion fibroblasts. Induction of cPLA(2) and PGHS-2 mRNA by dexamethasone was blocked by RU486. Dexamethasone did not affect PGHS-2, cPGES, and mPGES mRNA expression in amnion epithelial cells. In conclusion, amnion fibroblasts express a higher level of PGHS-2 mRNA and produced more PGE(2) per cell than amnion epithelial cells at term of human pregnancy. Glucocorticoids increase PGE(2) production only in the amnion fibroblasts mainly through induction of cPLA(2) and PGHS-2 expression.

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We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 minutes after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2–5 nM Kds for cocaine, and prevented cocaine’s entry into the brain by sequestering it in the plasma. Pharmacokinetic and radio-ligand binding assays supported designation of the highest producing clone (85) as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.