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Highly degenerate oligonucleotide primers designed from regions conserved between proteinaserine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encoding putative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein-serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP-dependent protein kinase (PKA) families and its gene has thus been termed pkb. Expression of the pkb gene is general but highest in brain, heart and lung. Translation of pkb RNA in vitro generated a 57-kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60-kDa protein which was partially purified by Mono Q anion-exchange chromatography. Column fractions containing PKB-immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase. [ABSTRACT FROM AUTHOR]

The article explores the association of high plasma levels of interleukin-18 (IL-18) with the documented natural killer (NK) cell failure in systemic-onset juvenile idiopathic arthritis (JIA). It notes the characterization of the phenotype and function of NK cells by staining and functional assays in vitro. According to the article, impaired NK cell function in systemic-onset JIA features a defect in the phosphorylation of IL-18RB.

Abstract: Phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (PKB/c-akt) have been implicated as critical mediators of cytokine-induced survival signals. In this study, we have utilized an IL-5 dependent hematopoietic cell line (TF-1) to investigate the signaling events involved in cytokine-dependent erythroblast survival. We demonstrate that IL-5 rescues TF-1 cells from apoptosis through a PI3K/PKB-dependent signaling pathway. Cytokine-withdrawal leads to activation of the Forkhead transcription factor FOXO3a and subsequent expression of the pro-apoptotic Bcl-2 family member Bim. Bim is itself sufficient to induce apoptosis in these cells. Importantly, activation of an inducible active FOXO3a mutant is alone sufficient for upregulation of Bim expression and induction of apoptosis. These data define a mechanism by which survival factors inhibit the default apoptotic pathway and can regulate TF-1 erythroblast survival. [Copyright &y& Elsevier]

In human neutrophils, both changes in intracellular Ca2+ concentrations, [Ca2+ ]i , and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca2+ ]i and its effector molecule calmodulin in human neutrophils. Increased [Ca2+ ]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3α (GSK-3α). Inhibition of calmodulin using a calmodulin antagonist N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N -formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3α phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca2+ /calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca2+ ]i . Taken together, our data indicate critical roles for changes in [Ca2+ ]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca2+ /calmodulin sensitive pathway in human neutrophils. [ABSTRACT FROM AUTHOR]